The Ecology and Glycobiology of Prymnesium parvum

Prymnesium parvum is a toxin-producing haptophyte that causes harmful algal blooms (HABs) globally, leading to large scale fish kills that have severe ecological and economic implications. A HAB on the Norfolk Broads, U.K, in 2015 caused the deaths of thousands of fish. Using optical microscopy and 16S rRNA gene sequencing of water samples, P. parvum was shown to dominate the microbial community during the fish-kill. Using liquid chromatography-mass spectrometry (LC-MS), the ladder-frame polyether prymnesin-B1 was detected in natural water samples for the first time. Furthermore, prymnesin-B1 was detected in the gill tissue of a deceased pike (Exos lucius) taken from the site of the bloom; clearing up literature doubt on the biologically relevant toxins and their targets. Using microscopy, natural P. parvum populations from Hickling Broad were shown to be infected by a virus during the fish-kill. A new species of lytic virus that infects P. parvum was subsequently isolated, Prymnesium parvum DNA virus (PpDNAV-BW1). Morphological analysis and genome sequencing revealed PpDNAV-BW1 to belong to the Megaviridae family of algal viruses. We propose that viral lysis of P. parvum may act as a novel release mechanism for intracellular toxins. The sialic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) has recently been shown to be important in viral infections of microalgae. LC-MS was used to demonstrate that P. parvum contains KDN. Candidate sequences for KDN biosynthesis from P. parvum were cloned and expressed and shown to produce cytidine monophosphate-activated KDN (CMPKDN). Using the newly characterized sequences in BLASTp analysis, we revealed that sialic acid biosynthesis is widespread amongst algae. Using bioinformatics, NDP-β-L-rhamnose biosynthesis was explored in P. parvum and across other algae. We propose that the haptophytes have acquired bacterial TDP-β-L-rhamnose biosynthetic genes from horizontal gene transfer and subsequently passed them on to some dinoflagellate species. Sugar-nucleotide profiling of two representative algae support this proposition

CuAAC click chemistry for the enhanced detection of novel alkyne-based natural product toxins

In the context of discovering and quantifying terminal alkyne-based natural products, here we report the combination of CuAAC click chemistry with LC-MS for the detection of polyether toxins (prymnesins) associated with harmful algal blooms. The added-value of the CuAAC-based approach is evident from our ability to detect novel prymnesin-like compounds in algal species with previously uncharacterised toxins

Isolation and characterization of a double stranded DNA mgavirus infecting the toxin-producing haptophyte Prymnesium parvum

Prymnesium parvum is a toxin-producing haptophyte that causes harmful algal blooms globally, leading to large-scale fish kills that have severe ecological and economic implications. For the model haptophyte, Emiliania huxleyi, it has been shown that large dsDNA viruses play an important role in regulating blooms and therefore biogeochemical cycling, but much less work has been done looking at viruses that infect P. parvum, or the role that these viruses may play in regulating harmful algal blooms. In this study, we report the isolation and characterization of a lytic nucleo-cytoplasmic large DNA virus (NCLDV) collected from the site of a harmful P. parvum bloom. In subsequent experiments, this virus was shown to infect cultures of Prymnesium sp. and showed phylogenetic similarity to the extended Megaviridae family of algal viruse

Discovery of an RmlC/D fusion protein in the microalga <i>Prymnesium parvum</i> and its implications for NDP-β-L-rhamnose biosynthesis in microalgae

The 6-deoxy sugar L-rhamnose (L-Rha) is found widely in plant and microbial polysaccharides and natural products. The importance of this and related compounds in host–pathogen interactions often means that L-Rha plays an essential role in many organisms. L-Rha is most commonly biosynthesized as the activated sugar nucleotide uridine 5-diphospho-L-rhamnose (UDP-L-Rha) or thymidine 5-diphospho-L-rhamnose (TDP-L-Rha). Enzymes involved in the biosynthesis of these sugar nucleotides have been studied in some detail in bacteria and plants, but the activated form of L-Rha and the corresponding biosynthetic enzymes have yet to be explored in algae. Here, using sugar-nucleotide profiling in two representative algae, Euglena gracilis and the toxin-producing microalga Prymnesium parvum, we show that levels of UDP- and TDP-activated L-Rha differ significantly between these two algal species. Using bioinformatics and biochemical methods, we identified and characterized a fusion of the RmlC and RmlD proteins, two bacteria-like enzymes involved in TDP-L-Rha biosynthesis, from P. parvum. Using this new sequence and also others, we explored L-Rha biosynthesis among algae, finding that although most algae contain sequences orthologous to plant-like L-Rha biosynthesis machineries, instances of the RmlC-RmlD fusion protein identified here exist across the Haptophyta and Gymnodiniaceae families of microalgae. On the basis of these findings, we propose potential routes for the evolution of nucleoside diphosphate -L-Rha (NDP-L-Rha) pathways among algae

Agl24 is an ancient archaeal homolog of the eukaryotic N-glycan chitobiose synthesis enzymes

Protein N-glycosylation is a post-translational modification found in organisms of all domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in Eukaryotes, although the enzyme catalyzing this reaction remains unknown. Here, we report the identification of a thermostable archaeal β-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed its function as an inverting β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed similarities of Agl24 to the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which are catalyzing the same or a similar reaction, respectively. Phylogenetic analysis of Alg14/13 homologs indicates that they are ancient in Eukaryotes, either as a lateral transfer or inherited through eukaryogenesis.</p

Insights into toxic prymnesium parvum blooms:The role of sugars and algal viruses

Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum, ambiguity still surrounds the responsible ichthyotoxins in P. parvum blooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvum occurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum, P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms

Does Council Tax Valuation Band (CTVB) correlate with Under-Privileged Area 8 (UPA8) score and could it be a better 'Jarman Index'?

BACKGROUND: Widespread scepticism persists on the use of the Under-Privileged Area (UPA8) score of Jarman in distributing supplementary resources to so-attributed 'deprived' UK general practices. The search for better 'needs' markers continues. Having already shown that Council Tax Valuation Band (CTVB) is a predictor of UK GP workload, we compare, here, CTVB of residence of a random sample of patients with their respective 'Jarman' scores. METHODS: Correlation coefficient is calculated between (i) the CTVB of residence of a randomised sample of patients from an English general practice and (ii) the UPA8 scores of the relevant enumeration districts in which they live. RESULTS: There is a highly significant correlation between the two measures despite modest study size of 478 patients (85% response). CONCLUSIONS: The proposal that CTVB is a marker of deprivation and of clinical demand should be examined in more detail: it correlates with 'Jarman', which is already used in NHS resource allocation. But unlike 'Jarman', CTVB is simple, objective, and free of the problems of Census data. CTVB, being household-based, can be aggregated at will

Assessing the toxicity and mitigating the impact of harmful Prymnesium blooms in eutrophic waters of the Norfolk Broads

Prymnesium parvum is a toxin-producing microalga, which causes harmful algal blooms globally, frequently leading to massive fish kills that have adverse ecological and economic implications for natural waterways and aquaculture alike. The dramatic effects observed on fish are thought to be due to algal polyether toxins, known as the prymnesins, but their lack of environmental detection has resulted in an uncertainty about the true ichthyotoxic agents. Using qPCR, we found elevated levels of P. parvum and its lytic virus, PpDNAV-BW1, in a fish-killing bloom on the Norfolk Broads, United Kingdom, in March 2015. We also detected, for the first time, the B-type prymnesin toxins in Broads waterway samples and gill tissue isolated from a dead fish taken from the study site. Furthermore, Norfolk Broads P. parvum isolates unambiguously produced B-type toxins in laboratory-grown cultures. A 2 year longitudinal study of the Broads study site showed P. parvum blooms to be correlated with increased temperature and that PpDNAV plays a significant role in P. parvum bloom demise. Finally, we used a field trial to show that treatment with low doses of hydrogen peroxide represents an effective strategy to mitigate blooms of P. parvum in enclosed water bodies

Health utility indices and equity considerations

The aim of this paper is to propose methods that incorporate equity concerns into cost utility analysis. The focus of the paper is on QALYs, but the results apply to health utility indices in general. Two interpretations of QALYs are considered: QALYs as (von Neumann Morgenstern) utilities and QALYs as measures of health. A justification is provided for aggregating consistently scaled “QALYs as utilities” over individuals. The conditions underlying unweighted aggregation of QALYs are identified. These conditions exclude two common types of equity concern. Algorithms are proposed that take into account equity concerns and that are relatively easy to apply