Christopher O. Miles

The evolutionary origins of three new Neotyphodium endophyte species from grasses indigenous to the Southern Hemisphere.

Members of the genus Neotyphodium are asexual, seedborne, protective fungal endophytes of cool season grasses that have likely evolved either directly from sexual Epichloë; species, or by the interspecific hybridization of distinct lineages of Epichloë; and Neotyphodium. We investigated the evolutionary origins of Neotyphodium endophytes from several grasses that are indigenous to the Southern Hemisphere using a multiple-gene phylogenetic approach. Intron regions of the genes encoding β-tubulin (tub2), translation elongation factor 1-α (tef1) and actin (act1) were amplified by polymerase chain reaction and sequenced. Phylogenetic analyses of these sequences, aligned with homologous sequences from Epichloë; spp., revealed the evolutionary origins of the Southern Hemisphere endophytes, where one lineage of apparently non-hybrid origin, and three lineages of unique interspecific hybrid origin were identified. On the basis of morphology, host range and evolutionary history, we propose three new species of Neotyphodium. Neotyphodium aotearoae was isolated from Echinopogon ovatus populations from New Zealand and Australia, and comprised a unique, apparently non-hybrid lineage within the Epichloë; species phylogeny. In contrast, an interspecific hybrid lineage was identified from two Australian Ec. ovatus populations, whose ancestry apparently involved lineages closely related to extant E. festucae and an E. typhina genotype similar to that of isolates from Poa pratensis. Endophytes infecting South African Melica racemosa and M. decumbens (dronkgras) appeared to be hybrids of E. festucae and N. aotearoae or close relatives. The names N. australiense and N. melicicola are proposed for these two hybrid lineages, respectively. The origin of N. tembladerae, an established endophyte species from South American Poa and Festuca spp., was also investigated. Neotyphodium tembladerae appeared to be of hybrid origin, involving E. festucae and an E. typhina genotype similar to that of isolates from Poa nemoralis. The results of this study highlight the widespread occurrence of interspecific hybrid Neotyphodium lineages on a global scale, and the extent of endophyte gene-flow between the Northern and Southern Hemispheres.

Isolation, structural determination and acute toxicity of pinnatoxins E, F and G.

Pinnatoxins and pteriatoxins are a group of cyclic imine toxins that have hitherto only been isolated from Japanese shellfish. As with other cyclic imine shellfish toxins, pinnatoxins cause rapid death in the mouse bioassay for lipophilic shellfish toxins, but there is no evidence directly linking these compounds to human illness. We have identified the known pinnatoxins A (1) and D (6), and the novel pinnatoxins E (7), F (8) and G (5), in a range of shellfish and environmental samples from Australia and New Zealand using LC-MS. After isolation from the digestive glands of Pacific oysters, the structures of the novel pinnatoxins were determined by mass spectrometry and NMR spectroscopy, and their LD(50) values were evaluated by ip administration to mice. Examination of the toxin structures, together with analysis of environmental samples, suggests that pinnatoxins F and G are produced separately in different dinoflagellates. Furthermore, it appears probable that pinnatoxin F (8) is the progenitor of pinnatoxins D (6) and E (7), and that pinnatoxin G (6) is the progenitor of both pinnatoxins A-C (1 and 2) and pteriatoxins A-C (3 and 4), via metabolic and hydrolytic transformations in shellfish.

Polyclonal antibodies to domoic acid, and their use in immunoassays for domoic acid in sea water and shellfish.

Ovine antibodies raised against conjugates linked through the secondary amino group of domoic acid (1) were used, together with activated-ester-derived conjugates of domoic acid (DA) as the plate coater, to develop a robust indirect competitive enzyme-linked immunosorbent assay (cELISA) for DA in shellfish and seawater. The ELISA was used to analyze shellfish samples for DA, and was compatible with several extraction procedures. The ELISA had a detection limit below 0.01 ng ml(-1), a limit of quantitation (LOQ) of 0.15 ng ml(-1) and a working range of 0.15-15 ng ml(-1) DA. The LOQ is equivalent to 38 ng g(-1) DA in shellfish flesh, assuming a 250-fold dilution during extraction. This is more than 500 times lower than the maximum permitted level (20 microg g(-1) flesh). The ELISA is designed for use alongside regulatory analyses, and, following formal validation, should be available for pre-screening of regulatory shellfish flesh samples. The ELISA was also shown to be appropriate for analysis of DA in algal cultures and in samples of seawater, and thus has the potential to provide early warning of developing algal blooms.

Formation of azaspiracids-3, -4, -6, and -9 via decarboxylation of carboxyazaspiracid metabolites from shellfish.

The azaspiracid (AZA) class of phycotoxins has been responsible for extended closures of shellfisheries in various locations around Europe, where levels of AZA1-3 are regulated in shellfish. Since their discovery in 1995, AZAs have been the focus of much research, resulting in the discovery of numerous analogues. During studies of procedures for processing of AZA-contaminated mussels ( Mytilus edulis ), an unusual phenomenon was observed involving AZA3. In uncooked tissues, AZA3 levels would increase significantly when heated for short periods of time in the absence of water loss. A similar increase in AZA3 concentrations occurred during storage of shellfish tissue reference materials for several months at temperatures as low as 4 degrees C. Concentrations of AZA1 and AZA2 did not change during these experiments. Several possible explanations were investigated, including an AZA3-specific matrix effect upon heating of tissues, release of AZA3 from the matrix, and formation of AZA3 from a precursor. Preliminary experiments indicated that toxin conversion was responsible, and more detailed studies focused on this possibility. LC-MS analysis of heating trials, deuterium labeling experiments, and kinetic studies demonstrated that a carboxylated AZA analogue, AZA17, undergoes rapid decarboxylation when heated to produce AZA3. Heat-induced decarboxylation of AZA19, AZA21, and AZA23 to form AZA6, AZA4, and AZA9, respectively, was also demonstrated. This finding is of great significance in terms of procedures used in the processing of shellfish for regulatory analysis, and it exemplifies the role that chemical analysis can play in understanding the contribution of metabolic processes to the toxin profiles observed in shellfish samples.

A convenient and cost-effective method for monitoring marine algal toxins with passive samplers

Passive sampling disks were developed based on the method of MacKenzie, L, Beuzenberg, V., Holland, P., McNabb, P., Selwood, A. [2004. Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves. Toxicon 44, 901-918] and protocols were formulated for recovering toxins from the adsorbent resin via elution from small columns. The disks were used in field studies to monitor in situ toxin dynamics during mixed algal blooms at Flødevigen in Norway. Examples are given from time-integrated sampling using the disks followed by extraction and high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis for azaspiracids, okadaic acid analogues, pectenotoxins, yessotoxins and spirolides. Profiles of accumulated toxins in the disks and toxin profiles in blue mussels (Mytilus edulis) were compared with the relative abundance of toxin-producing algal species. Results obtained showed that passive sampling disks correlate with the toxin profiles in shellfish. The passive sampling disks were cheap to produce and convenient to use and, when combined with HPLC-MS or enzyme-linked immunosorbent assay (ELISA) analysis, provide detailed time-averaged information on the profile of lipophilic toxin analogues in the water. Passive sampling is therefore a useful tool for monitoring the exposure of shellfish to the toxigenic algae of concern in northern Europe.

Isolation of paspaline B, an indole-diterpenoid from Penicilium paxilli

Abstract The known indole-diterpenoids paspaline and 13-desoxypaxilline were isolated from Penicillium paxilli Bainier for the first time. In addition, paspaline B , a new indole-diterpenoid, was isolated and identified as paspalin-30-al by a combination of mass spectral and 1D and 2D NMR techniques. Full assignments for the 13 C NMR chemical shifts of paspaline and paspaline B were obtained, resulting in a major revision of the published 13 C NMR chemical shifts for paspaline. The structure of paspaline B suggests that it may closely follow paspaline in the biosynthesis of the tremorgenic indole-diterpenoid paxilline.

Pinnatoxin G is responsible for atypical toxicity in mussels (Mytilus galloprovincialis) and clams (Venerupis decussata) from Ingril, a French Mediterranean lagoon

Following a review of official control data on shellfish in France, Ingril Lagoon had been identified as a site where positive mouse bioassays for lipophilic toxins had been repeatedly observed. These unexplained mouse bioassays, also called atypical toxicity, coincided with an absence of regulated toxins and rapid death times in mice observed in the assay. The present study describes pinnatoxin G as the main compound responsible for the toxicity observed using the mouse bioassay for lipophilic toxins. Using a well-characterised standard for pinnatoxin G, LC-MS/MS analysis of mussel samples collected from 2009 to 2012 revealed regular occurrences of pinnatoxin G at levels sufficient to account for the toxicity in the mouse bioassays. Baseline levels of pinnatoxin G from May to October usually exceeded 40 μg kg(-1) in whole flesh, with a maximum in September 2010 of around 1200 μg kg(-1). These concentrations were much greater than those at the other 10 sites selected for vigilance testing, where concentrations did not exceed 10 μg kg(-1) in a 3-month survey from April to July 2010, and where rapid mouse deaths were not typically observed. Mussels were always more contaminated than clams, confirming that mussel is a good sentinel species for pinnatoxins. Profiles in mussels and clams were similar, with the concentration of pinnatoxin A less than 2% that of pinnatoxin G, and pteriatoxins were only present in non-quantifiable traces. Esters of pinnatoxin G could not be detected by analysis of extracts before and after alkaline hydrolysis. Analysis with a receptor-binding assay showed that natural pinnatoxin G was similarly active on the nicotinic acetylcholine receptor as chemically synthesized pinnatoxin G. Culture of Vulcanodinium rugosum, previously isolated from Ingril lagoon, confirmed that this alga is a pinnatoxin G producer (4.7 pg cell(-1)). Absence of this organism from the water column during prolonged periods of shellfish contamination and the dominance of non-motile life stages of V. rugosum both suggest that further studies will be required to fully describe the ecology of this organism and the accumulation of pinnatoxins in shellfish.

Identification of a sapogenin glucuronide in the bile of sheep affected by Panicum dichotomiflorum toxicosis.

Abstract Throughout the world, several hepatogenous photosensitisation diseases of ruminants are characterised by the presence of birefringent crystals in and about the bile ducts. These include photosensitisations caused by Tribulus terrestris (1) (geeldikkop of sheep in South Africa), Nurthecium ossifrugum (2).(alveld of lambs in Norway), Agave lecheguilla (3) (4), Brachiaria decumbens (5) and several Panicum species such as P miliaceum (6), P. coloratum (7), P. schinzii (8) (9), and P. dichotomiflorum (10).

Lactone Ring of Pectenotoxins: a Key Factor for their Activity on Cytoskeletal Dynamics

Background: Pectenotoxins are a group of natural products from marine origin that can accumulate in shellfish and intoxicate humans. Recently, novel homologues such as pectenotoxin-11 (PTX-11) and pectenotoxin-2 seco acid (PTX-2SA) have been identified. Their toxic potential towards experimental animals has been evaluated however their interaction with cellular systems is almost unknown. This is the first report showing (i) the biological activity of PTX-11 and PTX-2SA on actin cytoskeleton and morphology of living cells and (ii) the structure- activity relationship for this family of toxic compounds. Methods: Fluorescent phalloidin was utilized to quantify and visualize any modification in polymerized actin. Fluorescence values were obtained with laser-scanning cytometer and cells were imaged through confocal microscopy. For structure-activity evaluations, pectenotoxin-1 (PTX-1) and pectenotoxin-2 (PTX-2) was also analyzed. Results: Data showed that PTX-11 triggered a remarkable depolymerizing effect on actin cytoskeleton and also modifications in the shape of cells. In contrast, PTX-2SA did not evidence the same effects. Conclusion: Our findings point out that (i) the actin cytoskeleton is a common target for PTX-11, PTX-2 and PTX-1, but not for PTX-2SA, and (ii) this difference in activity is related to the presence or absence of an intact lactone ring in their structures.

Biotransformation of zearalenone and zearalenols to their major glucuronide metabolites reduces estrogenic activity

Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungi. Once ingested, ZEN may be absorbed and metabolised to α- and β-zearalenol (α-ZOL, β-ZOL), and to a lesser extent α- and β-zearalanol (α-ZAL, β-ZAL). Further biotransformation to glucuronide conjugates also occurs to facilitate the elimination of these toxins from the body. Unlike ZEN and its metabolites, information regarding the estrogenic activity of these glucuronide conjugates in various tissues is lacking. ZEN-14-O-glucuronide, α-ZOL-14-O-glucuronide, α-ZOL-7-O-glucuronide, β-ZOL-14-O-glucuronide and β-ZOL-16-O-glucuronide, previously obtained as the major products from preparative enzymatic synthesis, were investigated for their potential to cause endocrine disruption through interference with estrogen receptor transcriptional activity. All five glucuronide conjugates showed a very weak agonist response in an estrogen responsive reporter gene assay (RGA), with activity ranging from 0.0001% to 0.01% of that of 17β-estradiol, and also less than that of ZEN, α-ZOL and β-ZOL which have previously shown estrogenic potencies of the order 17β-estradiol>α-ZOL>ZEN>β-ZOL. Confirmatory mass spectrometry revealed that any activity observed was likely a result of minor deconjugation of the glucuronide moiety. This study confirms that formation of ZEN and ZOL glucuronides is a detoxification reaction with regard to estrogenicity, serving as a potential host defence mechanism against ZEN-induced estrogenic activity.