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Paralytic shellfish poisoning is a worldwide problem induced by shellfish contaminated with paralytic shellfish toxins. To protect human health, a regulatory limit for these toxins in shellfish flesh has been adopted by many countries. In a recent study, mice were dosed with saxitoxin and tetrodotoxin mixtures daily for 28 days showing toxicity at low concentrations, which appeared to be at odds with other work. To further investigate this reported toxicity, we dosed groups of mice with saxitoxin and tetrodotoxin mixtures daily for 21 days. In contrast to the previous study, no effects on mouse bodyweight, food consumption, heart rate, blood pressure, grip strength, blood chemistry or hematology were observed. Furthermore, no histological findings were associated with dosing in this trial. The dose rates in this study were 2.6, 3.8 and 4.9 times greater, respectively, than the highest dose of the previous study. As rapid mortality in three out of five mice was observed in the previous study, the deaths are likely to be due to the methodology used rather than the shellfish toxins. To convert animal data to that used in a human risk assessment, a 100-fold safety factor is required. After applying this safety factor, the dose rates used in the current study were 3.5, 5.0 and 6.5 times greater, respectively, than the acute reference dose for each toxin type set by the European Union. Furthermore, it has previously been proposed that tetrodotoxin be included in the paralytic shellfish poisoning suite of toxins. If this were done, the highest dose rate used in this study would be 13 times the acute reference dose. This study suggests that the previous 28-day trial was flawed and that the current paralytic shellfish toxin regulatory limit is fit for purpose. An additional study, feeding mice a diet laced with the test compounds at higher concentrations than those of the current experiment, would be required to comment on whether the current paralytic shellfish toxin regulatory limit should be modified. [ABSTRACT FROM AUTHOR]

Regulatory limits for toxins in shellfish are required to ensure the health of consumers. However, these limits also impact the profitability of shellfish industries making it critical that they are fit for purpose. Since human toxicity data is rarely available, the setting of regulatory limits is dependent on animal data which can then be extrapolated for use in the assessment of human risk. The dependence on animal data to keep humans safe means that it is critical that the toxicity data used is robust and of high quality. Worldwide, the protocols used in toxicity testing are varied, making it hard to compare results and adding confusion over which results better reflect the true toxicity. In this study, we look at the effect of mouse gender, i.p. dose volume, mouse body weight and feeding protocols (both acute and sub-acute) on the toxicity of saxitoxin. This allowed the effect of different variables used in toxicity testing to be understood and showed that the feeding protocol used in both acute and sub-acute studies greatly influenced the toxicity of saxitoxin in mice. Therefore, the adoption of a standard protocol for the testing of shellfish toxins is recommended. [ABSTRACT FROM AUTHOR]

Fungal endophytes in perennial ryegrass are essential to New Zealand's pastoral system due to anti-insect effects. However, endophytes also produce compounds which can be detrimental to animals. Furthermore, as these toxins have been detected in the milk and fat of animals grazing common-toxic (containing lolitrem B) or AR37 endophyte-infected herbage they could enter the human food chain. To assess the risk to human health mice were fed for 90 days with three dose rates of lolitrem B and of AR37. Parameters indicative of animal health were measured as well as chemical, hematological and histological analysis of samples collected on day 90. Since endophyte toxin residues have been detected in milk, they could be transferred from mother to offspring via breast milk. To evaluate possible effects on reproduction two complete generations of mice were fed lolitrem B or AR37. At the dose rates given no adverse effects were observed in either study. The 100-fold safety factor to allow the use of animal data in human health assessments was applied and by considering the concentrations of lolitrem B or AR37 metabolites which could be ingested by a consumer it is highly unlikely that they pose any risk to human health. [ABSTRACT FROM AUTHOR]

Epichloë endophytes in grass associations express a myriad of secondary metabolites which can affect the health of grazing animals and reduce the impact of insect pests on pasture. The ideal endophyte-grass association must have a favourable chemical profile such that the impact on animal health is minimised while the beneficial, deterrent effect on insect pests is maximised. A number of endophyte-perennial ryegrass associations have been successfully commercialised but research is on-going to further improve production in farming systems. Secondary metabolites expressed by endophyte-infected tall fescue include lolines, an animal-safe class of compound which imparts a potent effect on insects. Since endophyte-infected perennial ryegrass does not express lolines, a tall fescue endophyte, AR501, was inoculated into perennial ryegrass in an attempt to improve the insect resistance of this pasture type. In addition to animal safety, it is imperative that consideration is given to the safety of humans consuming animal products derived from livestock grazing the novel pasture. Although pure loline alkaloids have previously been tested on mice it is essential that the entire AR501 endophyte-infected perennial ryegrass matrix is tested since this will result in the exposure of both known and unknown secondary metabolites to mice. Three treatment groups each containing 6 male and 6 female mice were fed diets containing AR501 endophyte-infected perennial ryegrass seed (30%), perennial ryegrass seed containing no endophyte (30%) or a diet without seed (control) for 3 weeks. Mice fed control diet ate more than either of the treatment groups fed a diet containing seed. Male mice fed diet containing Nil endophyte seed ate more than those eating AR501 endophyte-infected perennial ryegrass seed although there was no difference observed in the food intake of female mice. While a few statistically significant differences were observed in the haematology and serum biochemical data, in every instance the difference was restricted to only one gender so is considered unlikely to be of toxicological significance. Mice fed AR501 endophyte-infected perennial ryegrass seed remained healthy throughout the experimental period despite consuming 62,000 mg/kg lolines and 4600 mg/kg peramine per day as well as the wide array of other unknown secondary metabolites expressed by this endophyte. Although animal products may contain additional metabolites as a result of animal metabolism, this experiment raises no food safety concerns for AR501 endophyte-infected perennial ryegrass. [Display omitted] • Endophytes are used to confer beneficial insect pest protection to their host plants. • Endophytes produce a myriad of known and unknown secondary metabolites. • Secondary metabolites consumed by animals could go up the food chain to be consumed by humans. • AR501 endophyte-infected ryegrass seed was fed to mice for 21 days. • The lack of toxicity observed raises no food safety concerns for these compounds. [ABSTRACT FROM AUTHOR]

Both adults and larvae of Listronotus bonariensis damage ryegrass (Lolium spp.), a dominant component of New Zealand pasture. Damage can be reduced by Epichloë festucae var. lolii, a biotrophic fungal symbiont of ryegrass, through the production of alkaloids with deterrent and/or toxic effects to insect herbivores including L. bonariensis. Here we report results from two pot trials and a field trial comparing endophyte strains with different alkaloid profiles that have been introduced to reduce or eliminate adverse effects on stock. Adult L. bonariensis feeding scars, number of eggs and the proportion of tillers with larval damage and the severity of that damage were recorded. Samples of pseudostem and leaf blades were analysed for alkaloids. AR1 endophyte reduced adult feeding, oviposition, and larval damage whereas AR37 had no effect on adults but was highly effective in reducing larval damage. Reduction in adult feeding and oviposition by different NEA strains was governed by concentrations of peramine and ergovaline. For plants infected with AR37, alkaloid concentrations that determined the extent of larval damage were influenced by host species and ploidy; tetraploid L. perenne was more severely damaged than its diploid equivalent; L. multiflorum cultivars were more susceptible than the L. hybridum and L. perenne cultivars. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to compare the mode of action of the commonly used BK inhibitor paxilline with that of the more recently discovered lolitrem B. Similarities and differences in characteristics of inhibition between the two compounds were investigated. We have previously shown that lolitrem B does not affect the BK channel G–V, in contrast to the rightward shift produced by paxilline. These different effects on the voltage-dependence of activation suggest different modes of action for these two compounds. In this study we show that inhibition by both paxilline and lolitrem B is characterized by an open state preference for BK (hSlo) channels. Both compounds had a 3-fold higher apparent affinity under conditions likely to favour the open state, suggesting they have a similar BK conformational preference for binding. Furthermore, both compounds had a calcium concentration-dependence to their inhibitory effects. The G–V shift induced by paxilline was calcium concentration-dependent. [Copyright &y& Elsevier]

The heart generates and propagates action potentials through synchronized activation of ion channels allowing inward Na+ and Ca2+ and outward K+ currents. There are a number of K+ channel types expressed in the heart that play key roles in regulating the cardiac cycle. Large conductance calcium-activated potassium (BK) ion channels are not thought to be directly involved in heart function. Here we present evidence that heart rate can be significantly reduced by inhibiting the activity of BK channels. Agents that specifically inhibit BK channel activity, including paxilline and lolitrem B, slowed heart rate in conscious wild-type mice by 30% and 42%, respectively. Heart rate of BK channel knock-out mice (Kcnma1-/-) was not affected by these BK channel inhibitors, suggesting that the changes to heart rate were specifically mediated through BK channels. The possibility that these effects were mediated through BK channels peripheral to the heart was ruled out with experiments using isolated, perfused rat hearts, which showed a significant reduction in heart rate when treated with the BK channel inhibitors paxilline (1 μM), lolitrem B (1 μM), and iberiotoxin (0.23 μM), of 34%, 60%, and 42%, respectively. Furthermore, paxilline was shown to decrease heart rate in a dose-dependent manner. These results implicate BK channels located in the heart to be directly involved in the regulation of heart rate. [ABSTRACT FROM AUTHOR]

Abstract: Lolitrem B is an indole-diterpenoid neurotoxin which is the main causative agent of ryegrass staggers, an animal disease associated with tremors and incoordination. It is also a potent inhibitor of large conductance calcium-activated potassium (BK) channel activity (IC50 =4 nM). Furthermore, we have recently shown that the motor function deficits induced by lolitrem B are specifically mediated by BK channels, making the toxin a valuable tool for investigating the molecular function and physiological roles of these channels. To determine what structural features of BK channel agents are required for high potency, the effect of lolitrem B and seven structurally-related lolitrems on BK channel activity has been measured. Concentration–responses and conductance–voltage (G–V) relationships were determined for each compound and related to the different structure types. This study has identified seven new BK channel inhibitors and has allowed the identification of two key structural features required for high potency BK channel activity by lolitrems. [Copyright &y& Elsevier]

Regulatory limits for shellfish toxins are required to protect human health. Often these limits are set using only acute toxicity data, which is significant, as in some communities, shellfish makes up a large proportion of their daily diet and can be contaminated with paralytic shellfish toxins (PSTs) for several months. In the current study, feeding protocols were developed to mimic human feeding behaviour and diets containing three dose rates of saxitoxin dihydrochloride (STX.2HCl) were fed to mice for 21 days. This yielded STX.2HCl dose rates of up to 730 µg/kg bw/day with no effects on food consumption, growth, blood pressure, heart rate, motor coordination, grip strength, blood chemistry, haematology, organ weights or tissue histology. Using the 100-fold safety factor to extrapolate from animals to humans yields a dose rate of 7.3 µg/kg bw/day, which is well above the current acute reference dose (ARfD) of 0.5 µg STX.2HCl eq/kg bw proposed by the European Food Safety Authority. Furthermore, to reach the dose rate of 7.3 µg/kg bw, a 60 or 70 kg human would have to consume 540 or 630 g of shellfish contaminated with PSTs at the current regulatory limit (800 µg/kg shellfish flesh), respectively. The current regulatory limit for PSTs therefore seems appropriate. [ABSTRACT FROM AUTHOR]

Abstract: 44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC–MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC–MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada. [Copyright &y& Elsevier]

Abstract: The effects of the mycotoxin lolitrem B on the function of hSlo large conductance calcium-activated potassium channels expressed in HEK293 cells have been investigated using inside-out membrane patches. Lolitrem B potently inhibited hSlo potassium currents activated by depolarising voltage pulses in the presence of 10μM free calcium. At a concentration of 100nM, lolitrem B rapidly and completely inhibited outward potassium currents. The concentration that produced half-maximal inhibition was 3.7nM, indicating a high apparent affinity for hSlo channels. This is the first time that a molecular site of action has been identified for a compound of the lolitrem structural class of indole diterpene and identifies a novel BK channel blocker. [Copyright &y& Elsevier]

Understanding the toxicity and production rates of the various secondary metabolites produced by Gambierdiscus and cohabitating benthic dinoflagellates is essential to unravelling the complexities associated with ciguatera poisoning. In the present study, a sulphated cyclic polyether, gambierone, was purified from Gambierdiscus cheloniae CAWD232 and its acute toxicity was determined using intraperitoneal injection into mice. It was shown to be of low toxicity with an LD50 of 2.4 mg/kg, 9600 times less toxic than the commonly implicated Pacific ciguatoxin-1B, indicating it is unlikely to play a role in ciguatera poisoning. In addition, the production of gambierone and 44-methylgambierone was assessed from 20 isolates of ten Gambierdiscus, two Coolia and two Fukuyoa species using quantitative liquid chromatography–tandem mass spectrometry. Gambierone was produced by seven Gambierdiscus species, ranging from 1 to 87 pg/cell, and one species from each of the genera Coolia and Fukuyoa, ranging from 2 to 17 pg/cell. The production of 44-methylgambierone ranged from 5 to 270 pg/cell and was ubiquitous to all Gambierdiscus species tested, as well as both species of Coolia and Fukuyoa. The relative production ratio of these two secondary metabolites revealed that only two species produced more gambierone, G. carpenteri CAWD237 and G. cheloniae CAWD232. This represents the first report of gambierone acute toxicity and production by these cohabitating benthic dinoflagellate species. While these results demonstrate that gambierones are unlikely to pose a risk to human health, further research is required to understand if they bioaccumulate in the marine food web. [ABSTRACT FROM AUTHOR]

Epoxyjanthitrems I–IV (1–4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichloë festucae var. lolii. Although structures for epoxyjanthitrems I–IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests. [ABSTRACT FROM AUTHOR]

In identifying endophytes for use in pastures in New Zealand there have been two strategies used, either exclusion of ergopeptine and lolitrem alkaloids or, retaining some ergopeptine expression to enhance the stability of natural defences against invertebrate pests. Both have their strengths and weaknesses. It is a matter of balancing these to ensure the end-user has access to a product that satisfies their needs – a persistent pasture with low to nil animal toxicity in terms of animal production and welfare. The range and intensity of ergot alkaloids in grazed pasture on both pasture and ruminants is reviewed, with emphasis on New Zealand. Ergot alkaloids associated with pasture are produced by associations between certain Epichloë endophyte strains and temperate grasses. Ergot alkaloids have been shown to improve persistence of pasture through providing resistance/deterrence to insect pests as well as deterring grazing animals. However, ergovaline is toxic to grazing animals. Some commercially available ryegrass-endophyte associations can produce ergovaline concentrations close to those found in associations between ryegrass and the standard endophyte. It is feasible to eliminate ergot alkaloids from pasture grasses in New Zealand as endophyte strains are commercially available that do not express ergovaline and yet still provide excellent pest resistance. [ABSTRACT FROM AUTHOR]

Epichloë endophytes have been used successfully in pastoral grasses providing protection against insect pests through the expression of secondary metabolites. This approach could be extended to other plant species, such as cereals, reducing reliance on pesticides. To be successful, the selected endophyte must express secondary metabolites that are active against cereal insect pests without any secondary metabolite, which is harmful to animals. Chanoclavine is of interest as it is commonly expressed by endophytes and has potential insecticidal activity. Investigation of possible mammalian toxicity is therefore required. An acute oral toxicity study showed the median lethal dose of chanoclavine to be >2000 mg/kg. This allows it to be classified as category 5 using the globally harmonized system of classification and labelling of chemicals, and category 6.1E using the New Zealand Hazardous Substances and New Organisms (HSNO) hazard classes, the lowest hazard class under both systems of classification. A three-week feeding study was also performed, which showed chanoclavine, at a dose rate of 123.9 mg/kg/day, initially reduced food consumption but was resolved by day seven. No toxicologically significant effects on gross pathology, histology, hematology, or blood chemistry were observed. These experiments showed chanoclavine to be of low toxicity and raised no food safety concerns. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) is a potent neurotoxin associated with human poisonings through the consumption of pufferfish. More recently, TTX has been identified in bivalve molluscs from diverse geographical environments, including Europe, and is therefore recognised as an emerging threat to food safety. A recent scientific opinion of the EFSA Panel on Contaminants in the Food Chain recognised the need for further data on the acute oral toxicity of TTX and suggested that, since saxitoxin (STX) and TTX had similar modes of action, it was possible that their toxicities were additive so could perhaps be combined to yield one health-based guideline value. The present study determined the toxicity of TTX by various routes of administration. The testing of three different mixtures of STX and TTX and comparing the experimentally determined values to those predicted on the basis of additive toxicity demonstrated that the toxicities of STX and TTX are additive. This illustrates that it is appropriate to treat TTX as a member of the paralytic shellfish group of toxins. Since the toxicity of TTX was found to be the same as STX by feeding, a molar toxicity equivalence factor of 1.0 for TTX can be applied. [ABSTRACT FROM AUTHOR]

Climate change is anticipated to lead to an increase in the occurrence and intensity of drought and fluctuations in insect cycles that will challenge modern pasture systems. Feeding by root aphids such as Aploneura lentisci Pass. can be a significant challenge to pastures. These below-ground living aphids are commonly found in New Zealand and Australia, feeding year-round on the roots of graminaceous plants such as perennial ryegrass (Lolium perenne L.). Some strains of the fungal endophyte Epichloë festucae var. lolii in perennial ryegrass can provide protection against root aphids and greater resilience under drought, contributing to higher persistency and growth than endophyte-free plants. However, the interaction between insect pressure and drought is not understood. This study examined the effect of drought on root aphid populations and plant performance in perennial ryegrass plants relative to endophyte status (±) and endophyte strain (AR37, NZCT) in a glasshouse experiment. Plants were cloned across the drought and well-watered treatments, and half of the plants were inoculated with root aphids, whilst half of the plants were treated with insecticide. Endophyte infection with strain NZCT and AR37 reduced root aphid numbers. Aphid populations were significantly higher in drought-stressed than in well-watered plants in both endophyte-infected and endophyte-free treatments. Under drought conditions, root aphid populations were increased 4-fold in NZCT and 8-fold in AR37 and endophyte-free plants in comparison with their well-watered counterparts. Root aphids reduced shoot dry weight by 16% in drought-exposed and 26% in well-watered plants in comparison with their insecticide-treated counterparts whilst reducing root biomass by 49%. Our results suggest that root aphids are likely able to exploit the higher availability of amino acids in the plant sap of drought-exposed plants. This study provides evidence that climate change-mediated impacts of root aphids could reduce production in perennial ryegrass-dominant pastures. However, field trials would be necessary to determine whether this effect is seen in situ, where numerous additional factors will be operating at the same time. [ABSTRACT FROM AUTHOR]

Argentine stem weevil adults (ASW, Listronotus bonariensis) feed on the leaves of agricultural grasses and their larvae mine the pseudostem, causing extensive damage that can result in plant death. Plants emit volatiles that serve as signals to host-searching insects and these odours can be altered by both herbivory and fungal endophyte-infection. This study investigated whether ASW adults utilise olfaction to identify their host plants, perennial ryegrass (Lolium perenne), and if conspecific herbivory or the presence of Epichloë festucae var. lolii fungal endophytes (strain wild-type or AR1) influenced such responses. Results from olfactometer bioassays established that ASW adults were able to utilise their olfactory response to orient towards volatiles released by perennial ryegrass and further, the weevils displayed a preference for plants previously damaged by conspecific weevils. However, there was no evidence that weevils had the ability to distinguish between endophyte-infected and endophyte-free plants using olfaction alone. Using a push–pull extraction technique, thirteen volatile compounds were identified in the blend released by perennial ryegrass. Endophyte and herbivory were found to alter these volatile compounds and quantities emitted by this forage grass. This study suggests that despite observing differences in the plant volatile blend, ASW do not perceive endophyte (wild-type and AR1) using olfaction alone and must rely on other cues, e.g. contact chemoreception or post-ingestional malaise, to detect the presence of a bioactive endophyte in an otherwise acceptable host plant. [ABSTRACT FROM AUTHOR]

The aphid Aploneura lentisci is widespread in Australia and New Zealand, living all year round on roots of its secondary grass hosts. The fungal endophyte (Epichloë festucae var. lolii), strain AR37 in Lolium perenne is known to greatly reduce populations and was a likely reason for the superior growth and persistence of this association previously observed in the field. Aphid populations were quantified in a field trial near Ballarat, comparing yields of perennial ryegrass infected with eight different endophyte strains and an endophyte-free (Nil) control in a common ryegrass background (Grasslands Samson (G. Samson)). AR37 and another endophyte strain, AR5, had significantly fewer aphids than all other endophytes. These differences were significantly related to yield increases taken before and after sampling that persisted until the end of the trial. In a pot trial comparing commercially available ryegrass-endophyte combinations with equivalent Nil controls, aphid numbers were lower on G. Samson AR37 and Banquet II with AR5 (Endo®5) than on all other cultivar-endophyte combinations. Compared with Nil controls, the common toxic strain in G. Samson, and two strains in Trojan also reduced aphid numbers. The AR5 endophyte produces the alkaloid ergovaline but high concentrations of this in roots of potted plants could not account for differences in root aphid numbers. Root concentrations of epoxyjanthitrems, the only known alkaloids produced by AR37, were low and unlikely to be the cause of resistance to A. lentisci. An aphid, Aploneura lentisci , that feeds on grass roots is widespread in pastures in New Zealand and Australia. This paper reports data confirming that perennial ryegrass infected with a strain of Epichloë fungal endophyte, AR37, is highly resistant to this aphid and shows that another endophyte strain, AR5, gives the same level of control. Suppression of high aphid populations by these endophytes in the field at Ballarat resulted in a 37% increase in pasture production over endophyte-free ryegrass over a 5-month period. [ABSTRACT FROM AUTHOR]

• Acute toxicity of 44-methylgambierone to mice was low (20−38 mg/kg). • 252 marine microalgal isolates analysed for 44-methylgambierone production. • 44-Methylgambierone production ubiquitous to Gambierdiscus spp. • Production of 44-methylgambierone by some Coolia species. Ciguatera fish poisoning (CFP) is prevalent around the tropical and sub-tropical latitudes of the world and impacts many Pacific island communities intrinsically linked to the reef system for sustenance and trade. While the genus Gambierdiscus has been linked with CFP, it is commonly found on tropical reef systems in microalgal assemblages with other genera of toxin-producing, epiphytic and/or benthic dinoflagellates – Amphidinium, Coolia, Fukuyoa, Ostreopsis and Prorocentrum. Identifying a biomarker compound that can be used for the early detection of Gambierdiscus blooms, specifically in a mixed microalgal community, is paramount in enabling the development of management and mitigation strategies. Following on from the recent structural elucidation of 44-methylgambierone, its potential to contribute to CFP intoxication events and applicability as a biomarker compound for Gambierdiscus spp. was investigated. The acute toxicity of this secondary metabolite was determined by intraperitoneal injection using mice, which showed it to be of low toxicity, with an LD 50 between 20 and 38 mg kg−1. The production of 44-methylgambierone by 252 marine microalgal isolates consisting of 90 species from 32 genera across seven classes, was assessed by liquid chromatography-tandem mass spectrometry. It was discovered that the production of this secondary metabolite was ubiquitous to the eight Gambierdiscus species tested, however not all isolates of G. carpenteri , and some species/isolates of Coolia and Fukuyoa. [ABSTRACT FROM AUTHOR]

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis. [ABSTRACT FROM AUTHOR]

The cyanobacterium Microcoleus autumnalis grows as thick benthic mats in rivers and is becoming increasingly prevalent around the world. M. autumnalis can produce high concentrations of anatoxins and ingestion of benthic mats has led to multiple dog deaths over the past two decades. M. autumnalis produces a suite of different anatoxin congeners including anatoxin-a (ATX), dihydroanatoxin-a, (dhATX), homoanatoxin-a and dihydrohomoanatoxin-a. Benthic mat samples often contain high levels of dhATX, but there is little toxicology information on this congener. In the present study, natural versions of dhATX and ATX were purified from cyanobacteria to determine the acute toxicity by different routes of administration using mice. Nuclear magnetic resonance spectroscopy was used to confirm the putative structure of dhATX. By intraperitoneal (ip) injection, the median lethal dose (LD 50) for dhATX was 0.73 mg/kg, indicating a reduced toxicity compared to ATX (LD 50 of 0.23 mg/kg). However, by oral administration (both gavage and feeding), dhATX was more toxic than ATX (gavage LD 50 of 2.5 mg/kg for dhATX and 10.6 mg/kg for ATX; feeding LD 50 of 8 mg/kg for dhATX and 25 mg/kg for ATX). The relative nicotinic acetylcholine receptor-binding affinities of ATX and dhATX were determined using the Torpedo electroplaque assay which showed consistency with the relative toxicity determined by ip injection. This work highlights that toxicity studies based solely on ip injection may not yield LD 50 values that are relevant to those derived via oral administration, and hence, do not provide a good estimate of the risk posed to human and animal health in situations where oral ingestion is the likely route of exposure. The high acute oral toxicity of dhATX, and its abundance in M. autumnalis proliferations, demonstrates that it is an important environmental contaminant that warrants further investigation. • Natural forms of anatoxin-a and dihydroanatoxin-a assessed for acute toxicity. • Putative structure of dihydroanatoxin-a confirmed by nuclear magnetic resonance spectroscopy. • Isomer composition for dihydro-anatoxins in Microcoleus autumnalis was variable. • Dihydroanatoxin-a is more toxic than anatoxin-a by oral ingestion. [ABSTRACT FROM AUTHOR]

Gambierdiscus and Fukuyoa species have been identified in Aotearoa/New Zealand's coastal waters and G. polynesiensis, a known producer of ciguatoxins, has been isolated from Rangitāhua/Kermadec Islands (a New Zealand territory). The warming of the Tasman Sea and the waters around New Zealand's northern subtropical coastline heighten the risk of Gambierdiscus proliferating in New Zealand. If this occurs, the risk of ciguatera fish poisoning due to consumption of locally caught fish will increase. Research, including the development and testing of sampling methods, molecular assays, and chemical and toxicity tests, will continue. Reliable monitoring strategies are important to manage and mitigate the risk posed by this emerging threat. The research approaches that have been made, many of which will continue, are summarised in this review. [ABSTRACT FROM AUTHOR]