Simon Colas

Benjamin Marie

Emilie Lance

Catherine Quiblier

Hélène Tricoire-Leignel

César Mattei
email: cesar.mattei@univ-angers.fr

Anatoxin-a: overview on a harmful cyanobacterial neurotoxin from the environmental scale to the molecular target

Keywords: cyanobacteria, anatoxin-a, nicotinic acetylcholine receptor, nervous system, algal blooms

Anatoxin-a (ATX-a) is a neurotoxic alkaloid, produced by several freshwater planktonic and benthic cyanobacteria (CB). Such CB have posed human and animal health issues for several years, as this toxin is able to cause neurologic symptoms in humans following food poisoning and death in wild and domestic animals. Different episodes of animal intoxication in the wild have incriminated ATX-a, as confirmed by the presence of ATX-a-producing CB in the consumed water or biofilm and/or the observation of neurotoxic symptoms, which match experimental toxicity in vivo.

Regarding toxicity parameters, toxicokinetics knowledge is currently incomplete and needs to be improved. The toxin is able to cross passively biological membranes and act rapidly on nicotinic receptors, its main molecular target. In vivo and in vitro acute effects of ATX-a have been studied and make possible to draw its mode of action, highlighting its deleterious effects on the nervous systems and its effectors, namely muscles, heart and vessels, and the respiratory apparatus. However, very little is known about its putative chronic toxicity. This review updates available data on ATXa, from the ecodynamic of the toxin to its physiological and molecular targets.

This work was supprted by ANSES project "Cyanariv" (2019-CRD-06; SJ N°364/19).

Introduction 1

In the context of global changes, with -among other consequences -the increase of N and P inputs due to agricultural activities, and the rise of temperatures and lowwater periods, the phytobenthic and phytoplanktonic communities are largely dominated by cyanobacteria (CB) [START_REF] O'neil | The rise of harmful cyanobacteria blooms: The potential roles of eutrophication and climate change[END_REF].

One of the deleterious consequences of the rapid proliferation of CB, often due to weak water circulation, is the production and secretion of cyanotoxins which develop a large range of toxicity mechanisms, the main ones being hepatotoxicity, dermatotoxicity and/or neurotoxicity caused by hepatotoxins (eg microcystins, nodularins), dermatotoxins (eg lyngbiatoxins) and/or neurotoxins (anatoxins, saxitoxins), respectively [START_REF] Sivonen | Cyanobacterial toxins. In "Toxic Cyanobacteria in Water: A guide to their public health consequences, monitoring and management[END_REF][START_REF] Osswald | Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a[END_REF][START_REF] Aráoz | Neurotoxic cyanobacterial toxins[END_REF][START_REF] Huang | Cyanobacterial bioactive metabolites-A review of their chemistry and biology[END_REF]. However, the ecological role of these toxins, in terms of biological benefits for the CB, remains still poorly understood. Some of the possible roles explored in the literature are (i) to limit CB predation by other organisms such as zooplankton and nekton, and/or (ii) to serve as chemical mediators involved in cellular communicationallelopathy and chemotaxiswhich can establish ecological relationships with other CB or other micro-or macro-organisms and reduce the development of competitors [START_REF] Kaebernick | Ecological and molecular investigations of cyanotoxin production[END_REF][START_REF] Holland | Interpreting the possible ecological role (s) of cyanotoxins: compounds for competitive advantage and/or physiological aide?[END_REF].

Figure 1 near here

In freshwater, the significant concentrations of cyanotoxins are consecutive to the proliferation of cyanotoxin-producing CB. Anatoxin-a (ATX-a) and related molecules are mainly secreted by freshwater benthic CB (Figure 1) (e.g., Phormidium, Kamptonema, Microcoleus, Oscillatoria) in weak water currents [START_REF] Edwards | Identification of anatoxin-a in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland[END_REF][START_REF] Gugger | First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis[END_REF][START_REF] Wood | Anatoxins are consistently released into the water of streams with Microcoleus autumnalis-dominated (cyanobacteria) proliferations[END_REF], and planktonic CB (e.g., Anabaena, Aphanizomenon, Cuspidothrix, Cylindrospermopsis, Cylindrospermum, Dolichospermum, Planktothirx, Raphidiopsis, Tychonema) in stagnant water [START_REF] Sivonen | Preliminary characterization of neurotoxic cyanobacteria blooms and strains from Finland[END_REF][START_REF] Viaggiu | Anatoxin-a toxin in the cyanobacterium Planktothrix rubescens from a fishing pond in northern Italy[END_REF][START_REF] Vehovszky | Isolated strains of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) produce anatoxin-a like neurotoxins[END_REF][START_REF] Hodoki | Anatoxin-a-producing Raphidiopsis mediterranea Skuja var. grandis Hill is one ecotype of non-heterocytous Cuspidothrix issatschenkoi (Usačev) Rajaniemi et al. in Japanese lakes[END_REF][START_REF] Shams | Anatoxin-a producing Tychonema (Cyanobacteria) in European waterbodies[END_REF][START_REF] Trainer | Integrative monitoring of marine and freshwater harmful algae in Washington State for public health protection[END_REF][START_REF] Ballot | Variability in the anatoxin gene clusters of Cuspidothrix issatschenkoi from Germany, New Zealand, China and Japan[END_REF] . This harmful cyanotoxin causes neurotoxic symptoms, sometimes lethal, in livestock, fish, pets and humans [START_REF] Osswald | Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a[END_REF]. [START_REF] Osswald | Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a[END_REF].

Chemistry of ATX-a

ATX-a (2-acetyl-9-azabycyclo[4.2.1.]non-2-ene -MW 165 Da, C10H15NO) is a watersoluble secondary bicyclic amine, structurally related to alkaloids (Figure 2). It mainly exists in cationic form (pKa 9.6) and proved to be photosensitive at alkaline pH,

where it degrades into non-toxic compounds (see section 3.2; [START_REF] Stevens | Stability studies on the cyanobacterial nicotinic alkaloid snatoxin-A[END_REF]. The natural isomer is (+)-ATX-a, and the production of (-)-ATX-a by cyanobacteria has not been described to date. In this review, we specify -when available -which toxin isomer is used experimentally.

Figure 2 near here ATX-a should not be confounded with ATX-a(s), which is also a neurotoxic cyanotoxin, but structurally related to organophosphates. ATX-a(s) will not be considered in this review. Several analogues of ATX-a have been detected in different field samples or in CB cultures [START_REF] Skulberg | Investigations of a neurotoxic oscillatorialean strain (Cyanophyceae) and its toxin. Isolation and characterization of homoanatoxin-a[END_REF][START_REF] James | Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection[END_REF].

The most common are homoanatoxin-a (HTX-a), dihydroanatoxin-a (dhATX-a) and dihydrohomoanatoxin-a (dhHTX-a) (Figure 2). HTX-a (2-(propan-1-oxo-1-yl)-9azabicyclo [4.2.1] non-2-ene -MW 179 Da, C11H18NO) is a methylated homologue of ATX-a with a comparable toxicity [START_REF] Devlin | Anatoxin-a, a toxic alkaloid from Anabaena flos-aquae NRC-44h[END_REF][START_REF] Wonnacott | Homoanatoxin: a potent analogue of anatoxin-a[END_REF][START_REF] Osswald | Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a[END_REF][START_REF] Aráoz | Neurotoxic cyanobacterial toxins[END_REF], whereas dhATX is ten times less potent than ATX-a [START_REF] Wonnacott | Nicotinic pharmacology of anatoxin analogs. II. Side chain structure-activity relationships at neuronal nicotinic ligand binding sites[END_REF]. These four congeners can be found simultaneously in the environment in variable proportions. According to recent studies, dhATX-a seems to be the most common congener in New Zealand rivers followed by dhHTX-a [START_REF] Mcallister | The rise of toxic benthic Phormidium proliferations: a review of their taxonomy, distribution, toxin content and factors regulating prevalence and increased severity[END_REF][START_REF] Wood | Anatoxins are consistently released into the water of streams with Microcoleus autumnalis-dominated (cyanobacteria) proliferations[END_REF].

Biosynthesis and transformation

Biosynthesis

On the biosynthesis of ATX-a and HTX-a, a partial pathway using glutamate as substrate and involving polyketide synthases (PKS) was firstly described [START_REF] Hemscheidt | Biosynthesis of anatoxin-a in Anabaena flos-aquae and homoanatoxin-a in Oscillatoria formosa[END_REF]. As such, the production of ATX-a results from the condensation to three units of acetate on the (S)-1-pyrroline-5carboxylate, which derived from glutamate, followed by a decarboxylation. HTX-a is thought to result from the consecutive methylation of ATX-a. Thereafter, the genes encoding PKS enzymes involved in ATX-a biosynthesis have been identified [START_REF] Cadel-Six | Identification of a polyketide synthase coding sequence specific for anatoxin-aproducing Oscillatoria cyanobacteria[END_REF]. A nucleotidic sequence of Oscillatoria sp. strain PCC 6506 encoding a PKS has been found only in the genome of strains producing ATX-a or HTX-a, and surprisingly, within a cluster of genes encoding probably various enzymes involved in the biosynthesis of secondary ATX-a metabolites [START_REF] Méjean | The genome sequence of the cyanobacterium Oscillatoria sp. PCC 6506 reveals several gene clusters responsible for the biosynthesis of toxins and secondary metabolites[END_REF].

Bioinformatics analysis of the cluster showed that these genes encoded three proteinsnamely AnaB (Prolyl-ACP oxidase), AnaC and AnaDwhich are involved in the production of a substrate for three PKS enzymes (AnaE, AnaF, AnaG).

Predicting the function of each of these gene products has allowed the description of a full biosynthetic pathway for ATX-a and HTX-a [START_REF] Méjean | Evidence that biosynthesis of the neurotoxic alkaloids anatoxin-a and homoanatoxina in the cyanobacterium Oscillatoria PCC 6506 occurs on a modular polyketide synthase initiated by L-proline[END_REF][START_REF] Méjean | Biosynthesis of anatoxin-a and analogues (anatoxins) in cyanobacteria[END_REF]. The cyclic amino acid proline is activated and attached to an acyl-carrying protein (AnaD) by an adenylation protein (AnaC). Then, the oxidation of this prolyl-ACP to 1pyrroline-5-carboxyl-ACP (P5CACP) is performed by the AnaB oxidase. This newly formed imine is processed by a first PKS (AnaE), which adds a reduced acetate unit.

The chain is then modified by the second PKS (AnaF), which probably adds an acetate unit and performs a Michael-type cyclization leading to the anatoxinic thioester linked to the ACP domain. The latest PKS (AnaG) is supposed to add an unreduced acetate unit. Subsequently, methylation is carried out to form the precursor of HTX-a. Without this methylation step, the latter PKS is the precursor of ATX-a. In fact, since the latter PKS does not have a thioesterase (TE) domain, TE type II (AnaA) performs hydrolysis to form carboxyATX-a or carboxy-homoATX-a, which lead to the corresponding compounds through spontaneous decarboxylation [START_REF] Méjean | Biosynthesis of anatoxin-a and analogues (anatoxins) in cyanobacteria[END_REF].

Transformation

ATX-a and HTX-a are unstable in natural condition, where they are partially or totally degraded and converted into low-or non-toxic metabolites dhATX-a and epoxyATX-a (Stevens et Kriger, 1991;[START_REF] James | Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection[END_REF]. ATX-a can be degraded through photolysis and non-photochemical reactions, each of them affecting its chemical stability. It is also less bioavailable by dilution and adsorption [START_REF] Stevens | Stability studies on the cyanobacterial nicotinic alkaloid snatoxin-A[END_REF]. ATX-a degrades more quickly at higher temperatures (-76% after 1-hour exposure at 100°C, pH 7) and alkaline pH (-48% after 60-days exposure at pH 9.5°C, 20°C) [START_REF] Stevens | Stability studies on the cyanobacterial nicotinic alkaloid snatoxin-A[END_REF][START_REF] Kaminski | Determination of anatoxina stability under certain abiotic factors[END_REF]. Based on analysis of reaction products by gas chromatography-chemical ionization mass spectrometry (GC/MS) from various experimental photolysis, several products were identified weighing 165 Da, corresponding to the MW of ATX-a. It was concluded that photolysis of ATX-a generated a complex mixture of rearrangement products involving different bridgehead positions [START_REF] Stevens | Stability studies on the cyanobacterial nicotinic alkaloid snatoxin-A[END_REF][START_REF] Kaminski | Determination of anatoxina stability under certain abiotic factors[END_REF]. The breakdown products resulting from both photolytic and non-photolytic degradation are less toxic than ATX-a, some even being inactive [START_REF] Stevens | Stability studies on the cyanobacterial nicotinic alkaloid snatoxin-A[END_REF]. However, UV-B irradiation and high temperatures degraded ATX-a in the same way, leading to similar metabolites [START_REF] Afzal | Anatoxin-a degradation by Advanced Oxidation Processes: Vacuum-UV at 172 nm, photolysis using medium pressure UV and UV/H2O2[END_REF][START_REF] Kaminski | Determination of anatoxina stability under certain abiotic factors[END_REF]. Its half-life in freshwater is estimated to be 5 days at neutral pH [START_REF] Smith | The persistence of anatoxin-a in reservoir water[END_REF], but under conditions of sunlight intensity and at pH 9, ATX-a has a half-life of 1-2 h [START_REF] Stevens | Stability studies on the cyanobacterial nicotinic alkaloid snatoxin-A[END_REF].

Micro-organisms are also able to degrade ATX-a, in different type of lake sediments which undergo cyanobacterial blooms, but also in lake sediments with no history of proliferation. In the Finnish eutrophic Lake Tuusulanjärvi the toxin concentration in water drops from 2.4 µg.mL -1 to 0 within 4 days [START_REF] Rapala | Biodegradability and adsorption on lake sediments of cyanobacterial hepatotoxins and anatoxin-a[END_REF]. It also has been shown that Pseudomonas sp. degrades ATX-a at a rate of 6-30 µg.mL -1 per 3 days [START_REF] Kiviranta | Production and biodegradation of cyanobacterial toxins-a laboratory study[END_REF].

Factors influencing ATX-a production

Environmental conditions have a significant impact on cyanotoxin biosynthesis [START_REF] Neilan | Environmental conditions that influence toxin biosynthesis in cyanobacteria[END_REF][START_REF] Boopathi | Impact of environmental factors on the regulation of cyanotoxin production[END_REF]. The amount of toxin can be up-regulated through an increase of the CB biomass, and/or an increase in toxin biosynthesis.

Both mechanisms have been observed simultaneously.

Average concentrations of ATX-a measured range from 0.1 to 0.6 μg/L of water samples [START_REF] Cerasino | Diversity and distribution of cyanobacterial toxins in the Italian subalpine lacustrine district[END_REF]. These concentrations can somehow reach much higher levels such as 172,640 µg.L -1 in 2008 in the Anderson lake (USA) [START_REF] Hamel | Freshwater Algae Control Program: Report to the Washington State Lake Legislature (2008-2009[END_REF]. In CB biofilms, ATX-a concentrations reach 1233 µg.g -1 dw in the Tarn River (France) and 712 µg.g -1 in the Oreti River (New Zealand) [START_REF] Mcallister | The rise of toxic benthic Phormidium proliferations: a review of their taxonomy, distribution, toxin content and factors regulating prevalence and increased severity[END_REF][START_REF] Echenique-Subiabre | Spatial and temporal variability in the development and potential toxicity of Phormidium biofilms in the Tarn River, France[END_REF]. In natural conditions, the cellular concentration of ATX-a was 2.5 pg.cell -1 [START_REF] Wood | The abundance of toxic genotypes is a key contributor to anatoxin variability in Phormidium-dominated benthic mats[END_REF]. This cell content of ATX-a can reach maximal values and 1683 μg.g -1 dw, and a production of nitrogen [START_REF] Gagnon | Effect of nitrogen on cellular production and release of the neurotoxin anatoxin-a in a nitrogen-fixing cyanobacterium[END_REF], but experimental conditions influence this level. A 23-day culture of the same CB ended up with a maximum of 1105 μg.L -1 production [START_REF] Selwood | Production of anatoxin-a and a novel biosynthetic precursor by the cyanobacterium Aphanizomenon issatschenkoi[END_REF]. These values, among others, illustrate the heterogeneous nature of ATX-a production.

Early studies demonstrated that light, temperature and growth phase influenced ATXa production by Anabaena flos-aquae NRC-44-1 strains [START_REF] Peary | Influence of light and temperature on growth and toxin production by Anabaena flos-aquae[END_REF]).

The highest toxicity was observed after 14 growing days at 22°C and 2000-7500 lux.

Similar results confirmed the relationship between physicochemical parameters, growth phase and ATX-a production by cells [START_REF] Rapala | Anatoxin-a concentration in Anabaena and Aphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxic Anabaena-strains -a laboratory study[END_REF][START_REF] Rapala | Assessment of environmental conditions that favor hepatotoxic and neurotoxic Anabaena spp. strains cultured under light limitation at different temperatures[END_REF]. The amount of ATX-a produced by cell appears to be correlated with the exponential growth phase [START_REF] Gagnon | Effect of nitrogen on cellular production and release of the neurotoxin anatoxin-a in a nitrogen-fixing cyanobacterium[END_REF]. In fact, the highest ATX-a intracellular levels are found during the first two weeks of CB growth before reaching a plateau and halving within a week [START_REF] Rapala | Anatoxin-a concentration in Anabaena and Aphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxic Anabaena-strains -a laboratory study[END_REF][START_REF] Gallon | Biosynthesis of anatoxin-a by Anabaena flos-aquae: examination of primary enzymic steps[END_REF][START_REF] Harland | Phormidium autumnale growth and anatoxin-a production under iron and copper stress[END_REF].

In addition, the synthesis of ATX-a has been shown to be down-regulated at high temperatures, regardless of cyanobacterial cell growth, in Anabaena and Aphanizomenon strains [START_REF] Rapala | Anatoxin-a concentration in Anabaena and Aphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxic Anabaena-strains -a laboratory study[END_REF]. Temperature has a significant impact on toxin concentrations. The highest intracellular ATX-a concentrations have been recorded between 19°C and 21°C [START_REF] Rapala | Anatoxin-a concentration in Anabaena and Aphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxic Anabaena-strains -a laboratory study[END_REF][START_REF] Rapala | Assessment of environmental conditions that favor hepatotoxic and neurotoxic Anabaena spp. strains cultured under light limitation at different temperatures[END_REF].

When temperatures rise too much, ATX-a production decreases to a minimum observed at 30°C [START_REF] Rapala | Anatoxin-a concentration in Anabaena and Aphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxic Anabaena-strains -a laboratory study[END_REF]. N and P concentrations also influence the production of ATX-a. It is suggested that cellular production of ATX-a may be stimulated by moderate N deficiency [START_REF] Gagnon | Effect of nitrogen on cellular production and release of the neurotoxin anatoxin-a in a nitrogen-fixing cyanobacterium[END_REF]. Besides, the available form of N may also play a role in ATX-a production. The toxin concentration is slightly higher when CB are in a medium with N2 or NH4-N compared to a medium enriched with NO3 or urea-nitrogen. However, no correlation between levels of toxin produced by CB and P concentrations has been observed. A deficiency in P seems only to limit the cyanobacterial growth [START_REF] Rapala | Anatoxin-a concentration in Anabaena and Aphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxic Anabaena-strains -a laboratory study[END_REF].

In addition to influencing toxin production, temperature and environmental composition also modulate the preferential biosynthesis of ATX-a or HTX-a [START_REF] Aráoz | Neurotoxins in axenic oscillatorian cyanobacteria: coexistence of anatoxin-a and homoanatoxin-a determined by ligand-binding assay and GC/MS[END_REF]. For instance, when grown on BG11 medium at 22°C without CO2 enrichment, Oscillatoria PCC 6506 preferentially produces ATX-a. Conversely, when grown in BG1110 (with 10 nM NaHCO3) at 25°C with CO2 enrichment, HTX-a is the dominant congener. Different strains of the same species may respond in multiple ways to the same environmental conditions. Two strains of Oscillatoria sp. (PCC 6506 and PCC 9029), although deriving from the same original isolate, exhibit different toxin production profiles. Strain PCC 6506, grown under standard conditions in non-gassed flasks, in both BG11 and 2N media, exclusively produced ATX-a. In contrast, PCC 9029 grown under the same conditions, produced mainly HTX-a [START_REF] Aráoz | Neurotoxins in axenic oscillatorian cyanobacteria: coexistence of anatoxin-a and homoanatoxin-a determined by ligand-binding assay and GC/MS[END_REF].

Finally, biotic interactions can also influence the production of ATX-a, or at least the abundance of CB possessing the ana gene cluster. Microbial assemblages in mat samples with the ATX-a biosynthetic gene cluster consistently present a lower relative proportion of Burkholderiales (Betaproteobacteria) species than did mats without ATX-producing genes, suggesting that certain non-cyanobacterial organisms select CB species with genes for toxin production [START_REF] Bouma-Gregson | Impacts of microbial assemblage and environmental conditions on the distribution of anatoxin-a producing cyanobacteria within a river network[END_REF]. In conclusion, abiotic and biotic parameters seem to play a crucial role in toxigenic CB biomass and toxin production.

Bioaccumulation and depuration

There is still very little work on the accumulation and excretion of ATX-a, so it does not provide a clear picture. Although ATX-a is found in the environmentwater and biofilms samples -, naturally exposed organisms do not seem so far to prominently bioaccumulate the toxin [START_REF] Osswald | Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a[END_REF][START_REF] Ferrão-Filho | Cyanotoxins: bioaccumulation and effects on aquatic animals[END_REF]. However, it is important to point out that the hypothesis of ATX-a bioaccumulation is recent and has been little explored. Toxico-kinetics in fish appears to be fairly rapid. Following oral exposure of adult female medaka fish to a No Observable Adverse Effect Level (NOAEL = 6.67 µg.g -1 of (±)-ATX-a), the toxin could not be detected in the liver after 12h, and in the gut and muscles after 3 days [START_REF] Colas | Toxicity, transfer and depuration of anatoxin-a (cyanobacterial neurotoxin) in medaka fish exposed by single-dose gavage[END_REF]. Besides, the depuration rate for the 12 first hours is 57, 100 and 90% in guts, livers and muscles, respectively, leading to the rapid excretion of ATX-a. This suggests that fish do not bio-accumulate ATX-a in these tissues.

In 2009 and 2010, environmental and biological samples (water, fish, plants) were investigated for the presence of ATX-a in various watering places in Nebraska and Washington (USA). Extracellular ATX-a was found but no trace detected in all fish samples studied, while other cyanotoxinsnamely β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid dihydrochloride (DABA)were present in the same analysed tissues [START_REF] Al-Sammak | Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants[END_REF][START_REF] Hardy | Cyanotoxin bioaccumulation in freshwater fish, Washington State, USA[END_REF]. When testing the ability of the rainbow trout (Oncorhynchus mykiss) to bioconcentrate ATX-a by balneation in water containing dissolved ATX-a up to 5 mg.L -1 , no significant difference between bioconcentration factors (BCF) of ATX-a in juvenile subjects exposed to different doses of the toxin for 96 h was observed: in fact, ATX-a accumulates in fish body with BCF ranging from 30 to 47 [START_REF] Osswald | Experimental determination of the bioconcentration factors for anatoxin-a in juvenile rainbow trout (Oncorhynchus mykiss)[END_REF].

The exposure of the mussel Mytilus galloprovincialis to cultures of ATX-a-producing CB for 15 days revealed very low assimilation rates, despite the great capacity of this bivalve to bioaccumulate different toxicants, such as heavy metals, drug residues or even microcystins (MCs) [START_REF] Vasconcelos | Uptake and depuration of the heptapeptide toxin microcystin-LR in Mytilus galloprovincialis[END_REF][START_REF] Osswald | Uptake and depuration of anatoxin-a by the mussel Mytilus galloprovincialis (Lamarck, 1819) under laboratory conditions[END_REF]. Moreover, only one day after being placed in a CB-free environment, mussels presented no detectable ATX-a in their tissues. This toxin, which was mainly found in the digestive tract, seems to be quickly eliminated within 24h.

Under natural conditions, ATX-a was detected for the first time in fish harvested in a Polish lake by Pawlik-Skowrońska et al. ( 2012). The highest amounts were mainly found in livers compared to muscles, omnivorous fish accumulating roughly the same amounts of ATX-a as carnivorous fish: omnivorous roach (Rutilus rutilus) exhibited concentrations of 11 to 18 µg/g FW in liver in autumn 2008. Moreover, the bream Abramis brama from a dam reservoir was found to accumulate 6.2 to 18.4 μg/g FW in liver in 2010 [START_REF] Pawlik-Skowrońska | Cyanotoxin diversity and food web bioaccumulation in a reservoir with decreasing phosphorus concentrations and perennial cyanobacterial blooms[END_REF]. The contents of ATX-a detected in fish tissues seemed to increase with the occurrence of the toxin in the environment.

Both intracellular and extracellular ATX-a were found and the intracellular form was predominant, i.e. 10 times in spring and 3 times in summer [START_REF] Pawlik-Skowrońska | Cyanotoxin diversity and food web bioaccumulation in a reservoir with decreasing phosphorus concentrations and perennial cyanobacterial blooms[END_REF].

ATX-a has also been detected in aquatic plants of the genus Myriophyllum [START_REF] Al-Sammak | Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants[END_REF]. This contamination may be due to the endosymbiotic relationships of CB with some other higher plants, but may also occur via the presence of cyanotoxins in the aquatic environment and their absorption by vegetal organisms [START_REF] Al-Sammak | Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants[END_REF]. Other aquatic organisms were challenged for ATX-a bioaccumulation, such as benthic Chironomus which, fed on MC-and/or ATXa-producing CB, accumulated cyanotoxins [START_REF] Toporowska | Accumulation and effects of cyanobacterial microcystins and anatoxin-a on benthic larvae of Chironomus spp. (Diptera: Chironomidae)[END_REF].

Accidental exposure

Animal intoxications

Aquatic organisms can be naturally exposed to ATX-a by the oral, dermal or respiratory route [START_REF] Kuiper-Goodman | Toxic Cyanobacteria in Water: A guide to their public health consequences, monitoring and management[END_REF][START_REF] Carmichael | Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins[END_REF][START_REF] Draisci | Identification of anatoxins in blue-green algae food supplements using liquid chromatography-tandem mass spectrometry[END_REF]. The ingestion of cyanotoxin-contaminated water is the cause of frequent cases of acute animal intoxication causing neurologic symptoms up to death [START_REF] Quiblier | A review of current knowledge on toxic benthic freshwater cyanobacteriaecology, toxin production and risk management[END_REF][START_REF] Wood | Toxic benthic freshwater cyanobacterial proliferations: Challenges and solutions for enhancing knowledge and improving monitoring and mitigation[END_REF]. Water originating from a source with a proliferation of CB may contain toxins released during cell lysis (e.g. MCs) or excreted by CB (e.g.

ATX-a) [START_REF] Chorus | Toxic cyanobacteria in water: a guide to their public health consequences, monitoring, and management[END_REF]. Acute risks to animal health increase with the direct ingestion of CB biomass or biofilms, or of water containing large quantities of cyanotoxins. Alternatively, chronic intoxication may happen with the frequent ingestion of small doses of cyanotoxins over a long period of time through drinking water or food consumption [START_REF] Svirčev | Molecular aspects of microcystin-induced hepatotoxicity and hepatocarcinogenesis[END_REF]. The irrefutable imputation of a poisoning to a single toxin often lacks evidence because the same species of CB can produce different kind of toxins. For instance, Anabaena spiroids produces ATX-a and also MCs; some Kamptonema species can produce both ATX-a and cylindrospermopsin [START_REF] Méjean | Evidence that biosynthesis of the neurotoxic alkaloids anatoxin-a and homoanatoxina in the cyanobacterium Oscillatoria PCC 6506 occurs on a modular polyketide synthase initiated by L-proline[END_REF][START_REF] Mazmouz | Biosynthesis of cylindrospermopsin and 7-epicylindrospermopsin in Oscillatoria sp. strain PCC 6506: identification of the cyr gene cluster and toxin analysis[END_REF].

Table 1 near here

Nevertheless, ATX-a has been implicated in numerous animal poisonings worldwide (Table 1). Notably, the death of dogs due to ATX-a poisoning has been reported in North America, in New Zealand, but also in France, Kenya and Germany among others [START_REF] Edwards | Identification of anatoxin-a in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland[END_REF][START_REF] James | Anatoxin-a in Irish freshwater and cyanobacteria, determined using a new fluorimetric liquid chromatographic method[END_REF][START_REF] Hamill | Toxicity in benthic freshwater cyanobacteria (blue-green algae): First observations in New Zealand[END_REF][START_REF] Gugger | First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis[END_REF][START_REF] Wood | First report of homoanatoxin-a and associated dog neurotoxicosis in New Zealand[END_REF]Pushner et al., 2008Pushner et al., , 2010;;[START_REF] Faassen | First report of (homo)anatoxin-a and dog neurotoxicosis after ingestion of benthic cyanobacteria in The Netherlands[END_REF][START_REF] Fastner | Fatal neurotoxicosis in dogs associated with tychoplanktic, anatoxin-a producing Tychonema sp. in mesotrophic lake Tegel, Berlin[END_REF]. In addition, ATX-a and ATX-a-secreting CB have been found in tanks used for domestic water supply [START_REF] Fawell | The toxicity of cyanobacterial toxins in the mouse: II Anatoxin-a[END_REF].

Human intoxication

The presence of cyanotoxins in drinking water is not uncommon and has been mentioned in Asia, Europe and Oceania in the last ten years [START_REF] Buratti | Cyanotoxins: producing organisms, occurrence, toxicity, mechanism of action and human health toxicological risk evaluation[END_REF].

The consumption of cyanotoxin-containing water poses a health issue as it arises acutely in regions where the supply of drinking or irrigation water is made by storage notably dams, reservoirs, wells, retention basinslikely to accumulate toxinproducing CB or cyanotoxins, and no or insufficient treatment of drinking water.

Human intoxications might happen through the consumption of water, but other routes of exposure could be implicated.

With only one exception [START_REF] Biré | First evidence of the presence of anatoxin-A in sea figs associated with human food poisonings in France[END_REF], there is no epidemiological data on human poisoning formally incriminating ATX-a, although in several cases it is suspected to be the cause of symptoms in humans, due to their neurotoxic type [START_REF] Stewart | Recreational and occupational field exposure to freshwater cyanobacteria--a review of anecdotal and case reports, epidemiological studies and the challenges for epidemiologic assessment[END_REF][START_REF] Weirich | Freshwater harmful algal blooms: toxins and children's health[END_REF]. Human accidental ingestion of water during aquatic activities may result in the ingestion of dissolved cyanotoxins or cyanotoxin-producing CB. A variety of symptoms have been described, in relation to cyanotoxin exposure through water drinking.

The time to onset of symptoms -depending on the toxin involved, its dose and the route of exposure -are highly variable: a few minutes or even a few hours for skin symptoms and neurological disorders, several hours for hepatotoxins. The most commonly reported symptoms are gastrointestinal signs (diarrhoea, vomiting), fever and skin irritations. Headache, sore throat, and dry cough can be observed.

Considering only the ingestion or inhalation of CB, hepatic toxicities and neurotoxicities are more frequently described [START_REF] Wood | Acute animal and human poisonings from cyanotoxin exposure -A review of the literature[END_REF][START_REF] Svirčev | Global geographical and historical overview of cyanotoxin distribution and cyanobacterial poisonings[END_REF].

Despite this risk associated with such intoxication, no recurrent cases of human death associated with swimming have been reported. The only exception occurred in July 2002, following a swim in water with a bloom of ATX-a producing CB, in Wisconsin in the United States: five teens developed gastrointestinal symptoms 48 hours after swimming, and one died from heart failure. Stool and blood tests revealed the presence of Anabaena flos-aquae CB. ATX-a has been incriminated, despite the delay between exposure and death. However, this case has not been the subject of a scientific report [START_REF] Weirich | Freshwater harmful algal blooms: toxins and children's health[END_REF], and the identification of ATX-a was subsequently questioned [START_REF] Carmichael | Human mortality from accidental ingestion of toxic cyanobacteriaa case re-examined[END_REF][START_REF] James | Anatoxins and degradation products, determined using hybrid quadruple time-of-flight and quadruple ion-trap mass spectrometry: forensic investigations of cyanobacterial neurotoxin poisoning[END_REF]. To date, there is only one human poisoning case formally involving ATX-a: the consumption of ascidians of the Microcosmus genus in the south of France which intoxicated 26 people. The symptoms observed, mainly neurotoxic (blurred vision, ataxia, dizziness, asthenia, headache, muscle cramps, paresthesia) are entirely compatible with exposure to ATX-a. This work identified ATX-a at concentrations of 193.7 to 1240.2 µg .kg -1 in the seafood consumed [START_REF] Biré | First evidence of the presence of anatoxin-A in sea figs associated with human food poisonings in France[END_REF].

Toxicity

Acute toxicity

The symptoms observed experimentally after acute exposure to ATX-a are predominantly neurotoxic [START_REF] Jakubowska | Toxic picoplanktonic cyanobacteria--review[END_REF]. There are no formal kinetic data for ATX-a in mammals. However, toxicity studies indicate that the toxin is rapidly absorbed after ingestion and widely distributed in highly vascularized organs such as liver, gut and muscles. Because it is a secondary amine, it rather crosses the blood-brain barrier in rats, which contributes to its acute toxicity, namely a decrease of locomotor activity [START_REF] Rowell | Evidence for functional activity of up-regulated nicotine binding sites in rat striatal synaptosomes[END_REF][START_REF] Stolerman | Behavioural effects of anatoxin, a potent nicotinic agonist, in rats[END_REF].

Table 2 near here

ATX-a generally causes paralysis of intoxicated organisms. In rodents, ATX-a is lethal for doses of about 0.1 to 15 mg.kg -1 , depending on the route of exposure and the enantiomer tested (Table 2). In mice, ATX-a IP exposure causes after few minutes' hyperventilation, tremors, mild convulsions, urination, hypersalivation, hyperactivity, muscle contractions, then a decrease in locomotor activity, muscle paralysis and respiratory arrest preceding death. The acute toxicity shows rapid effects (2 to 6 min after administration) with muscular and respiratory paralysis. At a sub-lethal dose, there is a retrocession of all symptoms observed. However, and although animals can recover, changes in behaviour could have significant effects on the reproduction and interactions of intoxicated animals in their biotope [START_REF] Osswald | Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a[END_REF]. There is a difference in toxicity between the two enantiomers and the route of exposure: (+)-ATX-a being significantly more toxic than (-)-ATX-a (Table 1), while the LD50 is about 60 times smaller through the IP route than through the oral route [START_REF] Wonnacott | The chemistry and pharmacology of anatoxin-a and related homotropanes with respect to nicotinic acetylcholine receptors[END_REF][START_REF] Metcalf | Cyanobacterial toxins (Cyanotoxins) in water. Review of current knowledge[END_REF].

Finally, a study carried out by administering CB extracts containing ATX-a shows a great difference in sensitivity of various organisms to the toxin: rodent mammals are much less sensitive to ATX-a than avian species (ratio oral LD90 2.1 -5.1), and the goldfish displays a much higher sensitivity than avian and rodent species (ratio oral LD90 2.9 -15.0) [START_REF] Carmichael | Muscle sensitivity differences in two avian species to anatoxin-a produced by the freshwater cyanophyte Anabaena flos-aquae NRC-44-1[END_REF]. This heterogeneity could explain the fact that certain species may naturally accumulate ATX-a, without effect on their behaviour and fitness and constitute subsequent animal reservoirs of ATX-a, capable of transferring higher amount of this toxin through the food web (see Table 2).

Subchronic and chronic toxicity

Subchronic toxicity by ATX-a force-feeding or added to drinking water has been investigating [START_REF] Osswald | Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a[END_REF]. Due to mortalities observed in groups with higher doses, the oral NOAEL has been estimated at 98 μg.kg -1 .d -1 in mice, but the uncertainty on this value remains very high [START_REF] Fawell | The toxicity of cyanobacterial toxins in the mouse: II Anatoxin-a[END_REF]. This study provides data on daily chronic oral intoxication with ATX-a in mice (0 to 2.46 mg of ATX-a.kg -1 during 4 weeks), with no particular symptom in chronic intoxication been retrieved [START_REF] Fawell | The toxicity of cyanobacterial toxins in the mouse: II Anatoxin-a[END_REF]. Subcutaneous injection of (+)-ATX-a (0.05 to 0.2 mg/kg) to rats produced tolerance with weekly administration in a food-reinforcement paradigm [START_REF] Jarema | Effects of weekly exposure to anatoxin-a and nicotine on operant performance of rats[END_REF].

Hence, no data are available on a putative subsequent genotoxicity or carcinogenesis of ATX-a. Reprotoxic effects in male mice have been observed with the racemic mixture of ATX-a. An intraperitoneal Lowest Observed Adverse Effect Level (LOAEL) value of 50 µg.kg -1 .d -1 was estimated in male mice after 7 days, on sperm count [START_REF] Yavasoglu | Toxic effects of anatoxin-a on testes and sperm counts of male mice[END_REF]. Furthermore, maternal toxicity is reported at 200 μg.kg -1 IP in mice but without generating malformation or mortality in foetuses.

Conversely, ATX-a effects on foetuses are observed in the hamster: foetal stunting was observed, without skeletal or soft tissue malformations [START_REF] Astrachan | Evaluation of the subacute toxicity and teratogenicity of anatoxin-a[END_REF][START_REF] Fawell | The toxicity of cyanobacterial toxins in the mouse: II Anatoxin-a[END_REF][START_REF] Macphail | Nicotine effects on the activity of mice exposed prenatally to the nicotinic agonist anatoxin-a[END_REF][START_REF] Rogers | Potential developmental toxicity of anatoxin-a, a cyanobacterial toxin[END_REF]. In pregnant female mice, no sign of toxicity was observed in the offspring. The NOAEL (teratogenicity at 28 days) is established at 2.46 mg of ATX-a.kg -1 .d -1 [START_REF] Fawell | The toxicity of cyanobacterial toxins in the mouse: II Anatoxin-a[END_REF], as no adverse effects were deduced from exposure of pregnant mices by gavage with this ATX-a dose (2.46 mg.kg -1 bw per day), nor abnormal foetal development.

This toxin seems in fact to exert very little effects on the development of other vertebrate organisms. Embryos of zebrafish (Danio rerio) exposed to 400 µg.L -1 ATXa exhibit variations in heart rate, but these variations are differently modulated depending on the stage of development, without being significantly different from the control group. At the pec-fin stage (55 h), corresponding to the pectoral fin blade development, heart rate was decreased to a mean of 9 % of control embryos, while at the protruding-mouth stage (80 h), it was increased to a mean of 12 %. All these observations were temporary, and the toxin did not seem to cause chronic effects on the development of embryos [START_REF] Oberemm | Effects of cyanobacterial toxins and aqueous crude extracts of cyanobacteria on the development of fish and amphibians[END_REF].

It can however be noticed that there is a difference between the effects on early developmental stages induced by the exposure to pure ATX-a or to cyanobacterial extracts of an ATX-a producing strain [START_REF] Osswald | Effects of cyanobacterial extracts containing anatoxin-a and of pure anatoxin-a on early developmental stages of carp[END_REF]. A challenge with pure ATX-a has almost no effect on the development of carp eggs (Cyprinus carpio). Only a decrease in larval length was observed at the highest concentration (640 µg.L -1 ), while all the parameters studied (time to mortality, mortality rate, time to hatching, hatching rate, skeletal malformations rate and larval length) were not affected by the increasing concentrations of CB extracts [START_REF] Osswald | Effects of cyanobacterial extracts containing anatoxin-a and of pure anatoxin-a on early developmental stages of carp[END_REF].

Molecular target

8.1. Acetylcholine and cholinergic pathways ATX-a acts as an agonist of nicotinic acetylcholine receptors expressed in cholinergic synapses, which are widely distributed in the nervous system of vertebrates [START_REF] Thomas | +)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors[END_REF][START_REF] Swanson | Molecular mechanisms of the potent and stereospecific nicotinic receptor agonist (+)anatoxin-a[END_REF][START_REF] Bertrand | The wonderland of neuronal nicotinic acetylcholine receptors[END_REF]. Acetylcholine (ACh) is the main neurotransmitter of the peripheral nervous system (PNS) and one of the most important in the central nervous system (CNS). In the CNS, ACh plays a crucial role in a very large number of functions, including cognition or central motor control [START_REF] Bertrand | The wonderland of neuronal nicotinic acetylcholine receptors[END_REF]. The peripheral cholinergic synapses are found at neuromuscular junctions and in the autonomic nervous system and its effectors (heart, viscera, exocrine and endocrine glands). ACh ensures (i) the transmission of motor pathways which results in the contraction of the skeletal muscles, (ii) the transmission of an excitatory message in ganglia of the parasympathetic and orthosymapthic systems and (iii) the post-ganglionic transmission of the parasympathetic pathway. Two types of acetylcholine receptors mediate its action in the postsynaptic cell: ionotropic receptors (called nicotinic receptors, nAChRs) and metabotropic receptors (called muscarinic receptors, denoted mAChRs). After activating these receptors, ACh is hydrolysed by acetylcholinesterase, an enzyme present in the synaptic cleft. This reaction makes it possible to recycle choline in the presynaptic cell [START_REF] Amenta | Pathways of acetylcholine synthesis, transport and release as targets for treatment of adult-onset cognitive dysfunction[END_REF]. Activation of nAChRs by ACh opens a central pore permeable to cations (Na + , Ca 2+ and K + ), which promotes the excitation of the postsynaptic cell. From a pharmacological point of view, all nAChRs can be exogenously activated by the plant alkaloid nicotine. Throughout the discovery of new ligands, several pharmacological profiles have been characterized so far, which can be linked to their structure. Belonging to the ligand-gated ion channel family, nAChRs are expressed as homo-or heteropentamers. To date, 17 subunits have been identified in vertebrates: α1-10, β1-4, γ, δ, and ε [START_REF] Pedersen | Evolution of vertebrate nicotinic acetylcholine receptors[END_REF]. They can organize in not less than 10 different subunit combinations, always including an α subunit which is crucial for the ligand binding. There are generally neuronal and muscle specific nAChR structures. The muscle nAChR stoechiometry is 1(2)β (in embryo) or 1(2)β (in adult). It is activated by two ACh molecules which bind to two sites located at 1/ and 1/ interfaces. The activation of nAChRs ends up in depolarization and eventually drives the muscle fibre contraction [START_REF] Martyn | Basic principles of neuromuscular transmission[END_REF].

The neuronal nAChRs are heteropentamers with a stoichiometry x(2)βy(3) or homopentamers such as 7(5). Heteropentamers and homopentamers carry two and five ACh binding sites at the / and /interface respectively [START_REF] Papke | Merging old and new perspectives on nicotinic acetylcholine receptors[END_REF]. The most widely expressed neuronal nAChRs areβ2 and 7 in the brain and β4 in the peripheral nervous system.

Neuronal and muscle nAChRs can also be expressed in non-neuronal and nonmuscle tissues, such as vascular endothelium, bones, adrenal medulla, immune cells... [START_REF] Zoli | Neuronal and extraneuronal nicotinic acetylcholine receptors[END_REF]. The large distribution of nAChRs in the body potentially explains the variety of symptoms associated with an inhibition or an over-activation of these receptors.

ATX-a activation of nAChRs

ATX-a is a potent agonist of nAChRs and to a lesser extent mAChRs (Aronstam and Wiktop, 1981). The agonist activity of ATX-a for mAChRs is weak and requires micromolar concentrations of ATX-a to compete with muscarinic agonists in binding assays [START_REF] Aronstam | Anatoxin-a interactions with cholinergic synaptic molecules[END_REF][START_REF] Aracava | Nicotinic acetylcholine receptors in cultured neurons from the hippocampus and brain stem of the rat characterized by single channel recording[END_REF], although, at neuromuscular junctions, (+)-ATX-a is a more powerful agonist than acetylcholine for stimulating muscle contraction, due to a greater affinity for nAChRs and a much a slower desensitization kinetics. On neuronal 7 receptors expressed in Xenopus oocytes, (+)-ATX-a exhibits an EC50 of 0.58 µM, much lower than acetylcholine EC50

(320 µM) and (-)-nicotine EC50 (24 µM), illustrating the greater potency of the toxin to interact with the receptor [START_REF] Amar | Agonist pharmacology of the neuronal alpha 7 nicotinic receptor expressed in Xenopus oocytes[END_REF][START_REF] Thomas | +)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors[END_REF]. Competitive inhibition assays on membranes of Torpedo ocellata electric organscontaining muscle-type nAChRsevidenced that ATX-a bind at sub-micromolar concentrations to the ACh site in a competitive way towards both ACh and tubocurarine, making the toxin an efficient agonist of the receptor [START_REF] Aronstam | Anatoxin-a interactions with cholinergic synaptic molecules[END_REF]. Despite its capacity to cross the blood-brain barrier, ATX-a lethal effect can be primarily explained by its potent binding to muscle nAChRs, inducing a sustained depolarizing neuromuscular block [START_REF] Carmichael | Toxicology and pharmacological action of anabaena flos-aquae toxin[END_REF].

Figure 3 near here

Indeed, in cholinergic synapses, ATX-a is not hydrolyzed by acetylcholinesterase (which naturally degrades ACh) and the nAChRs are therefore overstimulated (Figure 3). This excessive stimulation disrupts the response of the effector organ, which can be a nervous ganglion (autonomous nervous system), a neuron (CNS) or a muscle fibre (sustained contraction) [START_REF] Spivak | Anatoxin-a: a novel, potent agonist at the nicotinic receptor[END_REF][START_REF] Wonnacott | Homoanatoxin: a potent analogue of anatoxin-a[END_REF][START_REF] Thomas | +)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors[END_REF]. At the molecular level, ATX-a very effectively activates the muscle nAChR 1(2)β. Through its binding to these nicotinic receptors, ATX-a causes the opening of their cationic pore, generating an influx of Na + /Ca 2+ ions into the postsynaptic cell and an efflux of K + ions, which leads to membrane depolarization. Prolonged exposure to ATX-a causes desensitization of nAChRs, and blockade of neuromuscular transmission [START_REF] Spivak | Anatoxin-a: a novel, potent agonist at the nicotinic receptor[END_REF]. HTX-a acts in a similar way as ATX-a: their affinities for nAChRs are comparable [START_REF] Wonnacott | Homoanatoxin: a potent analogue of anatoxin-a[END_REF].

In addition to these blocking effects of neuromuscular transmission and taking into account the fact that nAChRs are present in autonomic nervous system, their activation at interneurons of parasympathetic ganglia induces the release of ACh which stimulates post-synaptic muscarinic receptors on organs. Consequently, ATXa affects the cardiovascular system: it causes a decrease in heart rate, an increase in blood pressure, and disruptions of gas exchanges (pO2 and pCO2), which leads to hypoxia and respiratory arrest, as well as acidosis observed in animal deaths (Adeymo and Sire'n, 1992). In the adrenergic system, ATX-a acts on post-ganglionic and adrenal nAChRs and stimulates the secretion of catecholamines (dopamine, adrenaline and noradrenaline) which display multiple effects in the CNS and PNS (cardiac function, metabolic functions). ATX-a crosses the blood-brain barrier and exerts its nAChR agonist activity (Figure 3), but less effectively than on neuromuscular junctions [START_REF] Carmichael | Two laboratory case studies on the oral toxicity to calves of the freshwater cyanophyte (blue-green alga) Anabaena flos-aquae NRC-44-1[END_REF][START_REF] Stolerman | Behavioural effects of anatoxin, a potent nicotinic agonist, in rats[END_REF]. The most deleterious effects of ATX-a are therefore due to excessive stimulation of the skeletal muscles of the respiratory tract, which can lead to respiratory arrest [START_REF] Spivak | Anatoxin-a: a novel, potent agonist at the nicotinic receptor[END_REF][START_REF] Aronstam | Anatoxin-a interactions with cholinergic synaptic molecules[END_REF][START_REF] Swanson | Molecular mechanisms of the potent and stereospecific nicotinic receptor agonist (+)anatoxin-a[END_REF].

Regulatory thresholds

Numerous animal intoxications involving CB-producing ATX-a have been published

and show that it is a harmful toxin well disseminated in the environment. The symptoms observed experimentally or on field emphasize its neurotoxic potency, but also its cardiovascular effects in acute exposure. Despite the risk, ATX-a is scarcely regulated. Guideline values have been proposed locally in some countries: 5 µg.g -1 in fish flesh in California; 40 and 100 µg L -1 in drinking water for livestock and dogs, respectively, in California; 3.7 µg.L -1 in drinking water in Quebec (used as a temporary acceptable maximum dose); 6 µg.L -1 and 2 µg.L -1 in drinking water for ATX-a and HTX-a respectively, in New Zealand (used as the provisional maximum permissible dose); 20 µg.L -1 and 3 µg.L -1 in drinking water in Ohio and Oregon, respectively [START_REF] Farrer | Health-based cyanotoxin guideline values allow for cyanotoxin-based monitoring and efficient public health response to cyanobacterial blooms[END_REF]. No threshold currently exists in Europe for ATX-a or HTX-a. Recently, ATX-a producers were also evidenced for the first time in Australia, highlighting the need for more frequent investigation of the distributions of toxinproducing CB in this region [START_REF] John | First report of anatoxin-a producing cyanobacteria in Australia illustrates need to regularly up-date monitoring strategies in a shifting global distribution[END_REF]. Given its harmful character, its growing frequency in CB blooms and its involvement in animal poisoning, regulatory ATX-a thresholds aim at being more carefully investigated.

Concluding remarks

Studies on ATX-a and its structural analogues have resulted in a relatively satisfactory characterization of the ecological causes and circumstances under which this toxin is produced by CB, present in the water and the food web. These factors, in particular of anthropogenic origin, which favour CB proliferation, comprise eutrophication and perturbations of river hydric regime. The acute toxicity of ATX-a has been the subject of fairly old work, and its neurotoxic tropism has been evidenced, even if the toxicokinetic data are still quite fragmented. ATX-a, by its chemical structure and its physicochemical properties, seems to be able to cross most biological membranesincluding the gastro-intestinal tract, the blood-brain barrier and the placentaand quickly reaches its molecular target. After causing deleterious effects in the nervous system, it is rapidly excreted, and no sequels are evidenced at sublethal doses. The toxin binds with high affinity to nAChR, which is considered so far as its sole target. This ionotropic receptor is expressed in the central and peripheral cholinergic pathways. When bound, ATX-a causes nAChR persistent opening, compromising the communication between neuronal and postsynaptic cells, at the level of the skeletal muscle, cardiovascular and CNS. The symptoms observed in animals on field or through experimental exposure can therefore be explained by this ATX-a/nAChR interaction. There is no human epidemiological data sufficiently reliable to link human intoxication to exposure to this toxin, with the exception of one case recently described in the south of France. We can anticipate that a human acute intoxication linked to ATX-a would be managed through symptomatic treatments. The chronic toxicity of ATX-a remains however not characterized, and a long-lasting exposure is still possible, due to the recurrent CB bloom occurrence. Finally, the differential sensitivity to ATX-a of various animal species still specifically deserves to be the subject of future studies. Indeed, some animals seem to resist to this toxin better than others and the toxicokinetic or toxicodynamic determinants of this "resistance" are not yet known. Regulatory thresholds for water contamination should be proposed to consider and prevent possible human intoxication.

Figure captions

Figure 1 :

 1 Figure 1 : ATX-a producing cyanobacteria. (A) Phormidium sp bloom in the Cher

Figure 3 :

 3 Figure 3: Cellular and molecular mode of action of ATX-a. (A) ATX-a binds and

  Figure

Table 1 :

 1 Main episodes of ATX-a intoxication in the wild. DAD, Diode Array Detector, DW Dry Weight; ELISA, Enzyme Linked ImmunoSorbent Assay; ESI, Electrospray Ionisation; FW, Fresh Weight; GC/ECD, GC Gas Chromatography with Electron Capture Detector; MS, Mass Spectrometry; HPLC, High-Performance Liquid Chromatography; MALDI-TOF, Matrix Assisted Laser Desorption Ionization -Time Of Flight; NI, not indicated; PDA, Photometric Diode Array.

	Year, Country	Intoxication and	Method of	Matrix	Congener and maximum	Cyanobacteria	Reference
	and Location	mortalities	detection		concentration	species
		episode				(benthic/planktonic)
	1981, USA, Black	11 cows died	GC/ECD	Water sample	2.5 g.kg -1 (DW) of ATX-a	Anabaena flos-aquae	Stevens and Krieger,
	Lake	2 dogs died				(planktonic)	1988
		1000 bats died, 24	GC-MS	Green slime from	ATX-a	Anabaena flos-aquae	Pybus et al., 1986
	1985, Canada,	mallards and		the carcasses		(planktonic)
	Steele Lake	American wigeons
		died
	1985-1986,	5 cows died	HPLC, GC-	Water sample	3.7 g.kg -1 (DW) of ATX-a	Anabaena spp.	Sivonen et al., 1990
	Finland, Lakes		MS			(Planktonic)
	Sääskjärvi and
	Säyhteenjärvi
	1987, Canada,	16 cows died	GC-MS	NI	ATX-a	Anabaerna flos-aquae
	Alberta					(Planktonic)

Abbreviations used: ACh Acetylcholine; ATX-a Anatoxin-a; CB Cyanobacteria; CNS Central nervous system; dhATX-a Dihydroanatoxin-a; dhHTX-a Dihydrohomoanatoxin-a; FW Fresh weight; HTX-a Homoanatoxin-a; IP Intraperitoneal; IV Intravenous; LD50 Lethal dose 50%; LD90 Lethal dose 90%; LOAEL Lowest observed adverse effect level; mAChR Muscarinic acetylcholine receptor; MC Microcystin; nAChR Nicotinic acetylcholine receptor; NOAEL No observable adverse effect level; PNS Peripheral nervous system; PKS Polyketide synthase; TE Thioesterase

Funding

This work was supported by ANSES project "Cyanariv" (2019-CRD-06; SJ N°364/19).

Declaration of competing interest

The authors declare no conflicts of interest.

 [START_REF] Smith | A rapid analysis of water for anatoxin a, the unstable toxic alkaloid from Anabaena flos-aquae, the stable non-toxic alkaloids left after bioreduction and a related amine which may be nature's precursor to anatoxin a[END_REF]1990-1991, Scotland, Loch Insh4 dogs died and one intoxicated HPLC-DAD, GC-MS Water sample ATX-a Oscillatoria spp.

(benthic) [START_REF] Edwards | Identification of anatoxin-a in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland[END_REF]Dog stomach content ATX-a 1992-1993-1994, Ireland, Caragh Lake 6 dogs died HPLC-FL, GC-MS Water sample 444 µg.L -1 (FW) of ATX-a Oscillatoria spp.

(benthic) [START_REF] James | Anatoxin-a in Irish freshwater and cyanobacteria, determined using a new fluorimetric liquid chromatographic method[END_REF][START_REF] James | Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection[END_REF]James et al., -1999James et al., -2000

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: