Zebrafish locomotor capacity and brain acetylcholinesterase activity is altered by Aphanizomenon flos-aquae DC-1 aphantoxins

Aquatic Toxicology 138–139 (2013) 139–149

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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Zebrafish locomotor capacity and brain acetylcholinesterase activity is altered by Aphanizomenon flos-aquae DC-1 aphantoxins De Lu Zhang a,b,∗∗ , Chun Xiang Hu b,∗ , Dun Hai Li b , Yong Ding Liu b,∗ a b

Department of Lifescience and Biotechnology, College of Science, Wuhan University of Technology, Wuhan 430070, PR China State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072, PR China

a r t i c l e

i n f o

Article history: Received 24 January 2013 Received in revised form 20 April 2013 Accepted 22 April 2013 Keywords: Aphantoxins Mechanosensory change Locomotor abnormality Acetylcholinesterase Brain Zebrafish

a b s t r a c t Aphanizomenon flos-aquae (A. flos-aquae) is a source of neurotoxins known as aphantoxins or paralytic shellfish poisons (PSPs) that present a major threat to the environment and to human health. Generally, altered neurological function is reflected in behavior. Although the molecular mechanism of action of PSPs is well known, its neurobehavioral effects on adult zebrafish and its relationship with altered neurological functions are poorly understood. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed by HPLC. The major analogs found in the toxins were the gonyautoxins 1 and 5 (GTX1 and GTX5; 34.04% and 21.28%, respectively) and the neosaxitoxin (neoSTX, 12.77%). Zebrafish (Danio rerio) were intraperitoneally injected with 5.3 and 7.61 ␮g STXeq/kg (low and high dose, respectively) of A. flos-aquae DC-1 aphantoxins. The swimming activity was investigated by observation combined with video at 6 timepoints from 1 to 24 h post-exposure. Both aphantoxin doses were associated with delayed touch responses, reduced head–tail locomotory abilities, inflexible turning of head, and a tailward-shifted center of gravity. The normal S-pattern (or undulating) locomotor trajectory was replaced by a mechanical motor pattern of swinging the head after wagging the tail. Finally, these fish principally distributed at the top and/or bottom water of the aquarium, and showed a clear polarized distribution pattern at 12 h post-exposure. Further analysis of neurological function demonstrated that both aphantoxin doses inhibited brain acetylcholinesterase activity. All these changes were dose- and time-dependent. These results demonstrate that aphantoxins can alter locomotor capacity, touch responses and distribution patterns by damaging the cholinergic system of zebrafish, and suggest that zebrafish locomotor behavior and acetylcholinesterase can be used as indicators for investigating aphantoxins and blooms in nature. Published by Elsevier B.V.

1. Introduction Eutrophication and blooms of toxigenic cyanobacteria in major freshwater bodies remain problematic due to the increased excretion of nutrients that accompanies socioeconomic development (Anderson et al., 2002; Paerl and Huisman, 2008). Worryingly, cyanobacteria blooms dominated by Aphanizomenon species are frequent sources of paralytic shellfish poisons (PSPs) (Jackim and Gentile, 1968; Mahmood and Carmichael, 1986; Pereira et al., 2000, 2004a; Ferreira et al., 2001; Costa et al., 2006; Liu et al., 2006a,b; Ballot et al., 2010; Ledreux et al., 2010). Encouragingly, however, PSPs have attracted much scientific and public attention worldwide

∗ Corresponding authors. Tel.: +86 27 68780866; fax: +86 27 68780866. ∗∗ Corresponding author at: Department of Lifescience and Biotechnology, College of Science, Wuhan University of Technology, Wuhan 430070, PR China. Tel.: +86 27 68780866; fax: +86 27 68780866. E-mail addresses: [email protected], [email protected] (D.L. Zhang), [email protected] (Y.D. Liu). 0166-445X/$ – see front matter Published by Elsevier B.V. http://dx.doi.org/10.1016/j.aquatox.2013.04.016

due to their fatal neurotoxicity and extensive geographic distribution (Hallegraeff, 2005, 2010). In recent decades, blooms dominated by Aphanizomenon flosaquae (A. flos-aquae) DC-1 have occurred almost every year in Dianchi Lake in the Yunnan province of China, because the lake provides a favorable environment for bloom development in terms of ongoing nutrient supply and preferred water temperature (Xing et al., 2007; Wu et al., 2010). The dominant species in the blooms has also been confirmed to synthesize neurotoxic PSPs (Liu et al., 2006a,b; Zhang et al., 2011, 2013). A. flos-aquae DC-1 and its secreted neurotoxic PSPs have attracted attention because of the prevalence of this dominant species in Dianchi Lake, which is an important source of freshwater for the population of over five million in the vicinity of Kunming city in Yunnan province, China (Liu et al., 2006a,b; Zhang et al., 2011, 2013). PSPs, which are synthesized by dinoflagellates, freshwater cyanobacteria and bacteria (Kotaki et al., 1985; Kotaki, 1989; Sugawara et al., 1997; Michaud et al., 2002; Prol et al., 2009; Ballot et al., 2010; Hackett et al., 2012), can accumulate in the aquatic biota at very high concentrations (Shimizu and Yoshioka, 1981;

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Sullivan et al., 1983; Llewellyn et al., 2004; Pereira et al., 2004a; Bricelj et al., 2005; Zaccaroni and Scaravelli, 2008), and can lead to morbidity and mortality associated with paralytic shellfish poisoning if humans consume PSP-contaminated foodstuffs (Ferrão-Filho and Kozlowsky-Suzuk, 2011; Faber, 2012; Rodrigues et al., 2012). However, there is still no good antidote for PSPs, with artificial respiration and fluid therapy being the only treatments available at present (Wiese et al., 2010). Regarding the effects of exposure to PSPs on fish, previous studies demonstrated that PSPs could accumulate in larval Sciaenops ocellatus from the PSP-contaminated cladoceran Moina mongolica, which in turn accumulates the toxin by filter-feeding on the dinoflagellate Alexandrium tamarense under laboratory conditions. Thus PSPs can be transferred from toxic algae to a high-trophiclevel fish (Kwong et al., 2006; Jiang et al., 2007). When PSPs enter fish, they were shown to delay the hatching of zebrafish embryos and lead to malformations and mortality (Oberemm et al., 1999; Lefebvre et al., 2004). They also accumulated in the muscles of the fish Geophagus brasiliensis (Clemente et al., 2010), leading to histopathological changes, lipoperoxidation (LPO), and inhibition of antioxidant activities in the muscles and brains of the fish (da Silva et al., 2011b; Clemente et al., 2010). Along with their biochemical and physiological effects, PSPs also affected swimming performance, prey capture, and predator avoidance in mummichog larvae, winter flounders larvae and sheepshead minnow larvae after consumption of toxic dinoflagellates, copepods and Coullana canadensis (Samson et al., 2008). These studies demonstrate that PSPs may have potent toxic effects in fish, by not only inducing biochemical and physiological effects, but also by causing behavioral changes in larval fish (Ferrão-Filho and Kozlowsky-Suzuk, 2011). Behavioral alterations in organisms are frequently assessed as an endpoint, because altered behavior is an important biomarker in toxicological studies (Cazenave et al., 2008; Ballesteros et al., 2009; Eissa et al., 2010). Typically, both internal and external environmental alterations in an organism can cause behavioral changes, which often reflect the interactions of an animal with its environment (Bégout Anras and Lagardére, 2004). Indeed, when an animal is exposed to environmental contaminants, behavioral alterations can be the first defensive response (Bégout Anras and Lagardére, 2004). The animal will often try to escape the contaminant focus to reduce its chance of death or the increased metabolic consumption incurred by maintaining physiological homeostasis, thus promoting survival, growth and reproduction (Schreck et al., 1997). However, it is sometimes impossible for organisms to escape the source, and they must then adapt or face the situation (van der Oost et al., 2003). The environmental alterations induce endogenous physiological, biochemical and genetic changes of organisms, and these accumulated endogenous changes can be seen through behavioral pathways (Dantzer et al., 2008; Miller, 2009; Macêdo et al., 2013). Unlike internal alterations, behavioral alterations are evident and observable whole-organism activities that do not require harming the animals to obtain data (Brewer et al., 1999). Behavioral studies are therefore preferable to other types of studies from an ethical point of view, and in terms of endangered species (Festa-Bianchet and Apollonio, 2003; Sutherland, 1998). Thus behavioral toxicology is also often proposed as a way to monitor ecologically relevant contaminants (Scott and Sloman, 2004). Fish are an important and sometimes essential part of the aquatic ecosystem, and are thus an appropriate experimental organism for evaluating the impact of stressors on freshwater systems and assessing the impacts of harmful natural and xenobiotic chemicals, due to their systematic position as vertebrates and at the end of the aquatic food chain in freshwater systems (Baganz et al., 1998). Zebrafish is an important model organism in the freshwater vertebrates (Brewer et al., 2001). Although zebrafish has been a popular subject of research in the past three decades, especially in

developmental biology and genetics, its brain function and behavior are not well understood compared to other model organisms, such as the rat, the mouse, or the fruit fly (Sison et al., 2006). Recently, however, interest in the use of zebrafish in the fields of behavioral science and neurobiology has increased (Sison et al., 2006). Zebrafish provide a popular study species because of their small size and tendency to swim in groups, thus enabling large numbers of fish to be housed in small laboratory rooms (Norton and Bally-Cuif, 2010). Their eggs are fertilized and develop externally, and a single female may produce 200–300 offspring every other day (Norton and Bally-Cuif, 2010). Although it is a relatively simple vertebrate species, the zebrafish is physiologically and anatomically homologous to other vertebrates, including humans, and the zebrafish brain is fundamentally similar to the brains of other vertebrates, including humans (Tropepe and Sive, 2003). This species is therefore suitable for investigating pathways and mechanisms in vertebrates, including human pathologies and clinical treatments (Shin and Fishman, 2002; Damodaran et al., 2006; Lieschke and Currie, 2007; Tierney, 2011). Zebrafish possess all the typical vertebrate neurotransmitters (Mueller et al., 2004; Panula et al., 2006), and their neuroendocrine system can provide vigorous physiological responses to stress (Alsop and Vijayan, 2008). Importantly, the DNA sequence homologies between zebrafish genes and other vertebrate genes, including those of mammals (Barbazuk et al., 2000; Reimers et al., 2004; Lassen et al., 2005), as well as the anatomical similarities between vertebrate brains, mean that the mechanisms responsible for complex functional properties of the mammalian brain can be modeled and investigated using zebrafish (Gerlai, 2003; Tropepe and Sive, 2003; Kily et al., 2008; Brittijn et al., 2009). Furthermore, behavioral analysis may represent the most objective and meaningful method of studying brain function (Blaser and Gerlai, 2006). Crucially, swimming behavior in fish involves the nervous system, and altered swimming and locomotor activities thus reflect alterations in the nervous system (Vogl et al., 1999). These factors were relevant to the present study, because the toxin has been confirmed as a neurotoxin (Liu et al., 2006a,b). To date, behavioral research in zebrafish as a model organism has focused on the effects of different chemicals, such as pesticides, sodium hypochlorite and ethanol, on psychological and social behavior (Baganz et al., 2004; Carvan et al., 2004; Airhart et al., 2007; Nimkerdphol and Nakagaw, 2008; MacPhail et al., 2009; Egan et al., 2009; Irons et al., 2010; Rosemberg et al., 2012). Concerning behavioral studies of PSPs, only several studies have investigated the larvae or embryo of zebrafish for reduced sensorimotor deficits, whereas studies have found reduced spontaneous and touch-activated swimming behavior in larval Pacific herring and increased aggressive behavior in the rainbow trout (Lefebvre et al., 2004, 2005; Bakke et al., 2010). These studies suggest that the zebrafish may have a sophisticated behavioral repertoire and that functional changes in the brain, induced by drugs of abuse or toxins, can be detected at the behavioral level. These results also demonstrate that behavioral studies using zebrafish can further promote the development of genetics and behavioral neuroscience (Granato et al., 1996; Norton and Bally-Cuif, 2010). To the best of our knowledge, the effects of sublethal doses of A. flos-aquae DC-1 aphantoxins, which are proven neurotoxic PSPs, on the locomotor behavior of fish have not yet been studied. Aphantoxin-induced zebrafish locomotor abnormalities, which reflect functional cholinergic system changes in the zebrafish brain, remain unclear. An integrated evaluation of locomotor behavioral abnormalities and cholinergic system function in the zebrafish brain could thus provide a more comprehensive insight into the sublethal responses of fish exposed to aphantoxins. Therefore, in the present study, aphantoxins were extracted and purified from cultured A. flos-aquae DC-

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1 isolated from Lake Dianchi. HPLC analysis confirmed the presence of PSPs, and sublethal doses of the toxin preparation were administered to zebrafish by intraperitoneal injection. We assessed the concomitant changes in locomotor behavior and brain cholinergic system function to provide evidence of aphantoxin neurotoxicity in the zebrafish brain in order to develop an early warning system for A. flos-aquae DC-1 PSP toxicity. 2. Materials and methods 2.1. Chemicals Reference standards for saxitoxin (STX) group toxins (dcSTX, STX, neoSTX) and gonyautoxin (GTX) group toxins (GTX1–5, dcGTX2 and 3) were purchased from the National Research Council of Canada, Halifax, NS, Canada. All other chemicals, unless indicated otherwise, were of the highest grade available from commercial sources. 2.2. Culture of A. flos-aquae DC-1 We collected samples of A. flos-aquae DC-1 from a Lake Dianchi water bloom in 2002 to 2006 (Liu et al., 2006a,b); details of sampling, isolation, identification and culture techniques are presented elsewhere (Pereira et al., 2000; Liu et al., 2006a; Zhang et al., 2013). Briefly, algal cells were first cultured in 50 mL sterilized BG11 medium (25 ± 2 ◦ C, 16 h:8 h light:dark cycle, light intensity 30 ␮mol photon m−2 s−1 ) for 20 days with manual agitation twice per day, then diluted into 2 L of sterilized BG11 medium and cultured under the same conditions for a further 20 days to generate stock cultures. Large-scale algal culture was performed by diluting stock cultures (2 L) into 4× 10 L sterile BG11 medium (cell concentration = ∼1 × 104 cells/mL). Cultures were incubated at 25 ± 2 ◦ C with continuous illumination by cool-white fluorescent tubes (40 ␮mol photon m−2 s−1 ) and oxygenation by aeration with sterilized air. Cultures were harvested after 35–40 days, which was the phase of highest toxin production (Liu et al., 2006b; Zhang et al., 2011, 2013). Cells were recovered by centrifugation (5640 × g, 10 ◦ C, 10 min) using a GL-10LM centrifuge (Hunan Xingke Scientific Instrument Co. Ltd., China) and lyophilized (Yamato Scientific Co., Tokyo, Japan), yielding a total of 17.87 g (dry weight) of cells. These cells were stored at −20 ◦ C before further analysis. 2.3. Extraction and purification of aphantoxins Extraction and purification of aphantoxins was performed accorded to previous methods (Liu et al., 2006a; Zhang et al., 2013) with slight modifications. Briefly, 1.93 g of lyophilized dry cells were mixed with 200 mL of 0.01 M acetic acid solution, sonicated (4 ◦ C; Sonifier, 250, MN, USA) for 15 min in an ice bath, stirred vigorously for 30 min (25 ◦ C) and then frozen/thawed twice to promote the release of toxins into the medium. Preparations were examined under the microscope to confirm >99% cell lysis. Lysates were clarified by centrifugation (5900 × g, 4 ◦ C, 10 min), supernatants were harvested and the pellets re-extracted again with 0.01 M acetic acid solution (4 ◦ C) and clarified as before. The two supernatants were combined and macromolecules were precipitated by the addition of 20 mL absolute ethanol (v:v, 9:1). After centrifugation (5900 × g, 4 ◦ C, 10 min), the supernatant was serially filtered through 5.0, 0.45 and 0.22 ␮m filter membranes under mild vacuum and concentrated to dryness using a rotary evaporator (R-210; Buchi, Switzerland). Residues were dissolved in 50 mL 0.01 M acetic acid, again evaporated to dryness, then dissolved in 25 mL 0.01 M acetic acid solution. The extract was passed through

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Sep-Pak C18 cartridges (Waters, USA) for purification. The eluate was collected and concentrated to dryness using a rotary evaporator (R-210-Buchi) and then dissolved in 3 mL 0.01 M acetic acid and stored at −20 ◦ C for subsequent HPLC analysis and toxicological studies. 2.4. Toxin concentrations determined from HPLC All chemicals used were HPLC or analytical grade. Toxin analysis of PSPs was carried out using a HPLC system (Shimadzu, Tokyo, Japan) with fluorescent detection using ion-pair chromatography with post-column derivatization, as reported previously (Garcia et al., 2004; Diener et al., 2006; Liu et al., 2006a) with slight modifications. Briefly, the stored frozen extract was ultrafiltered through 10,000 Da filters (Millipore, MA, USA), aliquots (10 ␮L) were analyzed in parallel with reference standards of STXs (dcSTX, STX, neoSTX) and GTXs (GTX1–5, dcGTX2 and 3) using a silica-based reverse-phase column (Shim-Pack, VP-ODS, 250 L × 4.6; Shimadzu) in a HPLC instrument equipped with fluorescence monitoring (LC20A, RF-10AXL; Shimadzu). Fluorescence detection involved an excitation wavelength of 330 nm and an emission wavelength of 390 nm. HPLC chromatography was performed using a Shimadzu LC-20AD liquid chromatography apparatus with an online Shimadzu RF-10AXL spectrofluorometric detector. The mobile phase and acidifying and oxidizing reagents were pumped by a series three different pumps (LC-20AT; Shimadzu). Data acquisition and data processing were performed with Shimadzu Class-CR10 software (Shimadzu). STXs and GTXs in extracts were identified by comparing chromatograms to those obtained with reference standards. For toxicity studies, toxin concentrations were determined using the factor response method (peak area/toxin concentration) obtained from the injection of reference standards (Diener et al., 2006). The overall toxicities of the samples were calculated as STX equivalents based on the amount of toxin and its relative toxicity compared to STX based on the specific toxicities (Oshima, 1995a,b; Usup et al., 2004). 2.5. Pre-experimental determination of toxin dosage A total of around 300 fish or so were used to establish toxin doses. Toxin dosage was determined based on previous methods (Bruce, 1985; Lu and Tomchik, 2002; Oshima, 1995a) with minor modifications. Briefly, a single zebrafish was injected (i.p.) with a starting dose (5 ␮L, content 9.28 ␮g/kg, 6.4 ␮g STXeq/kg toxicity) of crude aphantoxin (using a 50-␮L single-use sterile microsyringe). If this fish died within 24 h, another fish was injected with a lower dose (diluted with 0.01 M acetic acid); if the injected fish died within 24 h, another fish was injected with a lower dose. This procedure was continued until the injected fish survived for 24 h, at which point the next fish was injected with a higher dose. Repeating this procedure established the doses giving 0 or 100% death. This series of experiments led to the selection of two different toxin doses—7.73 and 11.13 ␮g/kg (5.3 and 7.61 ␮g STXeq/kg body weight as low and high dose, respectively)—where the lower dose caused behavioral signs of toxicity but no death, and the higher dose gave severe behavioral signs in all animals but only a low death rate. Control animals received vehicle alone (0.01 M acetic acid). 2.6. Toxin treatment of fish and preparation of brain samples A total of 510 healthy male zebrafish, all from the same conditions, and ∼110 days old, with a mean bodyweight of 0.4 ± 0.01 g, were supplied by the Zebrafish Center of State Key Laboratory of Freshwater Ecology and Biotechnology, Wuhan, China. These fish were acclimatized in several rectangular aquaria (47 cm × 35 cm × 24 cm, length × depth × width) containing 33 L

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aerated tap water from storage tanks (27 ± 1 ◦ C, 14 h:10 h light:dark) (Wu et al., 2012). The water of all aquaria was changed daily using the same storage water and individually aerated using air stones attached to air compressors. Two sides, the back and the bottom of the aquarium were partitioned or marked by a covering of gray cardboard (two sides) and transparent (back and bottom) paper, respectively, with many marked horizontal and vertical cross-lines at intervals of about 3.0 cm, to aid the determination of the swimming distance of the fish (the distance of 3.0 cm was selected because of fish body length). Fish were fed twice daily with live brine shrimp (ingredients: crude protein, >38%; crude fat, >3%; crude fiber, <5%; moisture, >8%; Guangzhou Golden Rainbow Aquarium and Pet Co. Ltd., China) and commercial dry flake fish food (ingredients: protein, 60%; fat, 20%; others, 20%; Guangdong Jinshan Feed and Bait Co. Ltd., China). After 10 days of acclimatization, zebrafish were randomly assigned into control, low- and high-dose groups for studying mechanosensory properties and head and tail movement (30 fish per group, 3 fish per aquarium for convenient study purposes), distribution preference in water, and brain acetylcholinesterase (AChE) (30 fish per aquarium to allow clear observations of activities; 50 fish per aquarium to provide enough brain samples, respectively). The aquaria as described above contained approximately 33 L aerated tap water. Comparisons between numbers during the pre-exposure test revealed that the specific numbers chosen for each experiment had no effect on behavior or AChE parameters. The three groups received i.p. injections of 30 ␮L 0.01 M acetic acid containing aphantoxins at 7.73 and 11.13 ␮g/kg (5.3 and 7.61 ␮g STXeq/kg) (low- and high-dose groups, respectively). Fish exposed to 30 ␮L 0.01 M acetic acid vehicle alone served as controls. Comparison of zebrafish behavior during pre-exposure test conditions with that of the controls during exposure confirmed that a single exposure to 30 ␮L 0.01 M acetic acid vehicle had no effect on behavior and other parameters. The fish were not fed following injection. After injection, the fish behavioral changes were observed by the same three highly trained observers (inter-rater reliability >0.85), who were blinded to the treatments and who manually scored different behavioral endpoints, combined with videotaping with one digital video surveillance camera (HG10, Canon, Japan) positioned directly above each aquarium at 1, 3, 6, 9, 12 and 24 h post-treatment (Levin et al., 2007; Gerlai et al., 2008). These videotapes were later viewed for further behavioral analysis (Gerlai et al., 2006). Each experiment was performed three times with duplicates. 2.7. Mechanosensory (touch response) screens A tactile test method that has previously been used to examine mechanosensory abnormalities in zebrafish embryo and larva was used to detect mechanosensory (touch response) impairments due to A. flos-aquae DC-1 toxin exposure (Lefebvre et al., 2004), with minor modifications. Briefly, at each timepoint, ten fish in each group were randomly examined three times using a tactile stimulus by manually scoring different behavioral endpoints, combined with videotaping analysis (Gerlai et al., 2008). Healthy or control zebrafish immediately swam away from the stimulus source with a traveling distance of more than one to two body lengths (>3–6 cm) when a tactile stimulus was applied. Conversely, touch responses were considered as abnormal using the following criteria (Lefebvre et al., 2004): (1) reduced response [traveled less than one body length (<3 cm)]; (2) delayed response (more than two touches required for response); and (3) no response (paralysis). The number of fish with abnormal touch responses was expressed as a percentage of the total number of fish examined during each timepoint.

2.8. Observation of head and tail locomotory behavior Unexpected locomotory abnormalities, especially of the head and tail, were observed while monitoring zebrafish exposure to aphantoxins in the above experiments. To further investigate these locomotory changes due to A. flos-aquae DC-1 aphantoxin exposure, we subsequently designed several experiments to study the headtail locomotory changes, based on previous methods (Altringham and Ellerby, 1999; Bierman et al., 2004; Lefebvre et al., 2005; Levin et al., 2007; Müller and Van Leeuwen, 2006). The head locomotory function of zebrafish, according to observation of control or healthy fish in the laboratory, is to direct the fish forward, to turn the head flexibly more than a 30◦ angle, to explore the environment in front, to look for food and so on, without swaying mechanically from side to side (Foreman and Eaton, 1993; Domenici and Blake, 1997). Conversely, abnormal movement of the head was considered as follows: (1) slow and inflexible head movement, with a reduced turning angle (◦ ) and orientation control of head (Hale, 2002; Bierman et al., 2004); and (2) head swaying mechanically from side to side (or from left to right) (Tiedeken and Ramsdell, 2009). At each timepoint, ten fish each group were randomly investigated (30 fish per group, 3 fish per aquarium) for head movement, combined with videotaping for 10 min with a HG10 camera. The number of zebrafish showing head locomotory abnormalities were recorded and the characteristics were described. The number of fish with abnormal head movements was expressed as a percentage of the total number of fish examined during each timepoint. Regarding zebrafish tail movements, according to observation in the laboratory, healthy (or control) zebrafish swayed their tails from side to side easily, followed by alternating muscular contraction and relaxation of each side of their bodies (Wardle et al., 1995; D‘août and Aerts, 1999). The normal tail movement of zebrafish is to provide the power to drive the fish forward (Altringham and Ellerby, 1999). The locomotory trajectories of the tail always form wavy-type or S-type patterns (Müller and Van Leeuwen, 2006). The following were considered to be abnormal tail movements: (1) tail activity limited to the caudal end or only the caudal fin but not the whole tail (Hale, 1999; Bierman et al., 2004); (2) the tail lacked easy and limber tail movement patterns and swayed mechanically from side to side (Müller and Van Leeuwen, 2006); and (3) the forward swimming distance was less than one body length following a mechanical tail movement (Lefebvre et al., 2004). At each timepoint, ten fish each group were randomly investigated (30 fish per group, 3 fish per aquarium) for tail movement, combined with videotape analysis using an HG10 camera. The number of fish showing tail locomotory abnormalities was recorded and the characteristics were described. The number of fish with abnormal tail movement was expressed as a percentage of the total number of fish examined during each timepoint. 2.9. Zebrafish distribution in water column of aquarium To assess the distribution of the zebrafish in the water, a total of 90 fish (30 fish per aquarium) were used. The zebrafish distribution (preferred swimming activity) in water was assessed in three rectangular aquaria (as above) maximally filled with ∼33 L aerated tap water (Wu et al., 2012) to a height of 30 cm. Each aquarium was divided into three virtual horizontal portions (5 cm top, 20 cm middle and 5 cm bottom) by 2 lines marking the outside walls, referred to the previously described (Gerlai et al., 2000; Spence et al., 2006; Levin et al., 2007; Kistler et al., 2011) but with some modifications. Control or healthy zebrafish swam mostly in the middle water (20 cm of water from 5 to 25 cm), with very few fish in the top (25–30 cm) and bottom (0–5 cm) water of the aquaria (Spence et al., 2006). Zebrafish are constantly active and always prefer to be swimming without stopping. Therefore, it was considered to be

2.10. Determination of AChE activity To assess brain AChE activity, a total of 150 fish were exposed to the same concentrations of aphantoxins (low- and high-dose groups) and 0.01 M acetic acid (control). At each timepoint, 5 whole brains per group were quickly removed from 5 euthanized fish on ice granules (−8 ◦ C), briefly washed three times in 0.86% ice-cold saline, frozen quickly in liquid nitrogen, and then stored at −40 ◦ C until analysis. Brain AChE activity was determined using a commercial AChE detection kit (Guangdong Dongsheng, China) according to the manufacturer’s instructions and the previous methods (Jansen et al., 2009). Briefly, ∼45 mg frozen whole brains (−40 ◦ C) were homogenized (homogenizer; IKA® , Werke, Germany) in 405 ␮L (w:v 1:9) 0.86% w/v ice-cold physiological saline (4 ◦ C ice bath, pH 7.2) for 30 s. Supernatants were centrifuged to harvest enzymatic stock extracts (5900 × g, 4 ◦ C, 10 min). The 50 ␮L enzymatic extracts were reacted with 500 ␮L substrate buffer (acetylcholine) and 500 ␮L sulfhydryl chromogenic agent at 37 ◦ C for 6 min, before stopping the reaction. The intensity of the yellow compound was monitored at 412 nm using a microplate reader (Tacon, Salzburg, Austria). The protein concentration of the enzymatic extracts was quantified by a standard method (Bradford, 1976). The enzyme activity was computed according to 1 activity unit (U/mg protein) (when 1 ␮mol of substrate was hydrolyzed per mg of protein per 6 min). Values were finally indicated as the percentage of activity from treated brains compared to controls (Genovese et al., 2007). 2.11. Statistical analysis Statistical analyses were performed using SPSS statistical software (SPSS 13.0, Chicago, IL, USA). All data on mechanosensory properties, head or tail movement, distribution preference in the water column, and brain AChE activities were based on three independent experiments. Statistical significances of differences between groups were assessed by one-way analysis of variance (ANOVA) combined with least significant difference (LSD) post hoc tests. p < 0.05 was taken to indicate statistical significance (p < 0.01 = high significance). 3. Results 3.1. Analysis of A. flos-aquae DC-1 toxins HPLC with Post-column Fluorescence Derivatization (HPLCFLD) analysis revealed that the extracted toxins of cultured DC-1 algae contained the three toxic components of neoSTX, GTX1, and GTX5, which were identical to their respective control standards. The total PSP toxin content from A. flos-aquae DC-1 was calculated to be 9.52 ng/mg dry cell weight. Moreover, HPLC-FLD also revealed that the extracted toxins had a purity of 69.57%. GTX1 was the predominant toxin, accounting for 34.04% of total PSPs, while GTX5

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Aphantoxins exposure (hours) Fig. 1. Mechanosensory changes of zebrafish in response to aphantoxin exposure over a 24 h period. Data are plotted as mean ± S.E.M. C, L and H indicate control, lowand high-dose groups, respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

and neoSTX represented 21.28% and 12.77%, respectively, of total PSPs. 3.2. Touch response deficits We compared vehicle-administered controls and toxin-exposed zebrafish to determine if aphantoxin exposure led to touch response (mechanosensory) deficits. As shown in Fig. 1, the touch responses of control fish were normal but exposed fish showed clearly reduced touch responses and even paralysis (p < 0.05). Touch response deficits in both high- and low-dose groups became apparent at 1 h post-exposure and continuously declined overtime, reaching a minimum at 12 h with 81.67% and 38.33%, respectively, versus controls. Thereafter, touch responses in both exposure groups largely recovered with a better recovery in the low-dose group; nonetheless, touch responses were still significantly different from those of the controls at 24 h (48.34% and 81.66% in the high- and low-dose groups, respectively, versus controls). 3.3. Head and tail locomotory dysfunction Head and tail movements in toxin-exposed and control zebrafish were studied to determine if toxin exposure led to head and tail locomotor dysfunction. As shown in Figs. 2 and 3, head-tail movements of controls were normal while exposed fish showed significant locomotor abnormalities (p < 0.05). Head movement and turning of exposed zebrafish became inflexible, swinging mechanically from side to side, while tail movement of the exposed groups showed a narrowed range, with reduced amplitude of swing and driving force. Furthermore, the locomotor center of gravity of the whole body shifted backwards (tailward).

Cumulative % of head movement

abnormal distribution when fish preferred to swim in the top or bottom waters, mostly staying there for more than 10 min (Kistler et al., 2011). Zebrafish swimming behavior and their distribution in the aquaria (30 fish per group in one aquarium) were observed for 10 min, combined with analyzing videotape at each post-exposure timepoint. The numbers of fish in the top, bottom and medium water at each timepoint were the average of fish numbers at five sub-time points by recording fish numbers at 2, 4, 6, 8 and 10 min of each 10 min measuring period at each timepoint, and the percentages of fish in the top, middle and bottom water at each timepoint was expressed according to the total number of exposed fish in each group.

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Fig. 3. Tail locomotor changes of zebrafish in response to aphantoxin exposure over a 24 h period. Data are plotted as mean ± S.E.M. C, L and H indicate control, low- and high-dose groups, respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

The tail movement pattern also exhibited a mechanical side-toside swing that acted in concert with the mechanical side-to-side swinging of the head. The combination of the head and tail movements formed an abnormal locomotor pattern where exposed zebrafish shook the head after wagging the tail. The mechanical side-to-side head and tail movements meant that the fish could hardly move forwards. These locomotor changes in both aphantoxin groups were evident at 1 h post-exposure and progressively worsened with an increased duration of aphantoxin exposure: the most serious suppression of head-tail movement occurred at 12 h, with 87.65% (high dose) and 79.89% (low dose) reductions in head movements and 74.11% (high dose) and 62.6% (low dose) reductions in tail movements versus controls. Thereafter, the head and tail locomotor inhibition of the zebrafish in the two exposure groups began to recover but they still were different from those of the controls at 24 h.

Fig. 5. Distribution changes of zebrafish in the bottom water in response to aphantoxin exposure over a 24 h period. Data are plotted as mean ± S.E.M. C, L and H indicate control, low- and high-dose groups, respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

respectively, versus controls. The number of fish in the middle water column reduced gradually with exposure duration. This abnormal distribution of exposed zebrafish was visible at 1 h post-exposure and worsened with toxin duration and aphantoxins poisoning degree. The most robust changes in water distribution preference were evident at 12 h, followed by the occurrence of a polarization state between the top and bottom of the aquarium with some of these exposed fish [45.37% (high dose), 33.33% (low dose)] mainly distributed in the top water, and some [45.06% (high dose), 33.45% (low dose)] in the bottom water column. In both high- and low-dose groups, only sporadic fish [9.57% (high dose), 33.22% (low dose)] showed activity in the middle column of water. Subsequently, the normal distribution of the exposed zebrafish was recovered gradually with faster recovery in the lowdose, though their patterns were still different from those of the control group at 24 h.

As shown in Figs. 4 and 5, control fish spent most of their time actively swimming in the middle water column with few fish entering in the top or bottom water regions. However, exposure to aphantoxins resulted in significantly more fish entering the upper or bottom water columns and fewer fish entering the middle water column (p < 0.05). Some of the fish concentrated in the top water of the aquarium and spent most of the time (more than 10 min) there, with averages of 26.45% and 17.5% in the high- and lowdose groups, respectively, versus controls. Some fish spent most of the time (more than 10 min) in the bottom water of the tank, with averages of 32.77% and 23.49% in the high- and low-dose groups,

Whole brains of aphantoxin-exposed and control zebrafish were examined to detect aphantoxin-induced changes in brain AChE activity. As shown in Fig. 6, brain AChE activities were unchanged in controls but were significantly reduced in aphantoxin-exposed groups (p < 0.01), with average reductions from controls of 39.96% and 26.72% in the high- and low-dose groups, respectively (Fig. 6). Brain AChE activities of exposed zebrafish were reduced at 1 h postexposure, and showed more robust reductions with increasing time after exposure and the degree of aphantoxin poisoning. The most evident reduction in AChE activity occurred at 12 h, with an average reduction from controls of 57.25% and 41.5% in the high- and low-dose groups, respectively. After that, AChE specific activities in

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3.5. AChE activity in the brain of exposed zebrafish

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the exposed zebrafish began to recover, with faster recovery in the low-dose group. However, they were still different from that of the control group at 24 h post-exposure. 4. Discussion 4.1. Components and isomers of A. flos-aquae DC-1 toxins In the present study, we confirmed the negative effects of A. flos-aquae DC-1 PSP toxins on zebrafish behavior (touch response, head-tail movement, and distribution preference in water) and brain AChE activity. We selected A. flos-aquae DC-1 toxins because they are one of the most frequently detected toxins in fresh water, and because they have been shown to exert potent neurotoxic effects on animals (Liu et al., 2006a,b; Ballot et al., 2010; Ledreux et al., 2010; Zhang et al., 2011, 2013). Zebrafish have emerged as an excellent freshwater vertebrate model for assessing neurobehavioral phenotypes associated with neurotoxin effects (Savio et al., 2012). Behavioral observation of zebrafish was chosen as the endpoint, because the regulation of instinctive behaviors is a complex phenomenon involving interactions among the central and peripheral nervous systems and the neuroendocrine system (Matsuda et al., 2011). Furthermore, the cholinergic system, which includes AChE, is one of the most important modulatory neurotransmitter systems, and has long been recognized to play key roles in many functions in the central nervous system, including in the control of locomotor and sensory functions (Anglister et al., 2008; Da Silva et al., 2010; Martins-Silva et al., 2011). There is currently little information available regarding the relationship between A. flos-aquae DC-1 neurotoxins and locomotor behavior of animals and the cholinergic system. We therefore sought to determine if some of the behavioral changes observed in this study could be attributed to the cholinergic system. We have shown in this study that A. flos-aquae DC-1 strains produced PSPs, which is in accord with previous findings (Pereira et al., 2000; Ferreira et al., 2001). Recently, several Aphanizomenon strains in freshwater bodies worldwide have been reported to secrete PSPs (Nogueira et al., 2004; Pereira et al., 2004a; Costa et al., 2006; Ballot et al., 2010; Wörmer et al., 2011). These results demonstrate that the dominant Aphanizomenon genus in blooms of worldwide freshwater bodies can produce PSPs (Banker et al., 1997; Olli and Heiskanen, 1999; Kanoshina et al., 2003). 4.2. Touch response (mechanosensory) deficits and paralysis We studied the mechanosensory deficits caused by aphantoxin administration in zebrafish and showed that sublethal doses of aphantoxins induced touch response reduction and even paralysis. This is in line with previous results showing that marine STX can induce delayed touch responses in larva of both zebrafish and Pacific herring (Lefebvre et al., 2004, 2005). However, little was known about the touch responses of adult fish after aphantoxin exposure. The observation that aphantoxins cause delayed touch responses or paralysis in zebrafish is not particularly surprising given that aphantoxins have been proven to act as neurotoxic PSPs and block the transmission of action potentials by inhibiting ion transport through voltage-gated sodium channels in neuronal membranes (Aráoz et al., 2010). However, accumulated aphantoxin toxicity reduced touch responses and even caused paralysis in time- and dose-dependent manners. In this study, the aphantoxininduced delayed touch response/paralysis was more noticeable from 1 h to 12 h, suggesting that this effect was time-dependent, as suggested by previous studies (Yokoo et al., 2000; Perreault et al., 2011). The aphantoxin-induced delayed touch response/paralysis

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started at 1 h post-exposure suggesting that the onset of aphantoxin toxicity was rapid, as indicated in previous reports (Lefebvre et al., 2005). Furthermore, there were evident differences in the delayed touch response/paralysis induced by high and low doses in this study, suggesting that the toxin also acted in a dose-dependent manner, as suggested by previous studies (Jellett et al., 1995; Lefebvre et al., 2005). The partial recovery of the touch response from 12 to 24 h suggests the action of metabolic biotransformation that can decrease toxicity by changing toxic moieties or by increasing excretion rates, as well as a toxin sequestering mechanisms in some organisms (Trainer and Bill, 2004; Lefebvre et al., 2005). 4.3. Head and tail locomotory abnormality We also studied the head and tail locomotory abnormalities following aphantoxin administration in zebrafish, demonstrating that sublethal doses of aphantoxins led to a significant locomotor activity reduction in zebrafish. The most serious of these changes was the clear reduction in head and tail flexibility, followed by the diminished amplitude of the swing and the propulsive force of the tail, the limited locomotor center of gravity of the body, and the pronounced mechanical side-to-side swing of both head and tail, which together indicate that the inhibitory effect of the toxin was serious during this period. Several studies have observed that toxicant exposure can lower animals’ locomotor activity (Magalhães et al., 2007; Ferrão-Filho et al., 2007; Cazenave et al., 2008). These results are in accordance with our findings that suggest that aphantoxins can suppress the swimming activity of zebrafish. This locomotor hypoactivity may have several explanations. One possible reason may be a physiological compensation mechanism, designed to lessen the metabolic costs of the biotransformation of the toxins or the detoxification required to reduce the probability of death (Magalhães et al., 2007). Another possible and important reason for the decrease is related to the neurotoxin and its mechanism of action because aphantoxins have been shown to contain the components of PSPs (Liu et al., 2006a,b) and damage zebrafish brain cells (Zhang et al., 2011, 2013) by blocking sodium channels in neurons and leading to a cessation of nerve impulses (Aráoz et al., 2010). 4.4. Distribution preferences of exposed zebrafish in water of aquaria We also studied zebrafish distribution preference in aquaria following neurotoxin administration. We found that aphantoxin administration caused dose- and time-dependent distribution changes. Our studies demonstrated that aphantoxins induce a significantly higher proportion of fish to enter the upper and bottom water columns. A proportion of fish concentrated in the top waters of the aquaria and spent more time (more than 10 min) there, consistent with previous reports (Levin et al., 2007; Grossman et al., 2010), while another proportion of fish spent more time (more than 10 min) in the bottom area of tank, also in accord with previous findings (Gerlai et al., 2008; Sison and Gerlai, 2011; Kist et al., 2011). It is possible that hypoxia due to oxygen consumption increase and respiratory dysfunction from toxin-induced inhibited innervation of the respiratory system led to the fish spending most of their time in the top water (Zhang et al., 2009). Spending most of the time in the bottom area of the tank, on one hand, can be interpreted as an avoidance reaction to the dangerous environment due to toxin-induced fear or anxiogenic effects (Kist et al., 2011). On the other hand, a more significant explanation is that decreased locomotory capacity due to impaired motor function leads to more time being spent on the bottom (Gerlai et al., 2008).

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The current results, combined with previous findings, demonstrate that the administration of sublethal doses of A. flos-aquae DC1 PSPs caused some fish to spend more time at the top of the water column as a result of hypoxia, while others spent more time at the bottom, because of impaired motor function. This suggests that zebrafish nervous system-derived regulation of respiratory function and locomotor function can be suppressed by PSPs. 4.5. AChE inhibition in the brain of exposed zebrafish We also assessed changes in brain AChE activity and locomotor behavioral changes of zebrafish exposed to aphantoxins. AChE activity has been widely used as a bioindicator of environmental exposure and there are a number of studies in the literature concerning the effects of environmental organic pollutants, heavy metals and other chemical toxicants on AChE activity in animals (Rao et al., 2005a,b; Rico et al., 2006, 2007; Rosemberg et al., 2010; Richetti et al., 2011; Pereira et al., 2012). However, this is the first study to correlate the effects of aphantoxins on brain AChE activity and the locomotor behavioral activity of exposed animals. The results presented here demonstrated the inhibitory action of aphantoxins on AChE activity in zebrafish brain, suggesting that the toxins can interfere with cholinergic neurotransmission. AChE inhibition may be a compensatory mechanism that attempts to decrease the hydrolysis of free ACh and thereby increase the concentration of this neurotransmitter in the synaptic cleft where it is essential for the generation of nervous impulses (Rosemberg et al., 2010). On the other hand, AChE inhibition, may be related to cell damage in the nervous system because AChE is an enzyme anchored mainly in the cell membrane in brain regions rich in cholinergic pathways (Rodrigues et al., 2009; da Silva et al., 2011a). A previous study also showed that AChE plays an important role in the preservation of the integrity of the cell (Santi et al., 2011). Meanwhile, our previous studies showed that exposure to aphantoxins caused ultrastructural changes and apoptotic gene upregulation in zebrafish brain (Zhang et al., 2011, 2013). These findings suggest that inhibition of AChE could be causing direct cellular injury of the brain. 4.6. Ecological implications of exposure to aphantoxins The decreased touch response, head-tail locomotory incoordination, and abnormal distribution preference of zebrafish reported in the study is understood to be the result of different physiopathological responses of an organism. However, these behavioral changes are also related to the ecological environment. If these behavioral changes were to occur in natural conditions, they would have negative impacts on the survival and development of wild species, by affecting a range of abilities, including prey capture, predator avoidance, migration, courtship and reproduction. In Dianchi Lake in China, A. flos-aquae DC-1-producing blooms can continue for several months (Liu et al., 2006a,b; Zhang et al., 2011, 2013). Although the toxin levels in Dianchi Lake itself have not been thoroughly quantified, toxin concentrations are likely to be highly variable, with elevated concentrations present in association with dense microlayers of phytoplankton, even at the end of blooms when toxin-containing A. flos-aquae DC-1 cells begin to die and lyse. Thus, the reduced touch response and locomotor activity, and abnormal distribution demonstrated in this study will disadvantage the organisms in the ecosystem and will, therefore, influence biocoenotic structures and functions. These findings will improve our understanding of the ecological consequences of toxic algal blooms, and should be expanded in further studies. In summary, we report that administration of two different sublethal doses of A. flos-aquae DC-1 toxin to zebrafish caused

rapid dose- and time-dependent changes in sensory and locomotor behavior. Both toxin doses induced delayed touch responses, reduced head-tail locomotor abilities, inflexible turning of the head, a backward-shifted center of gravity, altered mechanical locomotor patterns, and altered water distribution preference, demonstrating that the toxin can inhibit zebrafish sensory and locomotor activity. Toxin exposure led to significant and sequential inhibition of brain AChE activity, revealing a close relationship between behavioral impairment, AChE activity changes and toxin dose and duration, suggesting zebrafish locomotor behavior and brain AChE can be used as indicators of aphantoxins and its blooms in nature. These results provide direct evidence that freshwater cyanobacterial neurotoxins can exert toxic effects on sensory and locomotor behavior by inhibiting the zebrafish cholinergic system and reflect the neurotoxicity of these PSPs. Acknowledgements The authors thank Dr.Yongmei Liu and Yanping Su for providing Aphanizomenon flos-aquae DC-1 and analyzing the aphantoxin components, respectively. The authors also thank Drs. Hong Zhang, Juanjuan Xu, Shubin Lan, Li Wu, and Weiju Wu for their assistance with these experiments. This study was financially supported by the National Basic Research Program of China (2008CB418001) and the National High-tech Research and Development Program of China (2013AA065804). References Airhart, M.J., Lee, D.H., Wilson, T.D., Miller, B.E., Miller, M.N., Skalko, R.G., 2007. Movement disorders and neurochemical changes in zebrafish larvae after bath exposure to fluoxetine (PROZAC). Neurotoxicology and Teratology 29, 652–664. Alsop, D., Vijayan, M.M., 2008. Development of the corticosteroid stress axis and receptor expression in zebrafish. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 294, R711–R719. Altringham, D., Ellerby, D.J., 1999. Fish swimming: patterns in muscle function. Journal of Experimental Biology 202, 3397–3403. Anderson, D.M., Glibert, P.M., Burkholder, J.M., 2002. Harmful algal blooms and eutrophication: nutrient sources, composition, and consequences. Estuaries 25 (4b), 704–726. Anglister, L., Etlin, A., Finkel, E., Durrant, A.R., Lev-Tov, A., 2008. Cholinesterases in development and disease. Chemico-Biological Interactions 175, 92–100. Aráoz, R., Molgó, J., de Marsac, N.T., 2010. Neurotoxic cyanobacterial toxins. Toxicon 56 (5), 813–828. Baganz, D., Staaks, G., Steinberg, C., 1998. Impact of the cyanobacteria toxin, microcystin-LR on Behaviour of zebrafish, Danio rerio. Water Research 32 (3), 948–952. Baganz, D., Staaks, G., Pflugmacher, S., Steinberg, C.E.W., 2004. Comparative Study of Microcystin-LR-Induced Behavioral Changes of Two Fish Species. Danio rerio and Leucaspius delineatus Environmental Toxicology 19 (6), 564–570. Bakke, M.J., Hustoft, H.K., Horsberg, T.E., 2010. Subclinical effects of saxitoxin and domoic acid on aggressive behaviour and monoaminergic turnover in rainbow trout (Oncorhynchus mykiss). Aquatic Toxicology 99, 1–9. Ballesteros, M.L., Durando, P.E., Nores, M.L., Díaz, M.P., Bistoni, M.A., Wunderlin, D.A., 2009. Endosulfan induces changes in spontaneous swimming activity and acetylcholinesterase activity of Jenynsia multidentata (Anablepidae Cyprinodontiformes). Environmental Pollution 157, 1573–1580. Ballot, A., Fastner, J., Wiedner, C., 2010. Paralytic shellfish poisoning toxin-producing cyanobacterium Aphanizomenon gracile in Northeast Germany. Applied and Environment Microbiology 76 (4), 1173–1180. Banker, R., Carmeli, S., Hadas, O., Teltsch, B., Porat, R., Sukenik, A., 1997. Identification of cylindrospermopsin in aphanizomenon obalisporum (cyanophyceae) isolated from lake kinneret, Israel. Journal of Phycology 33, 613–616. Barbazuk, W.B., Korf, I., Kadavi, C., Heyen, J., Tate, S., Wun, E., et al., 2000. The syntenic relationship of the zebrafish and human genomes. Genome Research 10, 1351–1358. Bégout Anras, M.L., Lagardére, J.P., 2004. Measuring cultured fish swimming behaviour: first results on rainbow trout using acoustic telemetry in tanks. Aquaculture 240, 175–186. Bierman, H.S., Schriefer, J.E., Zottoli, S.J., Hale, M.E., 2004. The effects of head and tail stimulation on the withdrawal startle response of the rope fish (Erpetoichthys calabaricus). Journal of Experimental Biology 207, 3985–3997. Blaser, R., Gerlai, R., 2006. Behavioral phenotyping in zebrafish: comparison of three behavioral quantification methods. Behavior Research Methods 38 (3), 456–469. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 218–254.

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Further reading Bass, S.L., Gerlai, R., 2008. Zebrafish (Danio rerio) responds differentially to stimulus fish: the effects of sympatric and allopatric predators and harmless fish. Behav Brain Res 186, 107–117. Begout Anras, M.L., Lagardére, J.P., 2004. Measuring cultured fish swimming behaviour: first results on rainbow trout using acoustic telemetry in tanks. Aquaculture 240, 175–186. Carmichael, W.W., Drapeau, C., Anderson, D.M., 2000. Harvesting and quality control of Aphanizomenon flos-aquae from Klamath Lake for human dietary use. Journal of Applied Phycology 12, 585–595. Cirelli, C., Tononi, G., 2000. Differential expression of plasticity-related genes in waking and sleep and their regulation by the noradrenergic system. The Journal of Neuroscience 20, 9187–9194.

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Colwill, R.M., Raymond, M.P., Ferreira, L., Escudero, H., 2005. Visual discrimination learning in zebrafish (Danio rerio). Behavioural Processes 70, 19–31. Cooke, S.J., Chandroo, K.P., Beddow, T.A., Moccia, R.D., McKinley, R.S., 2000. Swimming activity and energetic expenditure of captive rainbow trout Oncorhynchus mykiss (Walbaum) estimated by electromyogram telemetry. Journal of Aquatic Research 31, 495–505. Dobbertin, A., Hrabovska, A., Dembele, K., Camp, S., Taylor, P., Krejci, E., Bernard, V., 2009. Targeting of Acetylcholinesterase in Neurons In Vivo: A Dual Processing Function for the Proline-Rich Membrane Anchor Subunit and the Attachment Domain on the Catalytic Subunit. The Journal of Neuroscience 29 (14), 4519–4530. Downes, G.B., Granato, M., 2006. Supraspinal input is dispensable to generate glycine- mediated locomotive behaviors in the zebrafish embryo. Journal of Neurobiology 66, 437–451. Engeszer, R.E., Ryan, M.J., Parichy, D.M., 2004. Learned social preference in zebrafish. Current Biology 14, 881–884. Fränzle, O., 2003. Bioindicators and environmental stress assessment. In: Markert, B.A., Breure, A.M., Zechmeister, H.G. (Eds.), Bioindicators and Biomonitors: Principles, Concepts and Applications. Elsevier, The Netherlands, pp. 41–85. García, C., Rodriguez-Navarro, A., Díaz, J.C., Torres, R., Lagos, N., 2009. Evidence of in vitro glucuronidation and enzymatic transformation of paralytic shellfish toxins by healthy human liver microsomes fraction. Toxicon 53, 206–213. Gastro, D., Vera, D., Lagos, N., García, C., Vásquez, M., 2004. The effect of temperature on growth and production of paralytic shellfish poisoning toxins by the cyanobacterium Cylindrospermopsis raciborskii C10. Toxicon 44, 483–489. Kayal, N., Newcombe, G., Ho, L., 2008. Investigating the fate of saxitoxins in biologically active water treatment plant filters. Environ. Toxicol. 23, 751–755. Kiehn, O., 2006. Locomotor circuits in the mammalian spinal cord. Ann. Rev. Neurosci. 29, 279–306. Li, R., Carmichael, W.W., Liu, Y., Watanabe, M.M., 2000. Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA genesequences. Hydrobiologia 438, 99–105. Li, R., Carmichael, W.W., Pereira, P., 2003. Morphological and 16S rRNA gene evidence for reclassification of the paralytic shellfish toxin producing Aphanizomenon flos-aquae LMECYA31 as Aphanizomenon issatschenkoi (Cyanophycaea). Journal of Phycology 39, 814–818. Llewellyn, L.E., 2006. Saxitoxin, a toxic marine natural product that targets a multitude of receptors. Natural Product Reports 23, 200–222. Loucks, E., Carvan III, M.J., 2004. Strain-dependent effects of developmental ethanol exposure in zebrafish. Neurotoxicology and Teratology 26, 745–755. Mann, K.D., Turnell, E.R., Atema, J., Gerlach, G., 2003. Kin recognition in juvenile zebrafish (Danio rerio) based on olfactory cues. The Biological Bulletin 205, 224–225. Ninkovic, J., Bally-Cuif, L., 2006. The zebrafish as amodel system for assessing the reinforcing properties of drugs of abuse. Methods 39, 262–274. Pereira, P., Li, R.H., Carmichael, W.W., Dias, E., Franca, S., 2004b. Taxonomy and production of paralytic shellfish toxins by the freshwater cyanobacterium Aphanizomenon gracile LMECYA40. European Journal of Phycology 39, 361–368. Pietri, T., Manalo, E., Ryan, J., Saint-Amant, L., Washbourne, P., 2009. Glutamate drives the touch response through a rostral loop in the spinal cord of zebrafish embryos. Developmental Neurobiology 69, 780–795. Pyron, M., 2003. Female preferences and male–male interactions in zebrafish (Danio rerio). Can J Zool 81, 122–125. Shimizu, Y., 2000. Chemistry and mechanism of action. In: Botana, L.M. (Ed.), Seafood and Freshwater Toxins: Pharmacology, Physiology and Detection. Marcel Dekker, New York, NY, USA, pp. 151–172. Sillar, K.T., 2009. Escape Behaviour: Reciprocal Inhibition Ensures Effective Escape Trajectory. Current Biology 19 (16), 697–699. Stevens, M., Peigneur, S., Tytgat, J., 2011. Neurotoxins and their binding areas on voltage-gated sodium channels. Frontiers in Pharmacology 2, 1–13, http://dx.doi.org/10.3389/fphar.2011.00071. Vale, P., 2008. Fate of benzoate paralytic shellfish poisoning toxins from Gymnodinium catenatum in shellfish and fish detected by pre-column oxidation and liquid chromatography with fluorescence detection. Journal of Chromatography A 1190, 191–197. Vitebsky, A., Reyes, R., Sanderson, M.J., Michel, W.C., Whitlock, K.E., 2005. Isolation and characterization of the laure olfactory behavioral mutant in the zebrafish Danio rerio. Developmental Dynamics 234, 229–242. Williams, F.E., WhiteD, Messer, W.S., 2002. A simple spatial alternation task for assessing memory function in zebrafish. Behavioural Processes 58, 125–132.