Project description:Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained in the Center for Environmental Biotechnology at the University of Tennessee. Fish husbandry, spawning, and experimental procedures were conducted with approval from the University of Tennessee Institutional Animal Care and Use Committee (Protocol #1690-1007). Water for holding fish and conducting experiments (hereafter referred to as fish water) consisted of MilliQ water (Millipore, Bedford, MA) with ions added: 19 mg/L NaHCO3, 1 mg/L sea salt (Instant Ocean Synthetic Sea Salt, Mentor, OH), 10 mg/L CaSO4, 10 mg/L MgSO4, 2 mg/L KCl. Embryos were obtained by spawning adult fish with no history of contaminant exposure. Fertilization of embryos took place at the same time (Â± 15 min.), such that larvae used in experiments were of similar age at the time of exposure. All activities (maintenance of adult fish, spawning, and experiments) were conducted in an environmental chamber with a temperature of 27Â± 1 ÂºC and 14:10h light:dark photoperiod. Experiment Overall Design: At 72 h post-fertilization, zebrafish larvae were exposed to lyophilized Microcystis and purified MC-LR at concentrations of 100 and 1,000 Âµg/L. Controls consisted of zebrafish system water (negative control) and zebrafish system water containing 0.05% ethanol (vehicle control). Larvae from both control groups as well as 100 Âµg/L MC-LR, 1,000 Âµg/L MC-LR, and lyophilized Microcystis were exposed in groups of 50 with three replicates and were sacrificed after 96 hours for total RNA extraction and subsequent microarray analysis. All larvae were exposed in beakers containing 100 ml of solution. Experiment Overall Design: Water samples for microcystin analysis and water quality measurements were taken during the experiment, and mortality and behavioral observations were recorded at 24-hour intervals. Microcystin analysis was conducted by protein phosphatase inhibition assay. Measured concentrations of microcystin-LR were 140 Â± 12 SD (low concentration) and 1,703 Â± 71 SD (high concentration). The concentration of microcystin-LR in the lyophilized Microcystis treatment was 4.5 Âµg/L. Water quality parameters measured included dissolved oxygen (6.7 mg/L), pH (6.9), total alkalinity (36 mg/L as CaCO3), total hardness (18 mg/L as CaCO3), and ammonia (<0.2 mg/L). No significant mortality or behavioral changes in larvae were observed during the exposure.

Project description:Cyanobacterial blooms are increasing in frequency and geographic distribution worldwide, driven by changes in various environmental factors and nutrient enrichment of waterbodies. These blooms produce a suite of potent toxins that threaten aquatic ecosystems, wildlife, and human health. (+)-Anatoxin-a (ATX-a) is one such toxin produced by several genera of freshwater cyanobacteria. It is an agonist of nicotinic acetylcholine receptors (nAchR), found primarily at the neuromuscular junctions. Binding of ATX-a to nAchR leads to prolonged receptor activation, continuous depolarization of the postsynaptic membrane, and sustained muscle contraction. Despite its neurotoxicity very little is known about its effects on early vertebrate development. During development, nAChR signaling plays a critical role in the maturation of neuromuscular junctions, motor neuron connectivity, and muscle function. We hypothesized that transient activation of nAchR by anatoxin-a exposure during sensitive developmental windows has the potential to cause altered neuromuscular activity and may have longer-term effects on motor system development. We investigated the developmental impacts of anatoxin-a exposure using zebrafish (Danio rerio) embryos to identify windows of susceptibility. Wild type (TL strain) zebrafish embryos were exposed to ATX-a (2ng per embryo or larvae) via microinjection at different developmental stages (0.25, 1, 2, 3, or 4 days post-fertilization (dpf)). Developmental phenotypes and behavioral responses were monitored post-exposure. ATX-a exposure at 2 and 3 dpf induced rapid-onset muscle contractions within seconds and there is a transient loss of touch reflex for 3-4 hours post-exposure. No other overt phenotypes were observed at other time points. Gene expression analysis revealed large number of differentially expressed genes at 6 and 24 hpf with most changes at 6hours post-exposure. Gene Ontology and zebrafish phenotype analysis showed enrichment of genes related to muscle and nervous system development, suggesting disruption of neuromuscular junction function. Using Tg(mnx1:eGFP) zebrafish to visualiz the effects of ATX-a exposure on the motor neurons, we observed transient neuritic or axonal beading suggesting an acute response to injury, which is resolved within 24hours. Our findings demonstrate that anatoxin-a exposure during specific developmental windows can cause acute cellular and molecular disruptions, particularly affecting neuromuscular systems. These results highlight the need to further investigate whether such transient effects have lasting consequences, especially given the increasing global prevalence of cyanobacterial toxins and their potential to impact aquatic life and human health. This work is supported by the Woods Hole Center for Oceans and Human Health (NIEHS P01 ES028938 and NSF OCE-2418297).

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Danio rerio

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