Project description:Microcystin-LR (MC-LR), the most toxic member of microcystin family, inhibits protein phosphatase PP2A, triggers oxidative stress and induces hepatotoxicity. Gene expression profiling of MC-LR treated larvae using DNA microarray analysis revealed effects in the retinal visual cycle and pigmentation synthesis pathways that have not been previously associated with MC-LR. Liver-related genes were also differentially expressed. The microarray data were confirmed by quantitative real-time PCR. Our findings provide new evidence that microcystin-LR exposure of zebrafish larvae modulates the retinal visual cycle and pigmentation synthesis pathways and ultimately alter larval zebrafish behavior
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 (± 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 27± 1 ºC 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 µg/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 µg/L MC-LR, 1,000 µg/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 ± 12 SD (low concentration) and 1,703 ± 71 SD (high concentration). The concentration of microcystin-LR in the lyophilized Microcystis treatment was 4.5 µg/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:We tested the hypothesis that the behavioral response to selenium (Se) follows a hormetic dose response pattern, manifested through the functions of selenoproteins within the brain. We measured anxiety-related behaviors in zebrafish (Danio rerio) at deficient, control and supplemented levels of dietary Se, and measured the transcriptional response of selenoprotein genes important for neuroprotection. We also used a microarray approach to assess the transcriptomic response of the midbrain to Se. The behavioral response to Se was characterized by hormesis, and the direction, magnitude, and shape of the hormetic responses were dependent on both sex and zebrafish population. Transcription of selenoproteins within the midbrain also responded to Se in a similar hormetic dose-dependent manner, with sex and population influencing the trajectory of the responses. The hormetic behavioral response to Se may therefore be manifested through selenoproteins in the brain, but the influence is not direct. We performed a microarray analysis comparing the midbrain-specific transcriptome between male zebrafish from two populations (Pargana: P and Transgenic Mosaic 1: T) fed either a control, Se deficient, or Se supplemented diet (17 total samples: 9 fish per population, 3 fish per diet: missing 1 P control sample).
Project description:We tested the hypothesis that the behavioral response to selenium (Se) follows a hormetic dose response pattern, manifested through the functions of selenoproteins within the brain. We measured anxiety-related behaviors in zebrafish (Danio rerio) at deficient, control and supplemented levels of dietary Se, and measured the transcriptional response of selenoprotein genes important for neuroprotection. We also used a microarray approach to assess the transcriptomic response of the midbrain to Se. The behavioral response to Se was characterized by hormesis, and the direction, magnitude, and shape of the hormetic responses were dependent on both sex and zebrafish population. Transcription of selenoproteins within the midbrain also responded to Se in a similar hormetic dose-dependent manner, with sex and population influencing the trajectory of the responses. The hormetic behavioral response to Se may therefore be manifested through selenoproteins in the brain, but the influence is not direct.
Project description:Transcriptional profiling performed from total eye RNA extracts of wildtype control fishes versus Prpf31 morpholino injected larvae (at ~72hpf) two-condition experiment: wildtype zebrafish versus MO-Prpf31 injected zebrafish eye RNA; 6 replicates each (extraction from 6 pools (~200 eyes each) of controls and 6 pools MO-Prpf31 (~200 eyes each))
Project description:Cyanobacteria produce various cyanotoxins, which can cause severe effects to other organisms. Microcystins, one group of such toxins, primarily produced by species of Microcystis, are strong hepatotoxins and inhibit potently protein phosphatases 1 and 2A. Microcystin is the most studied cyanotoxin, however, others are not investigated. Eutrophication of water bodies promotes the occurrence of toxic algal blooms and since a anthropogenic caused increase in eutrophication events can be observed, it is becoming increasingly important to study the consequences and to increase the knowledge on toxins associated with algal blooms. Recently a new cyanobacteria toxin from a Microcystis strain, CP1020, was described. CP1020 belongs to the class of cyanopeptolins and its toxicity was shown to be comparable to that of microcystin (Gademann et al., 2009). It is a strong protease inhibitor inhibiting trypsin in the picomolar range (IC50 = 670 pM) and effects survival of the freshwater crustacean Thamnocephalus platyurus (LC50) 8.8 M-NM-<M (Gademann et al., 2009). Nothing is known, however, about the toxicity of CP1020 to fish. Furthermore, no information is available on the toxic modes of action, in addition to the proteinase activity. Consequently our study has the aim to elucidate the modes of action of CP1020 on zebrafish eleuthero-embryos. By using a microarray technique, we will analyse alterations of global gene expression by CP1020 at two different concentrations. Thereby, we hope to elucidate the whole array of affected biological pathways to elucidate the mechanisms by which CP1020 affect fish. Gene expression in zebrafish eleuthero-embryos was measured after exposure for 96h to 100 ug/L and 1000 ug/L CP1020 or to the respective controls. A total of 12 arrays (Agilent 4 M-CM-^W 44 K Zebrafish microarray) were used, including four for the solvent control group, four for the 100 M-NM-<g/L and four for the 1000 M-NM-<g/L CP1020 dose group.
Project description:Zebrafish populations recently collected from the wild differ from domesticated populations in anxiety-related behaviors. We measured anxiety-related behaviors in wild and domesticated zebrafish populations and performed a multi-brain region transcriptional comparison using microarrays to try to understand the genetic changes that accompany behavioral adaptation to domestication.