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.
Biomaterial provider
Emily Rodgers
Treatment protocol
Ethanol Treated Preparation of Exposure Solutions The Microcystis treatment was prepared using lyophilized cells of Microcystis aeruginosa. M. aeruginosa PCC-7806 was cultured in BG-11 media. Live cultures of Microcystis were centrifuged to concentrate cells into a pellet, and pellets obtained after centrifuging 6 L of culture were combined. Cells were lyophilized for 48 hours using a freeze-dry system (Labconco, Kansas City, MO) and the total mass of algal cells obtained was 300 mg (50 mg lyophilized cells/L of live culture). For the exposure of larval zebrafish, lyophilized Microcystis was reconstituted to a nominal concentration of 50 mg lyophilized cells/L. Solutions for microcystin-LR (MC-LR) treatments were prepared by dissolving 1mg of purified MC-LR (Alexis Biochemicals, San Diego, CA) in 0.5 mL ethanol and diluting to appropriate concentrations using fish water. The concentration of ethanol in all treatments was = 0.05%, and a treatment of 0.05% ethanol was used as a vehicle control. Fish water served as the negative control.
Growth protocol
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.
Extracted molecule
total RNA
Extraction protocol
Total RNA Extraction Larvae were centrifuged for 10 min. at 13,000 rpm to separate larvae from exposure water, and pellets containing larvae were stored at –80ºC until RNA extraction was performed. Total larval RNA was extracted using the RNeasy mini extraction kit for animal tissues (Qiagen, Valencia, CA) and quantified using a spectrophotometer (Nanodrop, Wilmington, DE).
Label
Biotin
Label protocol
Ambion Message Amp II-Biotin Enhanced Method with 1µg Total RNA
Hybridization protocol
Protocol follows the Affymetrix Hybridization, Wash and Stain kit for Eukaryotic Target Hybridization for 49Format array using 15µg fragmented cRNA and 45°C hyb temp for 16 hours at 60rpm. Washed with Affymetrix 450 Fluidics using FS450_001.
Scan protocol
Affymetrix 7G GeneChip Scanner standard parameters for Zebrafish Array
Description
Emily D. Rogers, Theodore B. Henry, Julia S. Gouffon, Jackson McPherson, Gregory L. Boyer, Gary S. Sayler, Steven W. Wilhelm
