MeNQ (CAS Number 4245-76-5) was obtained from a commercial vendor (TCI America, product number M0749). According to manufacturer specifications, this MeNQ was 98% pure and was hydrated (25% of mass as water) for safety purposes. MeNQ was used as received from the vendor for preparation of exposure solutions. Water used for preparation of stock solutions and for the control exposures was tap water (City of Vicksburg, MS, municipal source) dechlorinated using carbon filtration for all toxicity bioassays. For the D. pulex bioassay, standard synthetic freshwater bioassay medium (moderately hard reconstituted water) was used (USEPA 2002). Bioassays were conducted at a target temperature of 25 °C for 11 d as this period was required to acquire 3 broods. Exposures were performed in 50 mL beakers with a test solution volume of 40 mL. One test animal was exposed in each beaker, and 10 replicate beakers were tested per treatment. The daily feeding ration mixture consisting of half Raphidocelis subcapitata and half yeast, cereal leaves, and trout chow fish food. Complete water renewals were conducted three times a week on Mondays, Wednesdays and Fridays. The reproductive output was quantified as the total number of neonates produced in three broods per parental adult that survived to assay completion. D. pulex were exposed to MeNQ at control (0 mg/L), 174, 346, 709, 1385, and 2286 mg/L (measured concentrations) as juveniles through three-broods of reproduction. The reproduction assays included 20 animals exposed individually per treatment. The animals remaining at the termination of the assay were randomly assigned to a set of 4 replicate groups of animals per treatment to serve as treatment replicates in the transcriptomic-expression experimental design (6 treatments x 4 replicates). REFERENCE: U.S. Environmental Protection Agency (2002) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. EPA-821-R-02-012, 5th ed. Office of Water, Washington, DC.
Growth protocol
Daphnia pulex were obtained from in-house cultures (originally purchased from EC Testing) and reared in accordance with guidance in Laird et al. (2015). REFERENCE: Laird JG, Kennedy AJ, Melby NL, Gong P. 2015. Development of a Chronic Toxicity Testing Method for Daphnia pulex, US Army, Engineer Research and Development Center Report. ERDC/EL SR-15-5. pp 1-25.
Extracted molecule
total RNA
Extraction protocol
The D. pulex samples were flash frozen in liquid nitrogen and stored at -80°C until RNA extractions were conducted. Frozen samples were homogenized using disposable mortar and pellet pestles for each sample (Kimble Kontes, Vineland, NJ). Total RNA isolation and on-column DNase digestion was conducted using the Qiagen RNeasy Mini Kit (Qiagen, Germantown, MD) following manufacturer’s recommendations. RNA quantity was assessed using a NanoDrop One Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA) and final quality was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) where electrophoresis gels were visually inspected for degradation prior to use in downstream applications.
Label
Cy3
Label protocol
The Agilent Low Input Quick Amp Labeling Kit (one color) protocol was followed using 65 ng of total RNA as starting material from each biological sample.
Hybridization protocol
An Agilent Technologies (Agilent Technologies, Santa Clara, CA, USA) single color custom 8 x 60K microarray format (Amadid #: 063815) was used for all investigations. A completely randomized design was utilized for microarray hybridizations where each individual D. pulex sample was selected at random (using a random number generator) for hybridization onto individual microarrays. The Agilent Low Input Hybridization Kit (one color) was used for microarray hybridizations following manufacturer’s recommendations. A total of 24 microarrays were hybridized which included four replicates for all MeNQ exposures including the control (0 mg/L), 174, 346, 709, 1385, and 2286 mg/L exposure treatments.
Scan protocol
An Agilent Technologies, High-Resolution Microarray Scanner (Model G2505C) was used to scan microarray images at 2 μm resolution. Data were extracted from microarray images using Agilent Feature Extraction software, version 10.7.3.1.
Data processing
Microarray data were normalized to the 75th percentile within each array followed by median scaling among all exposures using GeneSpring Software version GX 14.9 (Agilent Technologies). GeneSpring was also utilized for the expression analysis where a one-way ANOVA was conducted (p = 0.01) including Benjamini-Hochberg multiple-tests corrections. Post-hoc tests were also conducted where the SNK test was utilized to determine which transcripts had significant differential expression relative to the 0 mg/L control.
