Specialized exposure chambers resembling ant farms were constructed from clear acrylic (JR's Custom Acrylics, Vandalia, OH). The chamber dimensions were 35 X 17 X 2 cm and contained approximately 1190 mL HRW (34.5 cm water height); each was initially loaded with 20 D. magna. To isolate the exposures from ambient laboratory lighting, sets of 3 replicate exposure chambers were placed within five separate wooden boxes (36 X 70 X 70 cm) where each photoperiod treatment was administered. Each photoperiod box had a black interior and was equipped with a wide-spectrum fluorescent lamp (Exo Terra PT2151, Rolf C. Hagen Corp., Mansfield, MA) providing overhead lighting that was uniform across all three exposure chambers. The photoperiods within each photoperiod box were separately regulated by digital light timers (Woods Digital Indoor Timer #50008, Coleman Cable, Inc., Waukegan, IL). The light penetration within the test chambers ranged from 730 to 6750 lux, depending on depth. The photoperiod exposures were run twice providing two sets of three replicates for each photoperiod. Both experiments were conducted for 21 days, adapted from the standard ASTM method E1193-97 (ASTM 2012) duration, at 25 ± 1ºC within Darwin Environmental Chambers (Darwin, St. Louis, Missouri, USA). Complete (100%) water renewals using HRW were performed three times a week (Monday, Wednesday, Friday); viable neonates per adult were enumerated and removed from the experimental chambers at each water exchange. Thus, only the original individuals loaded into the chambers remained for the duration of the experiment. Water quality monitoring included temperature, pH, dissolved oxygen, specific conductivity and ammonia.
Growth protocol
Daphnia magna neonates (<24-h old), obtained from 16L:8D in-house cultures, were immediately used in the photoperiod experiments. Organisms were originally purchased from a commercial source in May 2009 (Aquatic Biosystems, Fort Collins, CO, USA; EPA Ohio, AROF2, Lot No. 070092DM) and in-house cultures were started using one individual to ensure testing utilized a single genotype. All culturing and test methods used hard reconstituted water (HRW), formulated according to USEPA (2002). Test organisms in cultures and experiments were provided a daily ration of 2X105 algae cells/mL (Raphidocelis subcapitata, formerly Selenastrum capricornutum) and 0.01 mL/mL yeast-cerophyl-trout chow (YCT) (Aquatic Biosystems) as prescribed by guidance (ASTM 2012; USEPA 2002).
Extracted molecule
total RNA
Extraction protocol
Frozen samples were homogenized using a pellet pestle (Kimble Kontes, Vineland, NJ) and mortar (Kimble Kontes). Total RNA isolation was conducted using the Qiagen RNeasy Mini Kit (Qiagen, Germantown, MD) following manufacturer’s recommendations. RNA quantity was estimated using a NanoDrop ND-1000 Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA) and final quantity and quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Only samples with a 28s/18s ratio ≥2.0 and RNA integrity number (RIN) ≥7.0 were used for downstream applications.
Label
Cy3
Label protocol
The Agilent Low Input Quick Amp Labeling Kit (one color) and hybridization protocol (Agilent Technologies) were utilized for microarray hybridizations following manufacturer’s recommendations using 80 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 15K microarray format (Amadid #: 237101) was used for all investigations. The experimental design for the microarray experiments utilized animals collected directly from the photo-period exposures. The present GEO dataset was collected from Experiment 2. Within each exposure replicate, two population sub-samples consisting of single, unique D. magna were used for hybridization to microarrays. These population sub-samples were selected at random (using a random number generator) from 2-6 D. magna sampled for the genomics investigation that met the RNA quality criteria described in the following paragraph. In summary, the hybridization included 5 photoperiods x 3 exposure replicates x 2 population subsamples for a total of 30 total microarray hybridizations per experiment
Scan protocol
An Agilent Surescan Microarray Scanner (G2505 C, Agilent Technologies Inc.) was used to scan microarrays at 2 μm resolution.
Data processing
An Agilent Technologies, High-Resolution Microarray Scanner (Model G2505C, Agilent Technologies, Santa Clara, CA, USA) was used to scan microarray images at 2 μm resolution. Data were extracted from microarray images using Agilent Feature Extraction software, version 9.5.1 (Agilent Technologies). Microarray data were normalized to the 75th percentile within each array followed by median scaling among all exposures using GeneSpring Software version GX12.5 (Agilent Technologies). Two-way ANOVAs (p = 0.01) were conducted in GeneSpring to investigate variance within and among the photoperiod treatments. Specifically, the primary variable of interest, photoperiod, was investigated in addition to the contribution of variance from population sub-sampling within each photo-period treatment. Post-hoc pair-wise tests consisting of moderated t-tests (p = 0.05) including ≥ 1.5 fold change cutoff were conducted to identify transcripts differentially expressed in each experimental photo-period (4L:20D, 8L:16D, 12L:12D, and 20L:4D) relative to the control (16L:8D).
