Source of animals
Fish exposure protocols received prior approval from the University of Southern Mississippi Institutional Animal Care and Use Committee (#121 R02) and attempts to adhere to the Guidelines for the Use of Fishes in Research proposed by the American Fisheries Society were made whenever possible. Sheepshead minnows (Cyprinodon variegatus) used in the current study were either derived from an outbred, laboratory- reared stock maintained at the Gulf Coast Research Laboratory (GCRL), University of Southern Mississippi (Ocean Springs, MS) or were collected from estuarine waters located near Point Aux Chenes Road in Ocean Springs, Mississippi and acclimated to dilution/culture for at least one week. Prior to exposure, fish were maintained at 27°C in 15 ‰ salinity artificial seawater.
Exposures for trimmer normalization libraries
Fertilized eggs were collected from adult, wild caught Sheepshead minnows (Cyprinodon variegatus), allowed to hatch in 15 ‰ salinity artificial seawater, transferred to Carolina bowls, and maintained for up to 7 days post hatch (dph). Larvae were removed from the Carolina bowls at 1, 3, 5, and 7 dph, preserved in RNA Later (Ambion, Austin, TX), and stored at -20°C until total RNA was extracted. Adult wild caught Sheepshead minnows female fish were injected with 50μL of chorionic gonadotropin followed by a second injection on the next day after the initial injection. The fish were maintained in normoxic conditions until the third day following the initial injection at which time the eggs were stripped from the females. The eggs were collected in a 50mL beaker containing just enough seawater to cover the eggs and fertilized using the dissected testes from male Sheepshead minnows. Viable embryos were maintained in 15 ‰ salinity artificial seawater, removed at 12, 24, 48, 72, and 96 hours post fertilization, homogenized in 750ul RNA STAT-60 (Tel-Test, Inc., Friendswood, TX), and stored at - 20°C until total RNA was extracted.
Exposures for larval SSH libraries
Fertilized eggs were collected from the adult, laboratory reared stock of Sheepshead minnows and were allowed to hatch in 15 ‰ salinity artificial seawater. Egg collections were monitored daily and larval fish were collected within 6 hours of hatching and transferred to their respective exposure treatment. Thirty-five viable larvae were placed in retention chambers (15cm diameter petri dishes with a 400 micrometer nylon mesh collar) and exposed to the experimental or the control treatment over a period of 7 days. Experimental treatments of the larval fish included 9.5mg/L Cr(VI), 0.3mg/L Cd(II), 40μg/L pyrene, chronic 1.5mg/L DO, or cyclic 1.5mg/L DO using an intermittent flow-through system. Control treatments of the larval fish included saltwater control (chromium and cadmium), DMSO (pyrene), and 6-8mg/L normoxia (chronic and cyclic DO). Exposure temperatures were maintained at 26-28°C under a 16 h light:8 h dark photoperiod. Larvae were sampled at 1, 3, 5, and 7 days post hatch and stored in RNA Later (Ambion, Austin, TX) at -80°C until total RNA was extracted.
Exposures for Embryonic SSH libraries
Adult female fish were injected with 50μL of chorionic gonadotropin followed by a second injection on the next day after the initial injection. The fish were maintained in normoxic conditions until the third day following the initial injection at which time the eggs were stripped from the females. The eggs were collected in a 50mL beaker containing just enough seawater to cover the eggs and fertilized using the dissected testes from male Sheepshead minnows. After one hour, eggs were examined for fertilization and 25 viable, fertilized embryos were placed in retention chambers (15cm diameter Petri dishes with a 400 micrometer nylon mesh collar) and exposed to the experimental or the control treatment over a period of 4 days. Experimental treatments of the embryonic fish included 40μg/L pyrene, chronic 1.5mg/L DO, or cyclic 1.5mg/L DO using an intermittent flow-through system. Control treatments of the embryonic fish included DMSO (pyrene) and 6-8mg/L normoxia (chronic and cyclic DO). Exposure temperatures were maintained at 26-28°C under a 16 h light:8 h dark photoperiod. Embryos were sampled at 24, 48, 72, and 96 hours post fertilization and homogenized immediately in 750μl RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) and stored at -80°C until total RNA was extracted.
Exposures for Adult SSH libraries
Adult fish (6-8 months old) were subjected to either hypoxia (1.5mg O2 /L) or normoxia (~8mg O2 /L) in 38L glass tanks using a semi- flow-through exposure system and a 16 h light:8 h dark photoperiod. Throughout the experiments, water temperature and salinity were maintained at 27± 1 °C and 15 ‰, respectively. To create the desired dissolved oxygen (DO) levels, nitrogen gas was used to deplete oxygen. Gas input was regulated by solenoids controlled by AquaController II units (Neptune Systems, San Jose, CA) receiving data from temperature and DO probes. Measurements of tank pH, temperature, salinity, and dissolved oxygen were recorded. Fish were separated into 8 tanks per exposure group (1.5mg DO/L hypoxia and ~8mg DO/L normoxia) at a density of 10 fish /tank. At 10 and 96 h, fish from 4 tanks per exposure group were removed, sacrificed, and dissected. Excised hepatic tissue was placed in RNA Later (Ambion, Austin, TX) and stored at -80°C until total RNA was extracted.
Molecular biology procedures
RNA extraction
For isolation of total RNA, larval, embryonic, or tissue samples were homogenized in 750μl of RNA STAT-60 (Tel-Test, Inc., Friendswood, TX). Total RNA was extracted by two rounds of reagent treatment according to the manufacturer’s protocol. Following the second extraction, RNA was precipitated overnight in 100% isopropanol, pelleted at 12,000 x g and 4°C for 1 h, and washed twice in 70% ethanol. The RNA pellets were resuspended in RNA Storage Solution (Ambion, Austin, TX) and subjected to TURBO DNasefree (Ambion, Austin, TX) treatment to remove any contaminating DNA. The purity and quantity of the resulting total RNA was determined using the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). All total RNA samples used in this study possessed a 260/280nm ratio of greater than 1.8. The quality of the total RNA samples (ie. 28S/18S ratio, DNA contamination, and RNA degradation) was determined using the RNA 6000 Nano Chip Kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA samples were pooled and ethanol precipitated in preparation for poly A + mRNA extraction and stored at - 80°C. Poly A + mRNA was extracted from pooled total RNA samples using the Oligotex mRNA Kits (Qiagen, Valencia, CA). Total RNA was subjected to two rounds of mRNA purification according to the manufacturer’s protocol. The quality of the mRNA purification (ie. removal of rRNAs) was determined using the RNA 6000 Nano Chip Kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The purity and quantity of the resulting mRNA was determined using the ND- 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). mRNA samples were pooled and ethanol precipitated in preparation for cDNA normalization, suppression subtractive hybridization, or cDNA library preparation and stored at -80°C.
Trimmer normalization libraries
Larval mRNA (0.5μg; 125ng/time point) and embryonic total RNA (1.0μg; 200ng/time point) were used to generate double stranded cDNA using the BD SMART™ PCR cDNA Synthesis Kit (Clontech, Mountain View, CA) following the manufacturer’s protocol for “SMART cDNA Synthesis for Library Construction”. Double stranded cDNA products were purified using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA) and 1300ng purified cDNA product was aliquotted and ethanol precipitated. SMART prepared double stranded cDNA was normalized using the Trimmer cDNA Normalization Kit (Evrogen, Moscow, Russia) by treatment with duplex-specific nuclease (DSN). Non-normalized cDNA samples were also prepared in the absence of DSN. Normalized and non-normalized cDNA products were amplified using the Advantage 2 PCR Kit (Clontech, Mountain View, CA) according to the Evrogen protocol. PCR products of the normalized and nonnormalized cDNA were stored at -20°C.
Suppression subtractive hybridization libraries
The PCR-Select cDNA Subtraction Kit (Clontech, Mountain View, CA) was used to prepare suppression subtractive hybridization (SSH) libraries of up- and down-regulated genes in larval, embryonic, and/or adult Cyprinodon variegatus following exposure to chromium, cadmium, pyrene, chronic hypoxia, or cyclic hypoxia. Two micrograms of poly A + was used to generate double stranded cDNA for all exposed and control treatments. Double stranded cDNA was restriction digested with Rsa I and used to generate both tester and driver cDNA for all samples. Forward and reverse subtractive hybridizations between exposed and control samples for each corresponding treatment were performed according to the manufacturer’s protocol and the final subtracted PCR products were stored at -20°C.
Cloning and plasmid DNA miniprep
PCR products generated from the Trimmer cDNA Normalization Kit (Evrogen, Moscow, Russia) and the PCR-Select cDNA Subtraction Kit (Clontech, Mountain View, CA) were cloned using the pGEM-T Easy Vector System (Promega, Madison, WI) and Electromax DH10B T1 Phage Resistant Cells (Invitrogen, Carlsbad, CA). Ampicillin resistant colonies were isolated by blue- white screening, cultured in 2X LB, and preserved by freezing at -80°C with glycerol. Plasmid DNA was extracted from selected colonies using the Montage Plasmid Miniprep96 Kit (Millipore) and following the manufacturer’s protocol. Plasmid DNA concentrations were determined using the ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and samples were stored at -20°C.
Sequencing
DNA sequences of the cloned PCR products were determined using the DTCS Quick Start Kit (Beckman Coulter, Fullerton, CA). One-half 20μl reactions were performed using 75ng of plasmid DNA template and a M13 (-40) forward primer according to the manufacturer’s protocol for DNA sequencing of plasmid DNA. Sequencing reactions were purified by ethanol precipitation and analyzed on a CEQ 8000 Genetic Analysis System using a 33-75B separation capillary array under the LFR-1 default settings.
Bioinformatic analysis
EST processing
Sequencher (Gene Codes, Ann Arbor, Michigan, USA), Aligner (CodonCode, Dedham, MA, USA), and CAP (source) were utilized for sequence cleansing and assembly. Low quality sequences and vector sequences were be quickly removed from each EST clone by using these applications. The cleaned EST sequences were used to find overlap assembly of contiguous sequences. Phred [8] was used to perform base-calling. Phred read DNA trace data, called bases, assigned quality values to the bases, and wrote the base calls and quality values to output sequence files. Quality values for the bases were later used by the sequence assembly program, Phrap [8], to increase the accuracy of assembled sequences. We employed CodonCode Aligner (http://www.codoncode.com/aligner/) to remove low quality bases at both ends by setting quality score QV ≥ 20 (or Pe ≤ 0.01). Vector and adaptor sequences were also trimmed because they can lead to incorrect assembly. We also used an in house implementation of VecScreen [11] to detect any partial vector contamination in our ESTs. Cap3 [12] was used to identify overlaps between sequences and assemble sequence fragments into a larger sequence [12]. Samples that can be joined together were assembled into contiguous sequences “contigs”. Four criteria were used to determine whether to accept or reject an alignment and overlap: (1) minimum percent identity ≥ 70%; (2) minimum overlap length ≥ 25 bps, (3) minimum alignment score; similar to previous but takes any mismatches into account, ≥ 20 bps; and (4) maximum gap size ≤ 15 bps. After assembly with Cap3, contigs with more than three ESTs were examined in Consed [13] to eliminate additional missassemblies not resolved by Cap3.
EST comparative analysis and functional assignment
We performed comparative analysis using NCBI blastx [14] with the unique sequences (including assembled contigs and singletons). A local implementation of BLAST server was used to search against the NCBI’s non-redundant peptide sequence database. We set up a cut off value and discarded hits with an E-value < 10-5. To assign putative functions to the unique sequences, we employed blast2go [15], [16], GOTM [17], GOfetcher [6] and GOstat [18] to extract the GO hierarchical terms of their homologous genes from the protein databases. Meanwhile, we also mapped the unique sequences to metabolic pathways in accordance with the KEGG [19]. Enzyme commission (EC) numbers [20] were obtained and used to putatively map unique sequences to specific biochemical pathways.
