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Transcriptional Response to Acute Thermal Exposure in Juvenile Chinook Salmon Determined by RNAseq.

Tomalty KM, Meek MH, Stephens MR, Rincón G, Fangue NA, May BP, Baerwald MR - G3 (Bethesda) (2015)

Bottom Line: Differentially expressed transcripts were annotated using Blast2GO and relevant gene clusters were identified.In addition to a high degree of downregulation of a wide range of genes, we found upregulation of genes involved in protein folding/rescue, protein degradation, cell death, oxidative stress, metabolism, inflammation/immunity, transcription/translation, ion transport, cell cycle/growth, cell signaling, cellular trafficking, and structure/cytoskeleton.These results demonstrate the complex multi-modal cellular response to thermal stress in juvenile salmon.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, University of California, Davis, California 95616 kmtomalty@ucdavis.edu.

No MeSH data available.


Related in: MedlinePlus

Experimental design.
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fig1: Experimental design.

Mentions: Chinook salmon eggs obtained from Merced River Hatchery in November 2010 were reared at the University of California, Davis, in partially re-circulated aerated well water chilled to 12° (±1°) and fed commercial salmon feed (soft-moist formulation, 2–10% body weight; Rangen Inc.) until the time of the experiment, approximately 5 months posthatch. Fish were raised in a single 160-liter circular flow-through tank for 1 month preceding the experiment (see Figure 1 for experimental design), supplied with aerated well water and with a 12-hr light–12-hr dark photoperiod. The night prior to the experiment 55 fish were randomly assigned to one of five treatment groups (11 fish per treatment) and were allowed to acclimate at 12° in the experimental chambers overnight. Experimental chambers consisted of 5-gallon buckets with large mesh windows in the sides to allow water to freely enter and exit the chamber when submerged. Each chamber included an air stone to ensure well-aerated treatment water as well as to prevent thermal stratification. Water in the larger tanks was also well-aerated to maintain oxygen saturation. Treatments consisted of a 3-hr exposure to 15°, 18°, 21°, or 25° (±0.5°), followed by a 1-hr of recovery period at 12° (±0.5°). These temperatures range from optimal to the upper thermal limit for Chinook salmon determined by critical thermal methodology (Myrick and Cech 1998). Three hours was chosen as an ecologically relevant time exposure, because it approximates potential exposure times to warm water during juvenile outmigration and passage through warmer river reaches (Michel et al. 2013). The experimental chambers were moved to tanks held at a constant temperature using submersible titanium heating elements (Finnex 300W), facilitating very rapid change in the temperature experienced by the fish. Controls were handled identically to the other four treatment groups but remained at 12° (±0.5°). Following recovery, fish were killed with buffered tricaine methanesulfonate (500 mg/L tricaine and 420 mg/L NaCO3), weighed and measured, and gill tissue was immediately collected [there was no significant difference in weight (P = 0.53) or length (P = 0.79) of fish between groups]. Samples were preserved in RNAlater solution (Life Technologies) and stored at −80° prior to RNA extraction. Replicates of this temperature experiment were performed on 3 consecutive days at 9:00 am, yielding three replicates of 11 individuals at each temperature. Treatment of all animals was in accordance with University of California, Davis, animal care and use protocol #17875. RNA was isolated from gill tissue of 165 individuals using the TissueLyser II bead mill (Qiagen) for tissue homogenization and TRIzol Reagent Solution (Applied Biosystems) according to the manufacturer’s protocol. RNA was quantified and quality checked using the 2100 Bioanalyzer (Agilent) and RNA 6000 Nano Kit (Agilent). All RNA had a RIN (RNA Integrity Number) of 9.5 or higher. RNA from the 11 individuals in each experimental replicate was proportionally pooled and used to generate sequencing libraries using the Illumina TruSeq RNA Sample Preparation Kit and associated protocol (TruSeq RNA Sample Preparation Guide, part #15008136, November 2010 release). The 15 libraries were barcoded and processed, three libraries to a lane, with 100-bp paired-end sequencing on the Illumina HiSeq2000 platform at the University of California, Berkeley Vincent J. Coates Genomic Sequencing Laboratory. Raw data can be found at BioProject, record GSE59756.


Transcriptional Response to Acute Thermal Exposure in Juvenile Chinook Salmon Determined by RNAseq.

Tomalty KM, Meek MH, Stephens MR, Rincón G, Fangue NA, May BP, Baerwald MR - G3 (Bethesda) (2015)

Experimental design.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4502368&req=5

fig1: Experimental design.
Mentions: Chinook salmon eggs obtained from Merced River Hatchery in November 2010 were reared at the University of California, Davis, in partially re-circulated aerated well water chilled to 12° (±1°) and fed commercial salmon feed (soft-moist formulation, 2–10% body weight; Rangen Inc.) until the time of the experiment, approximately 5 months posthatch. Fish were raised in a single 160-liter circular flow-through tank for 1 month preceding the experiment (see Figure 1 for experimental design), supplied with aerated well water and with a 12-hr light–12-hr dark photoperiod. The night prior to the experiment 55 fish were randomly assigned to one of five treatment groups (11 fish per treatment) and were allowed to acclimate at 12° in the experimental chambers overnight. Experimental chambers consisted of 5-gallon buckets with large mesh windows in the sides to allow water to freely enter and exit the chamber when submerged. Each chamber included an air stone to ensure well-aerated treatment water as well as to prevent thermal stratification. Water in the larger tanks was also well-aerated to maintain oxygen saturation. Treatments consisted of a 3-hr exposure to 15°, 18°, 21°, or 25° (±0.5°), followed by a 1-hr of recovery period at 12° (±0.5°). These temperatures range from optimal to the upper thermal limit for Chinook salmon determined by critical thermal methodology (Myrick and Cech 1998). Three hours was chosen as an ecologically relevant time exposure, because it approximates potential exposure times to warm water during juvenile outmigration and passage through warmer river reaches (Michel et al. 2013). The experimental chambers were moved to tanks held at a constant temperature using submersible titanium heating elements (Finnex 300W), facilitating very rapid change in the temperature experienced by the fish. Controls were handled identically to the other four treatment groups but remained at 12° (±0.5°). Following recovery, fish were killed with buffered tricaine methanesulfonate (500 mg/L tricaine and 420 mg/L NaCO3), weighed and measured, and gill tissue was immediately collected [there was no significant difference in weight (P = 0.53) or length (P = 0.79) of fish between groups]. Samples were preserved in RNAlater solution (Life Technologies) and stored at −80° prior to RNA extraction. Replicates of this temperature experiment were performed on 3 consecutive days at 9:00 am, yielding three replicates of 11 individuals at each temperature. Treatment of all animals was in accordance with University of California, Davis, animal care and use protocol #17875. RNA was isolated from gill tissue of 165 individuals using the TissueLyser II bead mill (Qiagen) for tissue homogenization and TRIzol Reagent Solution (Applied Biosystems) according to the manufacturer’s protocol. RNA was quantified and quality checked using the 2100 Bioanalyzer (Agilent) and RNA 6000 Nano Kit (Agilent). All RNA had a RIN (RNA Integrity Number) of 9.5 or higher. RNA from the 11 individuals in each experimental replicate was proportionally pooled and used to generate sequencing libraries using the Illumina TruSeq RNA Sample Preparation Kit and associated protocol (TruSeq RNA Sample Preparation Guide, part #15008136, November 2010 release). The 15 libraries were barcoded and processed, three libraries to a lane, with 100-bp paired-end sequencing on the Illumina HiSeq2000 platform at the University of California, Berkeley Vincent J. Coates Genomic Sequencing Laboratory. Raw data can be found at BioProject, record GSE59756.

Bottom Line: Differentially expressed transcripts were annotated using Blast2GO and relevant gene clusters were identified.In addition to a high degree of downregulation of a wide range of genes, we found upregulation of genes involved in protein folding/rescue, protein degradation, cell death, oxidative stress, metabolism, inflammation/immunity, transcription/translation, ion transport, cell cycle/growth, cell signaling, cellular trafficking, and structure/cytoskeleton.These results demonstrate the complex multi-modal cellular response to thermal stress in juvenile salmon.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, University of California, Davis, California 95616 kmtomalty@ucdavis.edu.

No MeSH data available.


Related in: MedlinePlus