Jensen FISH495
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Transcript of Jensen FISH495
Rapid Senescence in Sockeye Salmon (Oncorhynchus nerka)
By Leslie Jensen
Rapid Senescence
• Pacific salmon are semelparous and die of senescence soon after beginning reproductive behavior
• Individuals go from peak health to death in a matter of days to weeks– Rates vary within populations and among populations
• Loss of aggression, loss of strength and swimming ability, and physical decline
Physical Signs of Senescence
Frayed fins
Loss of coloration
WHAT CAUSES RAPID SENESCENCE IN SOCKEYE SALMON?
Research Question:
Study Site in Southwest Alaska: Hansen Creek
Small tributary of Lake Aleknagik
Nearly uniform top to bottom: 4m wide & 10cm deep riffles
Annual spawning population of 3,000 – 20,000 sockeye salmon
Stream life annually averages below 10 days (usually 7-14)
Study Site in Southwest Alaska: Iliamna Lake Ponds
Tributary of Lake Iliamna, the largest lake in Alaska (2,600 km2)
Small, variable population size (~500)
Larger individuals and longer reproductive lifespan (19.4 days in 2008)
Tissue Sampling
Tissues sampled:• Brain tissue• Liver tissue
Preserved in RNAlater solution and frozen
Salmon Entering Hansen Creek
Up to 40% of individuals may strand at stream mouth in a given year
Sampled for a “pre-senescent” baseline for gene expression
Laboratory Procedures
• RNA extraction and quantification of genes of interest (brain tissues)– Adrenocorticotropic hormone (ACTH)– Corticotropin-releasing hormone (CRH)– Heat shock protein (HSC71)
• Protein extraction and quantification (liver tissues)– Heat shock protein: HSP70
Why did I look at ACTH & CRH?
Both are genes involved in the cortisol production and regulation pathway
Cortisol = stress hormone•Suppresses immune system•Inhibits glucose uptake•Stimulates protein degredation
Anterior Kidney
Why heat shock proteins?
• Heat shock proteins = stress proteins– Chaperones: move other proteins around cells– Help with protein-folding & identify incorrectly-
folded proteins– Prepare old proteins to be degraded by cell
• Predicted to be associated with senescence
Gene Expression Analysis
Designed primers for ACTH, CRH, and HSC71
Quantified with Real-time PCR
Corrected for total RNA used by comparing 18s expression levels
No significant differences or major trends
• Large Variation & Low Sample Sizes
Pre-Senesncent Hansen Senescent
Ponds Senescent
0
1
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ACTH
Rela
tive
Expr
essio
n
Pre-Senescent Hansen Senescent
Ponds Senescent
05
10152025303540
HSC71
Rela
tive
Expr
essio
n
Pre-Senescent Hansen Senescent
Ponds Senescent
0
5
10
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20
25
CRH
Rela
tive
Expr
essio
n
Explanations for high variation?
• Factors I explored for each gene of interest:– Body size– Sex of individuals– Entry date or date of senescence– Stream life
3 4 5 6 7 8 90.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
R² = 0.903199410309668
In-stream Life (Days)
Rela
tive
Expr
essi
on P = 0.0036
CRH levels lower in slower-senescing individuals
Western Blot Protein Analysis
Western Blot using antibody for HSP70
Quantified total protein from each sample and HSP70 proportion
Pre-Senescent Hansen Senescent Ponds Senescent0
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Sampling Group
Band
Inte
nsity
Increased concentrations of HSP70 in senescent individuals
Summary of Findings
• High CRH levels indicate or result in more rapid senescence
• HSP70 at higher levels in senescent individuals– Slightly higher in the more rapidly senescing
Hansen Creek population than in Iliamna Ponds
Prospects for Future Research
• Follow up on CRH findings, directly measure cortisol levels
• Look at other genes that may be involved in rapid senescence