2. 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
5. 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)
6. 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)
7.
8. 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
9. 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
10. 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
11. 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
12. 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
14. 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
16. Western Blot Protein Analysis
Western Blot using antibody for HSP70
Quantified total protein from each sample and HSP70 proportion
17. Increased concentrations of HSP70
in senescent individuals
8000
7000
6000
Band Intensity
5000
4000
3000
2000
1000
0
Pre-Senescent Hansen Senescent Ponds Senescent
Sampling Group
18. 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
19. Prospects for Future Research
• Follow up on CRH findings, directly measure
cortisol levels
• Look at other genes that may be involved in
rapid senescence
Notas do Editor
Ran proteins on SDS-PAGE to separate proteins by molecular weightProteins transferred to a nylon membrane