Presentation on salmon senescence as part of FISH495 course

1. Rapid Senescence in Sockeye Salmon
(Oncorhynchus nerka)

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

3. Physical Signs of Senescence
Frayed fins
Loss of coloration

4. Research Question:
WHAT CAUSES RAPID SENESCENCE
IN SOCKEYE SALMON?

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)

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

13. No significant differences
or major trends
25
Relative Expression
20
CRH
3
ACTH
Relative Expression
15
10 2
5
0 1
Pre-Senescent Hansen Ponds
Senescent Senescent 0
40 Pre-Senesncent Hansen Ponds
Relative Expression
35
HSC71 Senescent Senescent
30
25
20
15
10
• Large Variation &
5
0 Low Sample Sizes
Pre-Senescent Hansen Ponds
Senescent Senescent

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

15. CRH levels lower in
slower-senescing individuals
35.0
30.0 R² = 0.903
Relative Expression
P = 0.0036
25.0
20.0
15.0
10.0
5.0
0.0
3 4 5 6 7 8 9
In-stream Life (Days)

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