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

2

3

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

15

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

1000

2000

3000

4000

5000

6000

7000

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