Jensen FISH495

Rapid Senescence in Sockeye Salmon (Oncorhynchus nerka)

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

Research Question: WHAT CAUSES RAPID SENESCENCE IN SOCKEYE SALMON?

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)

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 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

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

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)

Western Blot Protein Analysis Western Blot using antibody for HSP70 Quantified total protein from each sample and HSP70 proportion

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

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

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Jensen FISH495

by sr320 on Jun 11, 2009

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