Date of Award

Spring 2011

Rights

© 2011 Stephanie Podolski

Document Type

Thesis

Department

Biological Science

First Advisor

Markus Frederich

Second Advisor

Geoffrey Ganter

Third Advisor

Stine Brown

Abstract

Variations in water temperature, salinity, pH, and oxygen concentration are stressors that marine invertebrates face on a daily basis. Each of these physiological stressors creates a large cellular demand for energy. In mammals, energy metabolism is regulated by the enzyme AMP-activated protein kinase (AMPK), which is highly conserved during evolution. This project was designed to test the hypothesis that AMPK is present and activated by temperature, hypoxia, and anoxia stress in the lobster, Homarus americanus.

Animals were exposed to a rapid and progressive increase in temperature (6ºC per hour) beginning at 14 ºC. We measured lactate concentrations and AMPK activity and heat shock protein 70 (HSP70) levels in 2ºC increments (14-32ºC) in heart, muscle, and liver tissue. Lactate concentration remained at low control levels between 14 and 28ºC and increased significantly (ANOVA, p

Secondly, lobsters were exposed for a 24 hour time period to the sub-lethal temperature of 28°C. The prolonged exposure to heat led to a significant increase in AMPK liver activity up to 2.1±0.1 fold between 0 and 24 hours. AMPK did not significantly increase in the heart or muscle tissues. HSP70 levels remained constant in heart, liver, and muscle tissues.

Lastly, to characterize the role of AMPK during hypoxia lobsters were exposed for 24 hours to a low oxygen concentration of 4 kPa. The same measurements as described above were performed at 0, 4 and 24 hours. We found up to a 6-fold increase in AMPK activity and a nearly 40 fold increase in AMPK mRNA expression of heart tissue after 24 h of hypoxia. In the liver nearly a 1000 fold increase was found in AMPK mRNA expression. HSP70 mRNA and protein expression remained unchanged.

The data show that in lobsters AMPK activation is an early indicator of stress when cellular energy levels are depleted, as indicated by the concurrence of AMPK activation and lactate accumulation. The traditionally used marker, HSP70, was a less reliable indicator for stress. Future comparison with other crustacean species will indicate whether AMPK activation during stress is a more potent mechanism than HSP70 for assessing stress levels in other populations of invertebrates.

Comments

Honors thesis.

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