Diapause, Stress Tolerance and Molecular Chaperones in Artemia franciscana

Embryos of the crustacean, Artemia franciscana, develop into swimming larvae or they encyst and enter diapause, a physiological state characterized by developmental arrest and greatly reduced metabolism. Encysted embryos (cysts) are very stress tolerant, surviving extreme temperature, desiccation and years without oxygen when hydrated at ambient temperature. A. franciscana cysts therefore represent one of the most stress tolerant multicellular life forms on Earth. A. franciscana embryos exhibit differential gene regulation during diapause-destined development, producing proteins required for the initiation, maintenance and termination of diapause, with maintenance the period of greatest stress tolerance. The global objectives of the proposed research are to elucidate cell, molecular and biochemical mechanisms by which A. franciscana regulate diapause-specific development and determine how embryos survive severe physiological stress. Heat shock factor 1 (HSF1) which normally activates genes expressed during physiological stress will be tested to determine if it regulates gene expression during diapause. Of particular interest is an abundant, diapause-specific molecular chaperone, p26, which functions in the absence of an energy supply. p26 is thought to bind denaturing proteins in diapause embryos and protect them from irreversible denaturation. Consequently, substrate proteins of p26 will be identified. Several molecular chaperones such as Hsp40, Hsp70 and Hsp90 require energy for activity. The synthesis of these proteins will be investigated during diapause-destined embryo development, as will their protective function when energy is limiting, as occurs in diapause. Proteins that regulate diapause termination will also be identified. Thus, a central question addressed by the proposed research is how embryos of A. franciscana survive severe stressors which kill most other organisms rather quickly. Are molecular chaperones responsible for stress tolerance and, if so, how do they contribute to this process? Answering these questions will provide significant fundamental insights into diapause and the functions of essential cellular proteins such as the molecular chaperones. On an applied level, the proposed research may facilitate the use of A. franciscana in aquaculture where the organism is used to feed the larvae of commercially important fish and aquatic invertebrates. Hsp70, for example, plays a role in the resistance of A. franciscana to bacterial infection, indicating that the proposed work will lead to improvements in the culture of A. franciscana. Moreover, a better understanding of diapause has consequences for agriculture, forestry and medicine as it may allow for the development of methods for the control of insect pests able to survive winter and attempts at their eradication when in diapause, this of direct benefit to Canada.