Diapause, Stress Tolerance and Molecular Chaperones in Artemia franciscana

甲壳动物卤虫的胚胎发育成游泳幼虫或包裹并进入滞育，这是一种以发育停滞和新陈代谢大大降低为特征的生理状态。包囊胚胎(囊)具有很强的抗逆性，在常温下水合后能在极端温度、干燥和多年无氧条件下存活。因此，弗朗西斯卡纳孢囊是地球上最耐受压力的多细胞生命形式之一。弗朗西斯卡那胚胎在滞育发育过程中表现出不同的基因调控，产生启动、维持和终止滞育所需的蛋白质，而维持是最耐受逆境的时期。这项拟议的研究的总体目标是阐明A.franciscana调节滞育特定发育的细胞、分子和生化机制，并确定胚胎如何在严重的生理压力下存活。热休克因子1(HSF1)通常激活生理应激期间表达的基因，将进行测试，以确定它是否调节滞育期间的基因表达。特别令人感兴趣的是一种丰富的滞育特异性分子伴侣P26，它在没有能量供应的情况下发挥作用。P26被认为与滞育胚胎中的变性蛋白结合，并保护它们免受不可逆变性的影响。因此，将鉴定P26的底物蛋白。一些分子伴侣，如Hsp40、Hsp70和Hsp90，需要能量才能发挥作用。这些蛋白质的合成将在滞育胚胎发育过程中被研究，当能量有限时，它们的保护功能也将被研究，就像滞育发生的那样。调节滞育终止的蛋白质也将被识别。因此，这项拟议的研究提出的一个中心问题是，弗朗西斯卡那的胚胎如何在严重的应激条件下生存下来，这些应激条件相当迅速地杀死了大多数其他有机体。分子伴侣对压力耐受性负有责任吗？如果是的话，它们对这一过程有何贡献？回答这些问题将对滞育和必要的细胞蛋白功能(如分子伴侣)提供重要的基本见解。在应用层面上，拟议的研究可能有助于在水产养殖中使用弗朗西斯卡纳，这种生物被用来喂养具有重要商业价值的鱼类和水生无脊椎动物的幼体。例如，热休克蛋白70在法国假丝酵母对细菌感染的抵抗力中发挥作用，这表明拟议的工作将导致法国假丝酵母菌培养的改进。此外，更好地了解滞育对农业、林业和医学都有影响，因为这可能有助于制定控制能够越冬的虫害的方法，并试图在滞育期间根除虫害，这对加拿大有直接好处。

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.