Diapause, Stress Tolerance and Molecular Chaperones in Artemia franciscana

甲壳类动物（Artemia franciscana）的胚胎发育成游泳幼虫，或者包囊并进入滞育，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（囊肿）具有很强的耐压能力，在环境温度下水合时，可以在极端温度、干燥和无氧环境下存活数年。因此，A. franciscana 包囊代表了地球上最能耐受压力的多细胞生命形式之一。 A. franciscana 胚胎在滞育发育过程中表现出差异性基因调控，产生滞育开始、维持和终止所需的蛋白质，并维持最大的应激耐受期。拟议研究的全球目标是阐明弗朗西斯卡纳调节滞育特异性发育的细胞、分子和生化机制，并确定胚胎如何在严重的生理应激下生存。通常会激活生理应激期间表达的基因的热休克因子 1 (HSF1) 将被测试，以确定它是否在滞育期间调节基因表达。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被认为可以结合滞育胚胎中的变性蛋白并保护它们免于不可逆的变性。因此，p26 的底物蛋白将被鉴定。 Hsp40、Hsp70 和 Hsp90 等几种分子伴侣需要能量才能活动。这些蛋白质的合成将在滞育胚胎发育过程中进行研究，以及当能量有限时它们的保护功能（如滞育中发生的情况）。调节滞育终止的蛋白质也将被鉴定。因此，拟议研究解决的一个核心问题是，A. franciscana 的胚胎如何在严重的应激源中生存下来，而这些应激源会很快杀死大多数其他生物。分子伴侣是否负责应激耐受性？如果是，它们如何促进这一过程？回答这些问题将为滞育和分子伴侣等重要细胞蛋白的功能提供重要的基础见解。在应用层面上，拟议的研究可能会促进 A. franciscana 在水产养殖中的使用，其中该生物体被用来喂养具有商业重要性的鱼类和水生无脊椎动物的幼虫。例如，Hsp70 在 A. franciscana 对细菌感染的抵抗力中发挥着作用，表明所提出的工作将导致 A. franciscana 培养的改进。此外，更好地了解滞育会对农业、林业和医学产生影响，因为它可能有助于开发控制能够在冬季存活的害虫的方法，并尝试在滞育期间消灭它们，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.