Temporal neuronal control of diapause in the high-latitude fly, Drosophila littoralis.

为即将到来的冬天做好及时的准备对生存至关重要，特别是对昆虫等小动物来说。必须在恶劣环境条件出现之前及时建立粮食储备，必须停止繁殖，必须提高对寒冷和干旱胁迫的抵抗力。来自高纬度地区的昆虫在冬季处于滞育状态，在此期间，食物大量停止摄取，新陈代谢急剧降低，寿命延长。进入滞育的信号是夏秋季白天长度(光周期)的减少，并假设生物钟提供测量白天长度的内部参考值。然而，只有少数几种昆虫清楚地证明了这一点。此外，大脑中的生物钟网络如何与控制滞育的荷尔蒙中心通信在很大程度上是未知的。本项目的目的是(1)阐明生物钟在测量光周期反应的北方果蝇中的功能，以及(2)比较地破译生物钟与小果蝇和黑腹果蝇大脑间部/侧脑部激素中心之间的通讯通路。我们发现这两种苍蝇的分子时钟和时钟网络都很保守，但在神经肽的使用上有显著的差异。这些差异可能解释了北方小苍蝇强烈的光周期反应。来自芬兰(Kilpisjärvi，69°N)的苍蝇进入滞育的关键日长为~18小时，而来自更南部地区(高加索，42°N)的苍蝇的行为与黑腹蝇相似，有时在12小时的日长下仍保持繁殖。有趣的是，在生物钟神经元的神经肽表达和节律行为方面，南方小卷蝇与黑腹小卷蝇都表现出相似之处。这使得在神经肽表达、节律行为和来自不同纬度的小卷蝇滞育开始的临界日长之间进行了相关分析。此外，我们计划模拟北部和南部的斜纹夜蛾在黑腹果蝇中的神经肽表达模式，并研究转基因果蝇的滞育和节律行为。时钟神经元和大脑间/外侧脑部激素中心之间的突触和神经肽能联系将通过TransTango和Tango-Map MKII在黑腹大鼠中得到阐明。最终，我们将在Littoris中使用CRISPR/Cas9在选定的时钟和神经肽基因中产生突变，以阐明生物钟在日长测量中的作用。我们预计，我们的研究将对我们理解昼长测量和通过生物钟季节性控制冬季休眠做出重大贡献。

Timely preparation for the coming winter is essential for survival, especially for small animals such as insects. Food reserves must be built up in good time before the onset of hostile environmental conditions, reproduction must be stopped and resistance to cold and drought stress must be increased. Insects from high-latitudes spend the winter itself in a state of diapause, during which food intake largely ceases, metabolism is drastically lowered, and lifespan is increased. The signal to go into diapause is the decreasing day length (photoperiod) in summer-autumn, and it is assumed that the circadian clock provides the internal reference value for measuring day length. However, this has been clearly demonstrated only for few insect species. Furthermore, it is largely unknown how the circadian clock network in the brain communicates with the hormonal centers controlling diapause. The goal of this project is to (1) elucidate the function of the circadian clock in measuring daylength in the northern fly species Drosophila littoralis, which exhibits a strong and well-studied photoperiodic response, and (2) comparatively decipher the communication pathways between the circadian clock and hormonal centers in the pars intercerebralis/ lateralis in D. littoralis and D. melanogaster. We showed that the molecular clock and clock network are well conserved in both fly species, but that there are striking differences in the use of neuropeptides. These differences may explain the strong photoperiodic response of D. littoralis flies from the north. Flies from Finland (Kilpisjärvi, 69°N) enter diapause at a critical daylength of ~18h, while flies from more southern regions (Caucasus, 42°N) behave similarly to D. melanogaster and sometimes remain reproductive at a daylength of 12h. Interestingly, southern D. littoralis flies show similarities with D. melanogaster in both neuropeptide expression in circadian clock neurons and rhythmic behavior. This allows a correlative analysis between neuropeptide expression, rhythmic behavior, and critical daylength for diapause initiation in D. littoralis flies from different latitudes. Furthermore, we plan to simulate the neuropeptide expression pattern of northern and southern D. littoralis flies in D. melanogaster and investigate diapause and rhythmic behavior in the transgenic flies. The synaptic and neuropeptidergic connections between clock neurons and hormonal centers in the pars intercerebralis/lateralis will be elucidated in D. melanogaster by trans-Tango and TANGO-Map MKII. Ultimately, we will use CRISPR/Cas9 in D. littoralis to generate mutants in selected clock and neuropeptide genes to elucidate the role of the circadian clock in daylength measurement. We expect that our studies will contribute significantly to our understanding of daylength measurement and the seasonal control of winter dormancy by circadian clocks.