DataSheet2_Pigment Dispersing Factor Is a Circadian Clock Output and Regulates Photoperiodic Response in the Linden Bug, Pyrrhocoris apterus.pdf

Daily and annually cycling conditions manifested on the Earth have forced organisms to develop time-measuring devices. Circadian clocks are responsible for adjusting physiology to the daily cycles in the environment, while the anticipation of seasonal changes is governed by the photoperiodic clock. Circadian clocks are cell-autonomous and depend on the transcriptional/translational feedback loops of the conserved clock genes. The synchronization among clock centers in the brain is achieved by the modulatory function of the clock-dependent neuropeptides. In insects, the most prominent clock neuropeptide is Pigment Dispersing Factor (PDF). Photoperiodic clock measures and computes the day and/or night length and adjusts physiology accordingly to the upcoming season. The exact mechanism of the photoperiodic clock and its direct signaling molecules are unknown but, in many insects, circadian clock genes are involved in the seasonal responses. While in Drosophila, PDF signaling participates both in the circadian clock output and in diapause regulation, the weak photoperiodic response curve of D. melanogaster is a major limitation in revealing the full role of PDF in the photoperiodic clock. Here we provide the first description of PDF in the linden bug, Pyrrhocoris apterus, an organism with a robust photoperiodic response. We characterize in detail the circadian and photoperiodic phenotype of several CRISPR/Cas9-generated pdf mutants, including three null mutants and two mutants with modified PDF. Our results show that PDF acts downstream of CRY and plays a key role as a circadian clock output. Surprisingly, in contrast to the diurnal activity of wild-type bugs, pdf null mutants show predominantly nocturnal activity, which is caused by the clock-independent direct response to the light/dark switch. Moreover, we show that together with CRY, PDF is involved in the photoperiod-dependent diapause induction, however, its lack does not disrupt the photoperiodic response completely, suggesting the presence of additional clock-regulated factors. Taken together our data provide new insight into the role of PDF in the insect’s circadian and photoperiodic systems.

地球上的日复一日及年度循环的骑行条件迫使生物进化出时间测量装置。生物钟负责调节生理机能以适应环境中的日周期，而季节变化的预期则由光周期钟所支配。生物钟具有细胞自主性，依赖于保守时钟基因的转录/翻译反馈回路。大脑中时钟中心之间的同步是通过依赖时钟的神经肽的调节功能实现的。在昆虫中，最显著的时钟神经肽是色素分散因子（PDF）。光周期钟测量并计算昼夜长度，并根据即将到来的季节调整生理机能。光周期钟的确切机制及其直接的信号分子尚不清楚，但在许多昆虫中，生物钟基因参与了季节性反应。在果蝇中，PDF信号不仅参与生物钟的输出，还参与休眠调节，然而，黑腹果蝇微弱的光周期响应曲线是揭示PDF在光周期钟中作用的重大限制。在此，我们首次描述了在桑天牛，Pyrrhocoris apterus，这一具有强大光周期响应能力的生物中的PDF。我们详细描述了由CRISPR/Cas9技术生成的多个pdf突变体的生物钟和光周期表型，包括三个null突变体和两个PDF结构改变的突变体。我们的结果表明，PDF在CRY之后发挥作用，并在生物钟输出中扮演关键角色。令人惊讶的是，与野生型昆虫的日间活动相比，pdf null突变体表现出明显的夜间活动，这是对光/暗切换的非时钟依赖性直接反应所致。此外，我们显示PDF与CRY一起参与光周期依赖性休眠诱导，然而，其缺失并未完全破坏光周期响应，这表明存在其他时钟调节因子。综上所述，我们的数据为PDF在昆虫昼夜节律和光周期系统中的作用提供了新的见解。