Daily oscillation and enrichment patterns in the transcriptome of the behavior-manipulating parasite Ophiocordyceps kimflemingiae

The natural histories of various parasite-host interactions that involve adaptive manipulation of host behavior describe a time-of-day synchronization of certain events. One example is the manipulated biting behavior observed in Carpenter ants infected with Ophiocordyceps unilateralis sensu lato. Field observations in Thailand demonstrated that manipulated biting takes place around solar noon. Laboratory work with species from South Carolina, demonstrated a shift of this synchronized biting event to the early morning. We therefore hypothesize that biological clocks play an important role in these and other parasite-host interactions. To set the stage for our future research into this, we, here, aimed to explore if fungal species of the complex O. unilateralis s.l. have a functional molecular clock. We, therefore, performed RNA-Seq on time course samples of the recently sequenced species Ophiocordyceps kimflemingiae. Fungal blastospores grown in liquid media were harvested under cycling light-dark and continuous darkness conditions. Rhythmic mRNA levels were assessed with JTK_CYCLE. We found that O. kimflemingiae is not hugely rhythmic under our sampling conditions, but rhythmicity can be observed. Moreover, we identified functional homologs of known clock genes. In addition, our data indicated that regulation of transcription appears to take place during the light phase (day time). In contrast, the expression levels of a significant number of secreted enzymes, proteases, toxins and small bioactive compounds peaked during the dark phase (night time). This suggests that this fungal parasite might indeed rely on biological clocks for the precise daily timing of certain events and additionally validates further research into this. Overall design: Samples were taken of O. kimflemingiae cultures at 4h intervals both under light cycling (LD) and constant darkness (DD) conditions. These samples were used for RNA-Seq and gene expression was analyzed for rhythmicity with a circa 24h period. For each sampled time point one replicate was generated. Part of the data was verified with RT-QPCR.

各类涉及宿主行为适应性操纵的寄生虫-宿主互作自然史研究均表明，部分事件存在日节律同步现象。其中一个典型案例是被Ophiocordyceps unilateralis sensu lato（蛇形虫草广义类群）感染的弓背蚁所表现出的被操纵叮咬行为。泰国的野外观察显示，被诱导的叮咬行为发生于太阳正午前后；而针对南卡罗来纳州物种的实验室研究则发现，这类同步化叮咬事件的发生时间提前至清晨时段。据此，我们提出假说：生物钟在这类及其他寄生虫-宿主互作过程中发挥关键调控作用。为开展后续相关研究奠定基础，本文旨在探究O. unilateralis s.l.类群的真菌物种是否具备功能性分子生物钟。
为此，我们对新近完成全基因组测序的金弗莱明氏蛇形虫草（Ophiocordyceps kimflemingiae）的时间序列样本开展了RNA测序（RNA-Seq）实验。在循环光照-黑暗（LD）与持续黑暗（DD）培养条件下，收集液体培养基中生长的真菌芽生孢子。使用JTK_CYCLE工具对mRNA的节律性表达水平进行评估。结果显示，在本实验的采样条件下，O. kimflemingiae的基因表达节律性并不显著，但仍可观测到微弱的节律特征。此外，我们成功鉴定出已知生物钟基因的功能性同源物。进一步分析表明，转录调控主要发生于光照阶段（日间）；与之相反，大量分泌酶类、蛋白酶、毒素及小型生物活性化合物的表达峰值均出现在黑暗阶段（夜间）。该结果提示，这类真菌寄生虫或许确实依赖生物钟实现特定生命事件的精准每日时序调控，同时也为后续相关研究提供了有力支撑。
整体实验设计：在循环光照（LD）与持续黑暗（DD）条件下，每4小时采集一次O. kimflemingiae的培养物样本。所有样本均用于RNA测序，并以约24小时为周期分析基因表达的节律性。每个采样时间点设置一个生物学重复。部分实验数据通过RT-QPCR（实时定量PCR）进行了验证。