Mechanistic Evaluation of Daphnia magna’s Behavioral and Life History Responses to Photoperiod [Experiment 2]

Across evolutionary time, nearly all animal species have harnessed photoperiod to initiate processes that ultimately influence seasonal behavior and life history traits. In the freshwater cladoceran Daphnia magna, the effect of photoperiod on various life history traits has generally been investigated in conjunction with additional environmental stimuli. In the present study, we sought to untangling responses directly attributable to photoperiod in D. magna and identify the molecular processes underlying resultant behavioral and life history responses using functional analysis of global transcriptomic expression. D. magna were exposed to five different photoperiods immediately post-hatch for 21d where a standard long-day photoperiod of 16 hours light and 8 hours dark (16L:8D) served as the control relative to 4L:20D, 8L:16D, 12L:12L, and 20L:4D photoperiods. Entrainment to short-day photo-periods (4L:20D, 8L:16D, and 12L:12L) resulted in significantly increased light-avoidance behaviors relative to the control photoperiod where young Daphnia (7d old) displayed the most pronounced avoidance responses. Correspondingly, functional transcriptomics identified differential transcriptional expression of genes involved in glutamate signaling, which is critical in arthropod light-avoidance responses, as well as period circadian protein and proteins coding F-box/LRR-repeat domains, all of which contribute to establishing circadian rhythms in arthropods. Short-day photoperiods also induced increased metabolic rates which corresponded with broad-scale changes in transcriptional expression across multiple systems-level energy metabolism pathways. The most striking observation was increased male production across short-day photoperiods (4L:20D, 8L:16D, and 12L:12D). Transcriptional expression consistent with multiple putative mechanisms of male production were observed including expression suggestive of increased glutamate signaling; a response observed to induce male production in D. pulex via photo-period sensitive mechanisms. Overall, the results demonstrate the importance of photoperiod on behavior and life history trajectories in D. magna where we have now established multiple putative mechanistic pathways underlying several critical responses. The experimental approach was to investigate the effects of photoperiod on key life history traits, metabolic rates, photo-taxis responses and global transcriptomic expression in D. magna to characterize foundational responses to light. Separate groups of D. magna were simultaneously exposed to five different photoperiods representing varying seasonal day lengths; this included a comparative control of 16 hours light and 8 hours dark (16L:8D) that is the standard for acute (USEPA 1996a; USEPA 2002; OECD No. 202(2004)) and chronic (USEPA 1996b; ASTM E1193-97(2012) , OECD No. 211 (2012)) ecotoxicological methods and an array of alternative photoperiods including extreme short days (4L:20D), short days (8L:16D), equinox (12L:12L), and extreme long days (20L:4D). Since changes in life history strategy are likely to involve changes in energy utilization (Ananthasubramaniam et al 2015), overall metabolic rate was also investigated. Provided the inherently variable nature of life history and behavioral responses to stimuli, we repeated the photoperiod exposures in two temporally separated experiments on 17 October 2014 and 11 March 2015, hereafter referred to as Experiment 1 and Experiment 2, to assess the reproducibility of all measures and endpoints from one experiment to the next, and assessed if the two data sets could be combined for examination as one large dataset. In summary, 2 duplicate photoperiod experiments were conducted where each included 5 photoperiods x 3 exposure replicates. The present GEO dataset represents Experiment 2. In addition to life history endpoints, basic behavioral responses to photo-period were of interest; thus, instantaneous response to light as a stimulus where photo-taxis (“light oriented reactive swimming” Fraenkel et al 1961) was investigated in real-time light/dark preference assays and time series light acclimation assays. Lastly, an investigation of global-transcript expression was conducted to identify the genes and genetic pathways that were responsive to the five different, static photoperiod lengths and to establish potential relationships between affected genes/pathways and any observed changes in life history traits, metabolic rate and behavioral responses.

在演化时间尺度上，几乎所有动物物种都已利用光周期（photoperiod）启动一系列生理过程，最终调控其季节性行为与生活史性状。在淡水枝角类大型溞（Daphnia magna）中，光周期对各类生活史性状的影响通常会结合其他环境刺激开展研究。本研究旨在厘清大型溞中仅由光周期介导的特异性响应，并通过全局转录组表达的功能分析，解析驱动其行为与生活史响应的核心分子过程。

研究将刚孵化的大型溞暴露于5种不同光周期条件下21天，其中以标准长日照光周期（16小时光照/8小时黑暗，16L:8D）作为对照，其余处理组分别为4L:20D、8L:16D、12L:12L与20L:4D光周期。相较于对照光周期，适应短日照光周期（4L:20D、8L:16D与12L:12L）的个体表现出显著增强的避光行为，其中7日龄幼体的避光响应最为显著。

相应地，功能转录组学分析鉴定出谷氨酸信号通路（glutamate signaling）相关基因的差异转录表达——该通路在节肢动物（arthropod）避光响应中发挥关键调控作用——以及周期节律蛋白（period circadian protein）和编码F-box/LRR重复结构域（F-box/LRR-repeat domains）的蛋白，上述因子均参与节肢动物昼夜节律（circadian rhythms）的建立。短日照光周期还会诱导机体代谢速率升高，该现象与多系统能量代谢通路的大范围转录表达变化高度匹配。

最引人注目的观测结果是，短日照光周期（4L:20D、8L:16D与12L:12D）条件下雄性子代产量显著提升。研究观测到与多种潜在雄性产生机制相符的转录表达特征，包括提示谷氨酸信号通路增强的表达模式——该响应已被证实可通过光周期敏感机制在蚤状溞（Daphnia pulex）中诱导雄性产生。

综上，本研究结果证实了光周期对大型溞行为与生活史轨迹的重要调控作用，我们现已明确介导若干关键响应的多条潜在机制通路。本实验旨在探究光周期对大型溞关键生活史性状、代谢速率、趋光性（photo-taxis）及全局转录组表达的影响，以表征其对光照的基础响应模式。

研究同步将多组大型溞暴露于5种代表不同季节日照长度的光周期条件下：其中16L:8D的标准光周期作为对照，该光周期已被纳入急性（美国环境保护署[USEPA] 1996a; USEPA 2002; 经济合作与发展组织[OECD] 第202号标准[2004]）与慢性（USEPA 1996b; ASTM E1193-97(2012)、OECD第211号标准[2012]）生态毒理学测试方法；其余光周期包括极端短日照（4L:20D）、短日照（8L:16D）、昼夜平分点光照（12L:12L）与极端长日照（20L:4D）。

由于生活史策略的改变往往伴随能量利用方式的变化（Ananthasubramaniam等，2015），本研究同时对整体代谢速率进行了检测。鉴于生活史与行为对刺激的响应存在固有变异性，我们分别于2014年10月17日与2015年3月11日开展了两次时间分离的光周期暴露实验（下文分别称为实验1与实验2），以评估所有测量指标与终点在不同实验间的可重复性，并检验两组数据集是否可合并为一个大型数据集进行综合分析。

简言之，本研究共开展2组重复光周期实验，每组均包含5种光周期×3个暴露重复。本基因表达综合（Gene Expression Omnibus, GEO）数据集对应实验2。

除生活史终点外，本研究还关注生物对光周期的基础行为响应，因此通过实时光/暗偏好实验与时间序列光适应实验，探究了趋光性（即‘以光线为导向的定向游泳行为’，Fraenkel等，1961）这一对光照刺激的瞬时响应。最后，本研究开展了全局转录表达分析，以鉴定受5种固定光周期长度调控的基因与遗传通路，并明确受影响基因/通路与观测到的生活史性状、代谢速率及行为响应之间的潜在关联。