研究夜间人造光对自由放养动物影响的实验方法：实施，结果和未来研究的方向
Summary
夜间人造光（ALAN）具有广泛的生物效应。本文介绍了一种在监控行为时在巢箱内操作 ALAN 的系统，该系统由连接到电池、定时器和支持音频的红外摄像机的 LED 灯组成。 研究人员可以利用该系统来探索有关ALAN对生物体影响的许多悬而未决的问题。
Abstract
动物已经进化出光明和黑暗的自然模式。然而，人造光正越来越多地从人类基础设施和娱乐活动中引入环境。夜间人造光（ALAN）有可能对动物行为，生理学和适应性产生广泛影响，这可以转化为对种群和社区的更广泛影响。了解ALAN对自由放养动物的影响并非易事，因为存在诸如测量移动生物遇到的光照水平以及将ALAN的影响与其他人为干扰因素的影响区分开来等挑战。在这里，我们描述了一种方法，该方法使我们能够通过实验性地操纵巢箱内的光照水平来隔离人造光照射对个体动物的影响。为此，可以使用由附着在板上并连接到电池和定时器系统的发光二极管（LED）光组成的系统。该设置允许巢箱内的个体暴露在ALAN的不同强度和持续时间下，同时获得视频记录，其中还包括音频。该系统已被用于对自由放养的大山雀（Parus major）和蓝山雀（Cyanistes caeruleus）的研究，以深入了解ALAN如何影响成人的睡眠和活动模式以及发育雏鸟的生理学和端粒动力学。该系统或其适应性可用于回答许多其他有趣的研究问题，例如ALAN如何与其他干扰因子相互作用并影响生物能量平衡。此外，类似的系统可以安装在各种物种的巢箱，巢穴或洞穴中或附近，以操纵ALAN的水平，评估生物反应，并致力于建立种间视角。特别是当与其他监测自由生活动物的行为和运动的先进方法相结合时，这种方法有望为我们理解ALAN的生物学意义做出持续的贡献。
Introduction
动物已经进化出定义白天和黑夜的光明和黑暗的自然模式。因此，荷尔蒙系统中的昼夜节律协调休息和活动模式，并允许动物最大限度地提高健身1，2，3。例如，糖皮质激素中的昼夜节律在日常活动开始时达到峰值，通过对葡萄糖代谢和对环境应激源的反应，使脊椎动物在24小时内表现适当4。同样，在黑暗中释放的松果体激素褪黑激素与昼夜节律性的控制模式有关，并且还具有抗氧化特性5，6。昼夜节律性的许多方面的夹带，例如褪黑激素的释放，受到环境中光照水平的光接收的影响。因此，将人造光引入环境以支持人类活动，娱乐和基础设施有可能对自由放养动物的行为，生理和适应性产生广泛的影响7，8。事实上，夜间暴露于人造光（ALAN）的各种影响已被记录为9，10，并且ALAN已被强调为21世纪10年全球变化研究的优先事项。
由于多种原因，测量ALAN对自由放养动物的影响带来了不小的挑战。首先，在环境中移动的动物不断经历不同程度的光照。因此，如何量化单个动物暴露的光照水平？即使可以量化动物领地上的光照水平，动物也可能采用影响暴露模式的回避策略，从而要求同时跟踪动物的位置和光照水平。事实上，在大多数实地研究中，光照水平的平均值和变化是未知的11。其次，暴露于ALAN通常与暴露于其他人为干扰因素有关，例如噪声污染，化学品暴露和栖息地退化。例如，占据道路边缘栖息地的动物将暴露在路灯的光线，车辆交通的噪音和车辆排放的空气污染中。那么，如何有效地将 ALAN 的影响与混杂变量的影响隔离开来呢？能够对光照水平和反应变量进行良好测量的严格现场实验对于评估ALAN生物效应的严重程度以及制定有效的缓解策略至关重要11。
本文描述了一种实验方法，尽管并非没有限制（参见讨论部分），但有助于缓解（如果不是消除上述困难的话）。该方法需要使用发光二极管（LED）灯系统和安装在巢箱内的红外（IR）摄像机，在自由生活的昼夜鸟类大山雀（Parus major）的巢箱内实验性地操纵ALAN水平。该设置可以同时采集视频记录，包括音频，使研究人员能够评估对行为和发声的影响。大山雀利用巢箱繁殖，并在11月至3月期间睡在巢箱中。雌性在繁殖季节12期间也会睡在巢箱内。该系统也已在较小程度上用于研究ALAN对蓝山雀（Cyanistes caeruleus）的影响。第一个困难，涉及了解动物遇到的光照水平，由于个体愿意进入巢箱（或者在不动的雏鸟的情况下已经在巢箱中），光照水平可以由研究人员精确确定。第二个困难涉及与混杂变量的相关性，可以通过在类似环境中使用嵌套框和/或测量嵌套框附近混杂变量的水平来控制。此外，在空巢筑巢鸟类中，采用实验方法是强大的，因为巢箱或自然洞穴可以保护雏鸟和成虫免受ALAN13的影响，这也许可以解释为什么一些相关研究发现ALAN（或人为噪音）的影响很小14，而实验研究更经常发现明显的效果（见下文）。此外，可以采用重复测量实验设计，其中个体作为自己的控制，这进一步增加了统计能力和检测有意义的生物效应的概率。以下部分：（1）解释该系统的设计和实现的细节，（2）总结迄今为止使用该系统得出的重要结果，以及（3）提出未来可以在山雀和其他动物中追求的研究方向。
Protocol
该系统在动物实验中的所有应用均已获得安特卫普大学伦理委员会的批准，并根据比利时和佛兰德法律进行。方法符合ASAB / ABS在行为研究中使用动物的指南。比利时皇家自然科学研究所（ Koninklijk Belgisch Instituut voor Natuurwetenschappen;KBIN）为所有研究人员和人员提供许可证。 1. 创建实验系统 获取用于创建 ALAN 的广谱 LED。从 LED 大灯取出 LED 灯。使用单个 LED ?…
Representative Results
使用该系统发表的同行评审研究文章总结在 表2中。其他几份手稿正在编写中。这些研究解决了三个主要的研究问题。首先，该系统已被用于研究光照对成人睡眠行为和活动水平的影响。为此，采用了重复的测量实验设计，其中首先记录同一个体在自然条件下睡觉，然后记录在点燃的巢箱中睡觉。这些研究中使用的所有个体都装有PIT标签，允许研究人员使用手持式应答器阅读器验证同…
Discussion
这种基于巢箱的LED灯系统和配对的红外摄像机使研究人员能够评估有关ALAN生物效应的一系列有趣问题。此外，该系统还可以追求更多的研究方向。此外，将该系统的使用扩展到其他物种可能有助于促进对ALAN敏感性的种间差异的理解。下面提出了一些未来研究的非详尽可能性，希望本文将有助于激励这一重要领域的研究。结论简要重申了这种实验方法的优势，并解决了该系统的局限性。
<p class=…Divulgations
The authors have nothing to disclose.
Acknowledgements
我们涉及ALAN对鸟类的生物效应的研究计划已获得FWO法兰德斯（M.E.和R.P.，项目ID：G.0A36.15N），安特卫普大学和欧盟委员会（M.L.G，Marie Skłodowska-Curie奖学金ID：799667）的资助。我们感谢安特卫普大学行为生态学和生态生理学研究小组成员的智力和技术支持，特别是Peter Scheys和Thomas Raap。
Materials
| Broad spectrum; 15 mm x 5 mm; LED headlight | RANEX; Gilze; Nederlands | 6000.217 | A similar model could also be used |
| Battery | BYD | R1210A-C | Fe-battery 12 V 120 Wh ( lithium iron phosphate battery) |
| Dark green paint | Optional. To color nest boxes/electronic enclosures | ||
| Electrical tape | For electronics | ||
| Homemade timer system | Amazon | YP109A 12V | A similar model could also be used |
| Infrared camera | Koberts-Goods, Melsungen, DE | 205-IR-L | Mini camera; a similar model could also be used |
| Light level meter | ISO-Tech ILM; Corby; UK | 1335 | To calibrate light intensity |
| Mini DVR video recorder | Pakatak, Essex, UK | MD-101 | Surveillance DVR Recorder Mini SD Car DVR with 32 GB |
| Passive integrated transponder (PIT) tags | Eccel Technology Ltd, Aylesbury, UK | EM4102 | 125 Kh; Provides unique electronic ID |
| Radio frequency identification (RFID) Reader | Trovan, Aalten, Netherlands | GR-250 | To scan PIT tags and determine bird identity |
| Resistor | RS Components | Value depending on voltage battery and illumination | |
| SD card | SanDisk | 64 GB or larger | |
| SongMeter | Wildlife Acoustics; Maynard, MA | Optional. Provides a means of monitoring vocalizations outside of nest boxes | |
| TFT Color LED Portable Test Monitor | Walmart | Allows verification that the camera is on and recording the image correctly | |
| Wood | To construct nest boxes/electronic encolsures |
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Citer Cet Article
Grunst, M. L., Grunst, A. S., Pinxten, R., Eens, G., Eens, M. An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research. J. Vis. Exp. (180), e63381, doi:10.3791/63381 (2022).