一种自动测定果蝇在空间和时间温度变化中的性能的方法
Summary
在这里, 我们提出了一个协议, 自动确定果蝇的运动性能在变化的温度使用可编程温度控制竞技场, 产生快速和准确的温度变化的时间和空间。
Abstract
温度是影响物种分布和行为的普遍环境因素。不同种类果蝇对温度变化的生理耐受性和适应性有特定的反应。果蝇还拥有一个温度传感系统, 已成为了解变温动物温度处理的神经基础的基础。我们在这里提供一个温度控制的竞技场, 允许快速和精确的温度变化与时间和空间控制, 以探索个别苍蝇对变化的温度的反应。单独的苍蝇被放置在竞技场和暴露在预编程的温度挑战, 如均匀逐渐增加的温度, 以确定反应规范或空间分布的温度在同一时间确定偏好。自动跟踪个人, 允许量化速度或位置偏好。此方法可用于快速量化在大范围温度下的响应, 以确定果蝇或其他类似大小的昆虫的温度性能曲线。此外, 它还可用于基因研究, 以量化突变体或野生型苍蝇的温度偏好和反应。该方法可以帮助揭示热形态和适应的基础, 以及温度处理后的神经机制。
Introduction
温度是一个持续的环境因素, 影响生物体的功能和行为1。纬度和海拔高度的差异导致气候有机体类型的差异, 这导致了他们对温度2,3反应的进化选择。生物体通过形态学、生理和行为适应来响应不同的温度, 从而在其特定环境下实现最佳性能4。例如, 在果蝇中, 不同地区的人群在不同温度下有不同的温度偏好、体型、发育时间、长寿、繁殖力和行走性能2 ,5,6,7。不同起源蝇之间的多样性由遗传变异和塑性基因表达8,9部分解释。同样, 不同区域的果蝇种类在温度梯度上分布不同, 并显示耐极端热和冷试验的差异10、11、12。
果蝇最近也成为了选择的模型, 了解温度知觉的遗传和神经基础13,14,15,16,17。一般来说, 成人苍蝇通过在天线和通过温度传感器在大脑13,14,15,16的冷和热外围温度传感器感知温度,17,18,19,20. 热温度的外围受体表示Gr28b16或发热21, 而外围冷受体的特征是Brivido14。在大脑中, 温度由表达TrpA115的神经元处理。对这些途径突变体的行为研究正在提高我们对温度如何处理的理解, 并对不同地区果蝇种群的变化机制进行深入的了解。
在这里, 我们描述一个温度控制的竞技场, 产生快速和精确的温度变化。调查人员可以对这些变化进行预编程, 这样就无需人工干预即可实现标准化和可重复的温度操纵。飞行记录和跟踪与专门的软件, 以确定他们的位置和速度在实验的不同阶段。本协议中提出的主要测量方法是在不同温度下的行走速度, 因为它是一种生态学相关的生理性能指标, 可识别个体的热适应性5。与温度受体突变体一起, 这种技术可以帮助揭示细胞和生化水平的热适应机制。
Protocol
1. 飞行食品培养基的制备 将 1 l 的自来水倒入2升玻璃杯中, 加入一个磁力搅拌棒。将烧杯放在300摄氏度的磁性热板上, 直到达到沸腾温度。 搅拌在500发/分钟, 并添加以下内容:10 克琼脂, 30 克葡萄糖, 15 克蔗糖, 15 克玉米粉, 10 克小麦胚芽, 10 克大豆面粉, 30 克糖蜜, 和 35 g 活性干酵母。 当混合泡沫剧烈时, 将热板温度降低到120摄氏度, 同时继续搅拌。 将热板温度进一步降…
Representative Results
温度控制的竞技场 (图 1A) 由三个铜瓦组成, 其温度可通过可编程电路单独控制。每块铜瓦都有一个温度传感器, 可以向可编程电路提供反馈。电路激活电源以增加每个磁贴的温度。被动式热电元件充当恒定的加热元件, 以保持所需的温度, 而风扇冷却的散热片提供恒定的冷却。温度变化的幅度以非线性的方式决定了过程的速度。增加2摄氏度只需要0.1 ?…
Discussion
在这里, 我们提出了一个自动化的温度控制竞技场 (图 1), 在时间和空间上产生精确的温度变化。这种方法允许单独的果蝇暴露, 不仅是预先编程的逐渐增加的温度 (图 2和图 3), 但也对动态温度挑战, 其中每个瓷砖的飞行竞技场被加热独立于不同的温度 (图 4和 图 5)。</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分由格罗宁根大学的行为和认知神经科学计划的奖学金和来自墨西哥的委员会 Ciencia y 国家 (国家科学技术委员会) 的研究生奖学金, 授予安德烈帕迪拉, 以及 Hedderik Rijn 和吉恩-克里斯托弗. 比耶泰的时间研究奖。我们也感谢彼得 Gerrit 峨参与开发FlySteps跟踪器。
脚本TemperaturePhases、FlySteps和FlyStepAnalysis可以被发现为补充信息, 并在以下临时和公开可用的链接:
https://dataverse.nl/privateurl.xhtml？token=c70159ad-4d92-443d-8946-974140d2cb78
Materials
| Arduino Due | Arduino | A000062 | Software RUG |
| Electronics Board | Ruijsink Dynamic Engineering | FF-Main-02-2014 | |
| Power supply Boost | XP-Power 48. V 65 W | ECS65US48 | Set to 53 Volt |
| Power supply Tile Heating | XP-Power 15. V 80 W | VFT80US15 | |
| Power supply Cooling | XP-Power 15. V 130 W | ECS130U515 | |
| Peltier elements | Marlow Industries | RC12-4 | 2 Elements, controlled DC feed |
| Heat sink | Fisher Technik | LA 9/150-230V | Decoupled for vibration |
| Temperature sensors | Measurement Specialties | MCD_10K3MCD1 | Micro Thermistor Probe |
| Copper block/tiles | Ruijsink Dynamic Engineering | FF-CB-01-2014 | |
| Auminum ring | Ruijsink Dynamic Engineering | FF-RoF-02-2015 | |
| Tesa 4104 white tape 25 x 66 mm | RS Components | 111-2300 | White conductive tape |
| Red LEDs | Lucky Ligt | ll-583vc2c-v1-4da | Wavelength between 625 nm, 20 mAmp and 6 V |
| Warm white LED strip | Ledstripkoning | HQ-3528-SMD | 60 LEDs per meter |
| Switch Power Supply | Generic | T-36-12 | |
| Logitech c920 | Logitech Europe S.A | PN960-001055 | |
| QuickTime Player | Apple Computer | Recording program | |
| Tracking analysis software | R | Packages: pacman | |
| Tracking analysis software | MATLAB | ||
| Thermal Imaging | FLIR T400sc | ||
| Graphs and Statisticts Software | Graph Pad Prism | ||
| Sigmacote | Sigma-Aldrich | SL2-100ML | Siliconising agent |
| Fly rearing bottles | Flystuff | 32-130 | 6oz Drosophila stock bottle |
| Flypad | Flystuff | 59-114 | |
| Fly rearing vials | Dominique Dutscher | 789008 | Drosophila tubes narrow 25×95 mm |
| Incubator | Sanyo | MIR-154 | |
| Magnetic hot plate | Heidolph | 505-20000-00 | MR Hei-Standard |
| Agar | Caldic Ingredients B.V. | 010001.26.0 | |
| Glucose | Gezond&wel | 1019155 | Dextrose/Druivensuiker |
| Sucrose | Van Gilse | Granulated sugar | |
| Cornmeal | Flystuff | 62-100 | |
| Wheat germ | Gezond&wel | 1017683 | |
| Soy flour | Flystuff | 62-115 | |
| Molasses | Flystuff | 62-117 | |
| Active dry yeast | Red Star | ||
| Tegosept | Flystuff | 20-258 | 100% |
References
- Abram, P. K., Boivin, G., Moiroux, J., Brodeur, J. Behavioural effects of temperature on ectothermic animals unifying thermal physiology and behavioural plasticity. bioRxiv. , (2016).
- Rajpurohit, S., Schmidt, P. S. Measuring thermal behavior in smaller insects: A case study in Drosophila melanogaster demonstrates effects of sex, geographic origin, and rearing temperature on adult behavior. Fly. 10 (4), 149-161 (2016).
- Jezovit, J. A., Levine, J. D., Schneider, J. Phylogeny environment and sexual communication across the Drosophila genus. The Journal of Experimental Biology. 220 (1), 42-52 (2017).
- Sinclair, B. J., Williams, C. M., Terblanche, J. S. Variation in Thermal Performance among Insect Populations. Physiological and Biochemical Zoology. 85 (6), 594-606 (2012).
- Gibert, P., Huey, R., Gilchrist, G. Locomotor performance of Drosophila melanogaster: Interactions among developmental and adult temperautures, age, and geography. Evolution. 55 (1), 205-209 (2001).
- Trotta, V., et al. Thermal plasticity in Drosophila melanogaster: A comparison of geographic populations. BMC Evolutionary Biology. 6, 1-13 (2006).
- Klepsatel, P., Gálikova, M., De Maio, N., Huber, C. D., Christian, S., Flatt, T. Variation in thermal performance and reaction norms among populations of Drosophila melanogaster. Evolution. 67 (12), 3573-3587 (2013).
- Latimer, C. A. L., Wilson, R. S., Chenoweth, S. F. Quantitative genetic variation for thermal performance curves within and among natural populations of Drosophila serrata. Journal of Evolutionary Biology. 24, 965-975 (2011).
- Chen, J., Nolte, V., Schlotterer, C. Temperature-related reaction norms of gene expression: Regulatory architecture and functional implications. Molecular Biology and Evolution. , (2015).
- Kellermann, V., Overgaard, J., Hoffmann, A. A., Flojgaard, C., Svenning, J. -. C., Loeschcke, V. Upper thermal limits of Drosophila are linked to species distributions and strongly constrained phylogenetically. Proceedings of the National Academy of Sciences. 109 (40), 16228-16233 (2012).
- Andersen, J. L., Manenti, T., Sørensen, J. G., Macmillan, H. A., Loeschcke, V., Overgaard, J. How to assess Drosophila cold tolerance: Chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits. Functional Ecology. 29 (1), 55-65 (2015).
- Krstevska, B., Hoffmann, A. A. The effects of acclimation and rearing conditions on the response of tropical and temperate populations of Drosophila melanogaster and D. simulans to a temperature gradient (Diptera: Drosophilidae). Journal of Insect Behavior. 7 (3), 279-288 (1994).
- Frank, D. D., Jouandet, G. C., Kearney, P. J., Macpherson, L. J., Gallio, M. Temperature representation in the Drosophila brain. Nature. 519 (7543), 358-361 (2015).
- Gallio, M., Ofstad, T. A., Macpherson, L. J., Wang, J. W., Zuker, C. S. The coding of temperature in the Drosophila brain. Cell. 144 (4), 614-624 (2011).
- Hamada, F. N., et al. An internal thermal sensor controlling temperature preference in Drosophila. Nature. 454 (7201), 217-220 (2008).
- Ni, L., et al. A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila. Nature. 500 (7464), 580-584 (2013).
- Liu, W. W., Mazor, O., Wilson, R. I. Thermosensory processing in the Drosophila brain. Nature. 519 (7543), 353-357 (2015).
- Neely, G. G., et al. TrpA1 Regulates Thermal Nociception in Drosophila. Public Library of Science ONE. 6 (8), e24343 (2011).
- Zhong, L., et al. Thermosensory and non-thermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat sensor domains of a thermoTRP channel. Cell Reports. 1 (1), 43-55 (2012).
- Barbagallo, B., Garrity, P. A. Temperature sensation in Drosophila. Current Opinion in Neurobiology. 34, 8-13 (2015).
- Tang, X., Platt, M. D., Lagnese, C. M., Leslie, J. R., Hamada, F. N. Temperature integration at the AC thermosensory neurons in Drosophila. Journal of Neuroscience. 33 (3), 894-901 (2013).
- Petavy, G., David, J. R., Gibert, P., Moreteau, B. Viability and rate of development at different temperatures in Drosophila: A comparison of constant and alternating thermal regimes. Journal of Thermal Biology. 26 (1), 29-39 (2001).
- Diegelmann, S., Zars, M., Zars, T. Genetic dissociation of acquisition and memory strength in the heat-box spatial learning paradigm in Drosophila. Learning & Memory. 13 (1), 72-83 (2006).
- Zars, M., Zars, T. High and low temperatures have unequal reinforcing properties in Drosophila spatial learning. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology. 192 (7), 727-735 (2006).
- Zars, T., Wolf, R., Davis, R., Heisenberg, M. Tissue-specific expression of a type I adenylyl cyclase rescues the rutabaga mutant memory defect: in search of the engram. Learning & Memory. 7 (1), 18-31 (2000).
- Jones, M. A., Grotewiel, M. Drosophila as a model for age-related impairment in locomotor and other behaviors. Experimental Brain Research. 46 (5), 320-325 (2011).
Cite This Article
Soto-Padilla, A., Ruijsink, R., Span, M., van Rijn, H., Billeter, J. An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time. J. Vis. Exp. (140), e58350, doi:10.3791/58350 (2018).