Summary

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

Published: August 09, 2024
doi:

Summary

该协议建立了提取和量化对 秀丽隐杆线虫中挥发性信息素的反应的方法,为研究化学通讯和导航轨迹提供了工具。

Abstract

化学通讯对于有机体健康、繁殖和整体福祉至关重要。了解控制这些信号的分子途径、神经过程和计算仍然是一个活跃的研究领域。线虫秀 丽隐杆 线虫为研究这些过程提供了一个强大的模型,因为它会产生挥发性的性信息素。这种信息素由处女雌性或精子耗尽的雌雄同体合成,可作为雄性的引诱剂。

该方案描述了从几种 秀丽隐杆线 虫菌株(WT 菌株 N2、 daf-22fog-2)和 C. remanei 中分离挥发性信息素的详细方法。我们还提供了一种方案,用于量化雄性趋化性对挥发性信息素的反应。我们的分析利用趋化性指数 (CI)、到达时间 (AT) 和轨迹图等测量值来准确比较各种条件下的男性反应。这种方法允许在具有不同遗传背景或发育阶段的雄性之间进行稳健的比较。此外,此处概述的实验设置适用于研究其他化学引诱化学物质。

Introduction

化学通讯与繁殖成功之间的相互作用是整个动物王国的基本原则 1,2,3,4,5,6,7,8,9,10。性信息素触发了广泛的性二态性行为,这些行为对于寻找配偶、协调寻找和吸引伴侣所涉及的步骤以及最终促进物种的繁殖至关重要 11,12,13,14,15,16,17.在理解信息素信号传导方面已经取得了重大进展,但控制这些相互作用的分子机制、神经回路和计算过程往往仍未完全定义 18,19,20,21,22,23,24,25,26。

线虫秀丽隐杆线虫为剖析这些问题提供了一个强大的模型。值得注意的是,秀丽隐杆线虫表现出一种不寻常的繁殖策略——雌雄同体可以自我受精,但也可以与雄性杂交27282930313233。这种灵活性需要一个强大的通信系统来传达生殖状态。秀丽隐杆线虫以其特征明确的水溶性信息素蛔苷而闻名,它在发育、行为和社会互动中发挥着不同的作用。最近的发现揭示了线虫使用的一类独特的挥发性信息素。这些信息素由性成熟的秀丽隐杆线虫C. remanei 处女雌性以及精子耗尽的雌雄同体专门产生,可作为成年雄性的引诱剂 29,34,35。这种引诱物在其产生和感知中表现出显着的性二态性。雌性体细胞性腺控制这种挥发性信息素的合成,其产生动态地反映生殖状态,在交配时停止,并在几个小时后恢复29,34

了解线虫性信息素通讯可以深入了解化学通讯系统的进化、生殖状态和行为之间的相互作用以及性二态神经加工的潜在机制 24,26,36,37,38,39.研究表明,雄性两栖神经元 AWA 对信息素检测至关重要,其中 G 蛋白偶联受体 SRD-1 在雄性信息素检测中起关键作用24秀丽隐杆线虫非常适合研究动物化学通讯,尤其是性信息素信号,因为它依赖嗅觉系统进行配偶搜索。虽然对蛔蛔苷信号传导了解很多,但挥发性的性信息素系统提供了独特的比较机会 25,26,36,40,41,42,43,44,45,46,47,48,49,50
51,52,53,54,55,56,57。此外,秀丽隐杆线虫是一种强大的遗传模式生物,因为它具有完全测序的基因组、明确定义的细胞谱系和特征明确的嗅觉突变体。

然而,处理这种信息素所涉及的完整神经回路、将其感知转化为靶向寻找配偶行为的计算以及它的生物合成调节仍有待完全阐明。对这些过程的进一步研究对于理解控制动物交流和繁殖行为的不同机制至关重要。鉴定参与信息素合成、分泌和感知的关键基因有望揭示动物交流中的新型分子参与者。此处描述的分析为解决这些问题提供了基础。

Protocol

1. 从女性和雌雄同体中提取粗性信息素 秀丽隐杆线虫同步协议成年雌性或雌雄同体的准备每天监测培养板,直到存在大量成年雌性/雌雄同体并且 OP50 食物来源耗尽。使用 fog-2 C. elegans 和 WT C. remanei 雌性进行粗制性信息素提取,从交配的雌性中制备同步卵。注意:在本方案中,不产生自身精子的 秀丽隐杆线虫雾-2 …

Representative Results

化学吸引试验中挥发性信息素感知缺陷菌株的轨迹分析这种化学吸引测定可靠地区分 了秀丽隐杆线虫 的野生型和突变型菌株对挥发性信息素的反应。对 him-5 雄性的成功实验始终表明对信息素来源的强强趋化性。这反映在高趋化指数 (C.I.)(图 2),通常超过 0.5,表明对信息素来源有强烈的偏好。相反,使用信息素受?…

Discussion

该方案为从秀丽隐杆线虫中提取挥发性信息素提供了一种强大的方法,同时建立了一种强大的化学吸引测定法来测量雄性化学吸引反应。其他信息可在 WormLab 用户指南中找到(参见材料表);有关可视化蠕虫移动轨迹的基本代码,请参阅协议第 7.3.8.5 节。协议中的几个关键步骤对结果很重要。首先,蠕虫种群的仔细同步对于控制年龄和生殖状态至关重要…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢 Tingtao 周 博士为我们分析中使用的轨迹可视化设计和编写代码。这项工作得到了资金支持:R01 NS113119 (PWS)、Chen 高级博士后奖学金以及 Tianqiao 和 Chrissy Chen 神经科学研究所。

Materials

10 cm Petri dishes Falcon 25373-100 Falcon bacteriological Petri dish 100 x 15 mm
6 cm Petri dishes Falcon 25373-085 Falcon bacteriological Petri dish 60 x 15 mm
C. remanei (EM464) CGC
Centrifuge Eppendorf centrifuge 5418  Any brand should work.
Chemoattraction assay plates Homemade solution N/A 1.5% agar, 25 mM NaCl, 1.5 mM Tris-base, and 3.5 mM Tris-Cl
Cholesterol Alfa Aesar 57-88-5
Dissecting Microscope Leica LeicaMZ75  Any brand should work.
E. Coli OP50 CGC
Ethanol Koptec 64-17-5
fog-2(q71) (JK574) CGC
him-5(e1490)(CB4088) CGC
Household bleach Clorox Germicidal bleach concentrated  Bleach
M9 buffer Homemade solution N/A 3 g KH2PO4, 11.3 g Na2HPO4.7H2O, 5 g NaCl, H2O to 1 L. Sterilize by autoclaving. Add 1 mL 1 M MgSO4 after cool down to room temperature.
Magnesium Sulfate, Anhydrous, Powder Macron M1063-500GM-EA
Microwave TOSHIBA N/A  Any brand should work.
N2 CGC
NaOH Sigma-aldrich S318-3 1 M
NGM plates solution Homemade solution N/A 2.5 g Peptone, 18 g agar, 3 g NaCl, H2O to 1 L.Sterilize by autoclaving. Once the autoclave is done (2 h), wait until the temperature of the medium drops to 65 °C. Put on a hotplate at 65 °C and stir. Then add the following, waiting 5 min between each to avoid crystallization: 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL K3PO4 (1 M, pH=6), 1 mL Cholesterol ( 5 mg/mL in ethanol).
Parafilm Bemis 13-374-10 Bemis Parafilm M Laboratory Wrapping Film
Peptone VWR 97063-324
Pipet- aid Drummond Scientific  4-000-100  Any brand should work.
Plastic paper  Octago Waterproof Screen Printing Inkjet Transparency Film https://www.amazon.com/Octago-Waterproof-Transparency-Printing-Printers/dp/B08HJQWFGD
Potassium chloride Sigma-aldrich SLBP2366V
Potassium phosphate Spectrum 7778-77-0
Pipette Eppendorf SKU: EPPR4331; MFG#: 2231300006 20 – 200 µL, 100 – 1000 µL, any brand should work.
Rotator Labnet SKU: LI-H5500  Labnet H5500 Mini LabRoller with Dual Direction Rotator. Any brand should work.
Sodium chloride VWR 7647-14-5
sodium phosphate dibasic Sigma-aldrich SLCG3888
Tris-base Sigma-aldrich 77-86-1
Tris-Cl  Roche 1185-53-1
Tryptone VWR 97063-390
Vortex Scientific industries Vortex-Genie 2  Any brand should work.
WormLab system  MBF Bioscience N/A https://www.mbfbioscience.com/help/WormLab/Content/home.htm; https://www.mbfbioscience.com/products/wormlab/
Wormpicker Homemade  N/A made with platinum and glass pipet tips

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Cite This Article
Wan, X., Sternberg, P. W. Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans. J. Vis. Exp. (210), e67115, doi:10.3791/67115 (2024).

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