Summary

测试环境线索对交配行为的影响的方法<em>黑腹果蝇</em

Published: July 17, 2017
doi:

Summary

我们展示了一种分析影响果蝇黑腹果蝇交配行为的环境和遗传线索的测定方法。

Abstract

个人的性行为受基因型,经验和环境条件的影响。这些因素如何相互作用来调节性行为仍然很少了解。在黑腹果蝇中 ,环境线索(如食物供应)影响交配活动,提供易处理系统来调查调控性行为的机制。在黑腹果蝇中 ,环境线索通常通过化学感觉味觉和嗅觉系统感觉到。在这里,我们提出了一种测试环境化学物质对交配行为的影响的方法。该测定由含有食物介质和交配对的小交配竞技场组成。连续监测每对夫妇的交配频率24小时。在这里,我们介绍了该测定法通过加压空气系统从外部来源测试环境化合物的适用性,以及直接在交配领域操纵环境成分。你加压空气系统对于测试非常易挥发的化合物的影响是特别有用的,而直接在配合领域操作组分可以有助于确定化合物的存在。该测定可以适应于关于遗传和环境线索对交配行为和繁殖力以及其他男性和女性生殖行为的影响的问题。

Introduction

生殖行为通常具有高能量成本,特别是对于产生比男性更大的配子的女性,并且必须仔细选择条件来提高其发育后代。由于能源成本,繁殖与营养条件相关并不奇怪。这是真正的大多数,如果不是所有的动物包括哺乳动物,其可以青春期营养不良,且其性欲可通过食物,限制1的负面影响被推迟。

遗传模式生物体黑腹果蝇的繁殖也受营养条件的影响。男性法院在食物挥发2的存在较高的水平,并且女性更性酵母,鸡蛋的生产和后代存活3,4,5的主要营养素的存在接受。这个对食物的进化保守的生殖反应提供了机会,研究将环境食物可利用性与有遗传易感性和时效性生物体中的有性繁殖联系起来的机制。事实上,在果蝇的工作有牵连胰岛素通路作为食物和交配行为6之间连接的重要调节器。它也表明,交配行为本身改变女性的食品的偏好以及相关联的神经元化学感应7,8,9。

很明显,食物线索影响黑腹果蝇的生殖行为。这些影响似乎主要影响女性,特别是那些已经交配过的人5 。然而,为了测试环境条件的这些急性效应,经典地用于雌性交配行为的测定法可能会由于交配事件之间的长时间中断,不太适合。在经典的重建测定中,一个处女女性首先与男性交配,并立即分离出来,并在24到48小时后呈现了一个新的男性。此经典测定法已被用于取得了巨大成功,以确定修改的女性行为和阴响应12,13,14,15,16,17,18阳射精的组件。因此,这里证实的连续交配测定是经典交配测定法的补充,可用于研究环境条件对生殖行为的急性影响。

使用这里说明的交配行为的连续测定,我们以前表明,一对暴露于酵母的苍蝇会恢复在24小时观察期5,19,20,21 everal倍,而苍蝇不暴露于食品只会remate一次5。这一发现可在指示女性不初始交配后remate数天的黑腹果蝇文献的大部分的光被令人费解的(参考文献10综述,11)。然而,这种差异可以通过测定条件容易地解释,其中在提供新的交配机会之前女性被隔离一至几天。如果在这个小时长的观察期间该对不配合,那么女性的特征就是不接受。此外,由于来自野生捕蝇的数据显示,女性在其存储器官中含有4至6名男性的精子,所以交配频率高不应该是令人惊讶的。因此在dicating,女性自然remate几次22,23。

在这里,我们展示了使用这种连续交配测定来揭示苍蝇如何收集和组合关于环境条件的信息来调节交配频率。该测定允许测试相对大量的交配对进行遗传研究并测试挥发性和非挥发性环境线索的影响。该测定通常运行24小时,但可以延长至48小时,允许测试循环环境线索,例如光暗(LD)循环。我们通过测试来自加压空气系统中的酵母培养物的挥发性线索的影响与食品基质中非挥发性酵母营养物的可用性的结合来证明该测定。

加压空气系统将挥发性线索连续地泵入包含的配合场地sa食物底物和一对测试夫妇(其交配行为被监控)。为了进一步确定酵母影响交配的细节,我们测试酵母的主要挥发性化合物,即乙酸24 ,与蛋白胨(氨基酸)形式的与食品底物中酵母相当的氨基酸含量衍生自动物蛋白酶消化的酸)。这些实验一起证明了可以通过该测定来测试环境线索对黑腹果蝇交配行为的影响。

Protocol

环保接头盒 为了确保受控和易于清洁的测试区域,请设置一个120厘米x 64厘米x 85厘米的不锈钢厨柜,如图1A所示。 在天花板正下方的机柜背面钻一个孔,在侧面钻四个四孔,每个孔的直径为2厘米。在盒子的每一侧,距离盒子底部7厘米的高度和12.5厘米的孔之间钻第一组四个孔。在距离底部35厘米的高度处,在盒子的每一侧钻另外两套。 注意:四组四个孔用于摄像机?…

Representative Results

使用这种连续测定,可以在实验环境条件下确定具体的交配行为和交配频率。为了控制环境条件,我们将不锈钢厨柜改造成具有自己的光源和扩散的测试区域,确保了高度丰富的光线和从配套场地顶部的最少量的眩光( 图1A ) 。内部测试区域完全由不锈钢和玻璃包裹,可以用有机溶剂如己烷或乙醇清洗。此外,机柜配备有用作管道入口的孔,从加压空气系统…

Discussion

该协议描述了在24小时内测试交配行为的测定,同时连续控制耦合对被假设用于确定交配频率的环境线索。当培养基含有酵母时,可以响应于通过加压空气系统输送的酵母空气来增加交配频率( 图2B )。另外,用介质中仅含有琼脂,蛋白胨和乙酸气味的简化食品介质可以观察到交配频率中的类似反应( 图3B

通过这里展示的实验,只能对夫妻的一般…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢布卢明顿果蝇库存中心的苍蝇股票; C. Gahr,JT Alkema和S. van Hasselt早期尝试开发加压空气测定;碧玉博士为培养酵母提供建议;和Rezza Azanchi和Joel Levine最初开发了果蝇交配行为的延时监测。 JA Gorter得到了神经科学研究学校BCN / NWO研究生课程资助的支持。这项工作得到了荷兰科学研究组织(NWO)(参考文献:821.02.020)对JC Billeter的支持。

Materials

Cabinet
Stainless steel kitchen cabinet Horecaworld 7412.0105
White LEDs Lucky Light ll-583wc2c-001 Cold white, 20 mAmp and 2 V
Red LEDs Lucky Ligt ll-583vc2c-v1-4da Wavelength between 625 nm, 20 mAmp and 6 V
Resistor Royal Ohm CFR0W4J0561A50 560 ohm, 0.25 W, 250 V and 5 % tolerance
Smartphone light meter app Patrick Giudicelli Light/Lux Meter FREE, version 1.1.1
Power timer Alecto TS-121
Metal brackets Sharp angle 5 by 5 mm,  2 x 5450 and 1 x 1100 mm long
Frosted glass plate 1190 x 545 x 5 mm
Filter paper sheets LEE filters 220 White frost
Small fan Nanoxia Deep silence 4260285292828 80 mm Ultra-Quiet PC Fan, 1200 RPM
Big fan Nanoxia Deep silence 4260285292910 120 mm Ultra-Quiet PC Fan, 650-1500 RPM
Webcam camera Logitech 950270 B910 HD WEBCAM OEM, Angle: 78-degree, resolution: 5-million-pixel  
Camera software DeskShare Security monitor pro
Name Company Catalog Number Comments
Fly rearing
Fly rearing bottles Flystuff 32-130 6oz Drosophila stock bottle
Flypad Flystuff 59-114
Wild-type flies Canton-S
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%
Peptone (bacto) BD 211677
Acetic Acid Merck 1000631000 Glacial, 100%
Small petridish Greiner bio-one 627102 35 x 10 mm with vents
Paraffin film Bemis NA Parafilm
Name Company Catalog Number Comments
Yeast and pressurised air set-up
Big petridish Gosselin BP140-01 140 x 20.6 mm
Ultrapure water Millipore corporation MiliQ
Yeast extract BD 212750
Agar (pure) BD 214530 bacto
Glucose (0(+)-glucose monohydrate)  Merck 18270000004
Open caps Schott 29 240 28  GL45
Silicone septum VWR 548-0662
Barbed bulkhead fittings Nalgene 6149-0002
Large PVC tubing diameter: outer 1.2 cm and inner 0.9 cm
Small PVC tubing diameters: outer 0.8 cm and inner 0.5 cm
15 ml tube Falcon
Aquarium pump Sera precision Sera air 110 plus, AC 220-240 V, 50/60 Hz, 3 W and pressure >100 mbar
Activated charcoal Superfish A8040400 Norit activated carbon
Disposible filter unit Whatman 10462100
Serological pipettes VWR 612-1600
Syringe BD Plastipak 300013
Hot glue Pattex
Syringe filter Whatman FP 30/pore size 0.45 mm CA-S
Name Company Catalog Number Comments
Analysis
Statistics software R lme4 package

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Gorter, J. A., Billeter, J. A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster. J. Vis. Exp. (125), e55690, doi:10.3791/55690 (2017).

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