解剖和蜜蜂背血管的观察心功能的研究
Summary
The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.
Abstract
The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.
Introduction
这种方法的总体目标是允许研究者快速，轻松地观察和量化药理剂对蜜蜂的心脏速率的影响。蜜蜂，像其他昆虫，具有传播血淋巴，血液的昆虫当量，在整个体腔，被称为血腔的开放循环系统。血淋巴的循环为营养物质，免疫因子，废产物，以及神经激素和其他信号分子1的运输是必不可少的。循环是通过背容器中，沿昆虫的背中线，以及附件搏动器官延伸促进。背容器被分成两个功能上不同的部分，指定在腹部的心脏，并在胸部和头部的主动脉。在对胸部和头部的心脏泵血淋巴传播收缩，而附属器官搏动血淋巴确保流向四肢。
<p类=“jove_content”>可以使用多种方法，这取决于昆虫的大小，生理学，或生命阶段观察到昆虫的心脏功能。观察幼虫或更小的昆虫心脏速率的常见方法是利用活体成像2。这种方法是在成年蜂不太有用，但是，由于它可能很难通过腹壁来清楚地观察背脉管。在各种昆虫，包括蜜蜂记录心脏速率的建立的方法，是利用接触热成像，其利用施加到昆虫的外部，以检测心脏脉动3,4-热敏电阻。在成人蜜蜂心脏速率也已使用电生理技术来测量电阻抗信号4,5-记录。这种技术需要的电极插入到动物旁边的心脏和使用阻抗变换器来记录心跳4。同样，心电图已用于DETECŧ由心脏产生，并与蜜蜂的录像相结合，观察心脏活动6变化的电信号。一个显着的优势，这些方法是心脏速率在一个完整的，活蜂评估，而不是在一个解剖的标本，这有助于确保全方位的主题生理反应的可用性。这些方法的挑战包括会计主体，需要限制之外的变量和刺激，可能改变心脏率，以及测试药物制剂时，确定合适的给药方法的固定或麻醉。已用于研究蜂的心脏活动的另一种方法是，以部分解剖昆虫以暴露心脏，然后进行测量使用一个力位移传感器7背脉管收缩。在这个协议中，心脏不断与正在运行的生理盐水和测试沐浴合作mpounds可以溶解在该溶液应用到对象7。这种方法与前面描述的那些之间的显著区别在于腹神经索被去除，消除了对中枢神经系统已被证明在调节心脏速率5发挥作用。其结果是，在基线心跳，这通常是相当不稳定的，稳定在一个低得多的频率和幅度比在活昆虫5,7-通常观察到。什么所有这些方法的共同点在于，它们需要高度专业化和通常是昂贵的设备，除了专门知识的一定水平，以进行。或许最大的缺点是，没有这些方法均特别适用于涉及测试大量的受试者，如筛选潜在cardiomodulatory化合物文库的实验。
这里所描述的方法的最大优势是它的简单性。该协议是比较容易掌握，安装需要空间不大，只有最小的资金投入是必要的。该方法需要比一些蜜蜂，几个手术器械，等渗溶液更小，以及一个数字显微镜或用数码相机拍摄传统显微镜。蜜蜂解剖可视化背船只和数字视频使用前，用药剂治疗后，记录心脏速率。虽然录像实际上不是必要观察心脏速率的变化，这将大大增加吞吐量（ 即 ，可以在给定的时间量来处理对象的数目）。研究者可以一次录制了大量的视频，然后后来在更方便的时间攻入这些视频最大限度地提高效率。这种方法的另一个优点是，视频允许研究者重新开始，应在评分过程被中断，并使其更容易在Viewer，以便减少偏见蒙蔽治疗。
Protocol
Representative Results
Discussion
The protocol presented here provides a simple and effective approach to testing pharmacological compounds for their effects on honey bee heart rate. As observed in prior experiments that either transect the ventral nerve cord of a living insect5 or dissect out the ventral nerve cord when exposing the dorsal vessel7, the loss of central nervous system regulation results in a stable, low frequency heartbeat. The low frequency of beats allows the investigator to visually assess heart rate without havin…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Drs. Jeffrey Bloomquist and Daniel Swale for their technical comments and suggestions. This project was partially funded by the Department of Entomology and the College of Agriculture and Life Sciences at Virginia Tech.
Materials
| Dino-Lite Edge digital USB microscope | Dino-Lite | AM4815ZT | Any digital microscope or similar setup will suffice |
| Microscope stand | Dino-Lite | RK-10 | Any stand appropriate for the digital microscope |
| Laptop or PC | Necessary for digital microscope | ||
| Microdissection scissors (Vannas, 8cm, Straight, 5mm Blades) | World Precision Instruments | 14003 | Any similar scissors suitable for microdissection will suffice |
| Microdissecting Forceps, 10.2cm, Angled (2 pair) | World Precision Instruments | 504482 | Any similar forceps suitable for microdissection will suffice |
| Ringers solution 1/4 strength tablets | Sigma-Aldrich | 96724-100TAB | |
| Dissecting tray | Any surface suitable for microdissection | ||
| Single channel 10 µl pipette | Any device capable of accurately delivering 10 µl volume | ||
| Pipette tips | |||
| Small beaker or container of water | Used to rinse instruments between subjects | ||
| Hand tally counter | Office Depot | 295033 | Any similar product will suffice |
| Timer | Office Depot | 644219 | Any similar product will suffice |
| Deionized water | Preparation of Ringers solution and rinsing instruments |
References
- Klowden, M. J. Circulatory Systems. Physiological Systems in Insects, 3rd Edition. , 365-413 (2013).
- League, G. P., Onuh, O. C., Hillyer, J. F. Comparative structural and functional analysis of the larval and adult dorsal vessel and its role in hemolymph circulation in the mosquito Anopheles gambiae. J Exp Biol. 218 (Pt 3), 370-380 (2015).
- Wasserthal, L. T. Oscillating Hemolymph Circulation in the Butterfly Papilio-Machaon L Revealed by Contact Thermography and Photocell Measurements. J Comp Physiol. 139 (2), 145-163 (1980).
- Wasserthal, L. T. Interaction of circulation and tracheal ventilation in holometabolous insects. Adv Insect Physiol. 26, 297-351 (1996).
- Schwab, E. R., Chilson, R. A., Eddleman, C. D. Heartbeat Rate Modulation Mediated by the Ventral Nerve Cord in the Honey-Bee, Apis-Mellifera. J Comp Physiol B-Biochem Syst Environ Physiol. 161 (6), 602-610 (1991).
- Kaiser, W., Weber, T., Otto, D., Miroschnikow, A. Oxygen supply of the heart and electrocardiogram potentials with reversed polarity in sleeping and resting honey bees. Apidologie. 45 (1), 73-87 (2014).
- Papaefthimiou, C., Theophilidis, G. Octopamine–a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): Acceleration vs. inhibition. J Insect Physiol. 57 (2), 316-325 (2011).
- Castro, C. A., Hogan, J. B., Benson, K. A., Shehata, C. W., Landauer, M. R. Behavioral-Effects of Vehicles – Dmso, Ethanol, Tween-20, Tween-80, and Emulphor-620. Pharmacol Biochem Behav. 50 (4), 521-526 (1995).
- Papaefthimiou, C., Papachristoforou, A., Theophilidis, G. Biphasic responses of the honeybee heart to nanomolar concentrations of amitraz. Pestic Biochem Phys. 107 (1), 132-137 (2013).
- Roeder, T. Octopamine in invertebrates. Prog Neurobiol. 59 (5), 533-561 (1999).
- Johnson, E., Ringo, J., Dowse, H. Modulation of Drosophila heartbeat by neurotransmitters. J Comp Physiol B. 167 (2), 89-97 (1997).
