吸收荧光、磁性纳米微粒测定昆虫的吸液能力
Summary
液体喂养的昆虫有能力从多孔表面获取少量液体。该协议描述了一种方法, 直接确定昆虫的能力, 从多孔表面摄取液体使用荧光, 磁性纳米微粒的喂养解决方案。
Abstract
液体喂养的昆虫摄取各种液体, 它们存在于环境中, 如水池、薄膜, 或局限于小孔隙。液体采集的研究需要评估口器结构和功能关系;然而, 流体吸收机制的历史推断从结构结构的观察, 有时无人陪伴的实验证据。在这里, 我们报告了一种新的方法, 以评估液体摄取能力与蝴蝶 (鳞翅目) 和苍蝇 (双翅目) 使用少量的液体。昆虫用20% 的蔗糖溶液与荧光、磁性纳米微粒混合, 从特定孔径的滤纸中过滤。该作物 (用于储存液体的内部结构) 从昆虫中移除并放在共聚焦显微镜上。一个磁铁是由作物挥舞, 以确定存在的纳米颗粒, 这表明, 如果昆虫能够摄取液体。这种方法是用来揭示一个广泛的喂养机制 (毛细管作用和液体桥梁形成), 可能共享之间的鳞翅目和双翅目时, 从多孔表面喂养。此外, 该方法可用于研究各种供液昆虫的摄食机制, 包括那些在疾病传播和仿生学中重要的, 以及可能涉及纳米或微米导管的其他研究液体运输需要验证。
Introduction
许多昆虫群有口器 (proboscises) 适应在液体哺养, 例如花蜜, 腐烂的果子, 汁液流动 (例如双翅目1, 鳞翅目2, 膜翅目3), 木质部 (半翅目4), 泪花 (鳞翅目5) 和血液 (Phthiraptera6, 蚤7, 双翅目7, 半翅目8, 鳞翅目9)。昆虫在液体中觅食的能力与生态系统健康相关 (如授粉10)、疾病传播4、11、biodiversification2、12, 并研究的收敛演化13。尽管各种各样的食物来源, 一个主题在一些液体哺养的昆虫是能力获取少量液体, 可能被限制到微或纳米微粒、液体影片或者多孔的表面。
考虑到液体喂养昆虫的广泛多样性 (超过20% 的所有动物种类14,15) 和它们在各种食物来源上觅食的能力, 了解它们的摄食行为和流体摄取机制是在许多领域很重要。例如, 昆虫口器功能在仿生技术的发展中扮演了重要的角色,例如, 微流控设备可以执行一些任务, 如使用类似的方法获取少量的流体由昆虫16。然而, 研究流体吸收机制的一个基本问题是, 不仅要确定昆虫如何以液体为食, 而且还要获得支持这种机制的实验证据。仅使用行为 (例如, 用喙12、17进行探测) 作为喂养的指示器是不够的, 因为它不能确认液体的成功摄取, 也不提供一种方法来确定液体通过昆虫传播。此外, 对少量的流体进行实验更好地代表了自然的喂养方案, 其中流体是限制资源2,12。
用 X 射线相衬成像与帝王蝶 (Danaus plexippus L.) 来评估蝴蝶是如何从多孔表面的少量液体中觅食的12。帝王蝶通过表皮投射 (背 legulae) 沿喙间的空间进行毛细管作用, 将液体局限在小孔中进入食物管。进入的流体形成一个影片在食物渠道墙壁生长并且崩溃入一台液体桥梁由高原不稳定12,18, 然后被传送到蝴蝶的内脏由吮泵的行动在头。虽然 x 射线相衬成像是一种优化的工具, 可视化流体在昆虫内部12,19,20,21, 该技术是不容易获得和更方便方法是需要快速评估昆虫的能力, 吸收液体和摄取它们。
要确定D. plexippus的进给机制是否适用于其他鳞翅目和苍蝇 (双翅目) (两组都是从多孔表面上的液体中进食), Lehnert et al.13应用了一种技术, 用于评估昆虫在多孔表面的少量液体中的进食能力, 这里详细报道。虽然这里概述的协议是用于研究, 使用湿润和多孔表面, 方法可以改变的其他研究, 如那些解决池喂养机制。此外, 应用扩展到其他领域, 包括微和生物技术。
Protocol
1. 昆虫种类、溶液的配制和加料站设置 注: 白菜蝶 (菜青虫菜青虫, 粉蝶) 被选为具有代表性的鳞翅目物种, 因为它们已用于以前的流体摄取能力和口器形态学研究22,23。房子苍蝇 (家蝇, 蝇科) 和蓝瓶苍蝇 (Calliphora vomitoria l., 蝇) 使用, 因为它们经常被观察到多孔表面上的摄食13。 将P. ?…
Representative Results
研究液体喂养昆虫的吸水能力的模式需要确定进食发生的时间。此处所述的协议用于测试鳞翅目和双翅目中的极限孔径假设13。限制性孔隙大小假说指出, 如果孔径直径小于进料管道的直径12, 则液体喂养的昆虫无法从充液的孔隙中进食。从多孔表面进入的流体必须形成一个稳定的液体桥梁, 通过高原不稳定18, 以克服毛?…
Discussion
昆虫口器功能是从研究的大体形态学 (e. g, 鳞翅目的喙功能相关的饮水秸秆25,26);然而, 最近的研究, 纳入实验证据, 导致了一个范例的转变, 我们理解昆虫口器和结构-功能关系的复杂性2,12,13,22,27. 虽然现代成像技术, 如扫描电?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家科学基金会 (NSF) 的资助。IOS 1354956。我们感谢 Dr. 安德鲁 d. 沃伦 (马奎尔昆虫和生物多样性中心, 佛罗里达自然历史博物馆, 佛罗里达大学) 允许使用蝴蝶图像。
Materials
| 20% sucrose solution | Domino Sugar | Sugar needed to produce the sucrose solution with dH2O | |
| Phosphate Buffered Saline (PBS) | Sigma-Aldrich | P5493 | 10X concentration diluted to 1X in dH2O for insect dissections |
| Single depression concave slide | AmScope | BS-C6 | Slide is necessary for feeding stage setup |
| Filter paper | EMD Millipore | NY6004700 (60 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Filter paper | EMD Millipore | NY4104700 (41 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Filter paper | EMD Millipore | NY3004700 (30 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Filter paper | EMD Millipore | NY2004700 (20 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Filter paper | EMD Millipore | NY1104700 (11 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Filter paper | EMD Millipore | TCTP04700 (10 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Filter paper | EMD Millipore | TETP04700 (8 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Filter paper | EMD Millipore | TMTP04700 (5 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Filter paper | EMD Millipore | RTTP04700 (1 µm) | Nylon net filters and isopore filters needed to produce a porous surface for insect feeding |
| Iris microdissecting scissors | Carolina Biological Supply Company | 623555 | Scissors used for dissections |
| Insect pins (#1) | Bioquip Products | 1208B1 | Pins used during dissections and feeding trials |
| Extra-fine point dissecting forceps | Carolina Biological Supply Company | 624684 | Dissecting equipment |
| Leica M205 C Stereoscope | Leica Microsystems | M205 C | Stereoscope used for dissections |
| Inverted confocal microscope | Olympus | IX81 | Fluorescent microscope used to detect magnetic nanoparticles |
| Fisherbrand PTFE Disposable Stir Bar | Fisherscientific | S68067 | Magnet used to detect nanoparticles |
| Kimtech Science Kimwipes | Kimberly-Clark Professional | 34155 | Tissues used to secure insects during feeding trials |
| House fly (Musca domestica) pupae | Mantisplace.com | insects for experiments | |
| Blue bottle fly (Calliphora vomitoria) pupae | Mantisplace.com | insects for experiments | |
| Cabbage butterfly (Pieris rapae) larvae | Carolina Biological Supply Company | 144102 | insects for experiments |
| Finnpipette F1 | ThermoFisher Scientific | 4641080N | micropipette for dispensing liquids |
| Finntip 250 pipette tips | ThermoFisher Scientific | 9400250 | micropipette tips |
| Microscope Glass cover slides (=coverslips) (24 x 24 mm) | AmScope | CS-S24-100 | coverslips for viewing the insect's crop on confocal microscope |
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Citer Cet Article
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