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。

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。
<ol>
	<li>将P. 菜青虫作为来自昆虫供应商的幼虫, 并将它们放在人工饲料中 (见材料表), 直到它们在环境室中蛹和出现时, 设置为23° c 和18升: 6 d 光周期。顺序M。家蝇和C. vomitoria作为蛹, 并在与P. 菜青虫相同的环境条件下将它们后置。在喂食实验之前, 不要喂成年蝶和苍蝇。</li>
	<li>准备一个20% 蔗糖溶液 (控制) 和20% 蔗糖纳米颗粒溶液, 以测试流体摄取量。通过加入荧光磁性纳米微粒 (在直径约 20 nm) 的24到20% 蔗糖溶液 (1 毫克/毫升 dH2O 纳米颗粒与20% 蔗糖) 制备纳米粒子溶液解决方案, 1:1)。准备一个1x 溶液磷酸盐缓冲盐水 (PBS) (10x 稀释到1x 在 dH2O, pH 7.4), 用于解剖。</li>
	<li>设置一个喂食站, 它由一个带有夹具的手动机械手和一个独立的进给工作台 (一个平面平台) (图 1) 组成。在进料台上放置凹滑块, 并有尼龙网过滤器, 其孔径直径为11、20、30、41或60µm, 膜过滤器与孔大小直径为1、5、8或10µm, 用于喂食实验。</li>
</ol>
2. 喂养协议
<ol>
	<li>把昆虫的身体、腿和翅膀裹成一张折叠的纸巾。定位昆虫, 使只有头部和口器暴露。将昆虫的翅膀放在两个显微镜幻灯片之间, 它们由机械手的钳夹在一起, 使昆虫悬浮在喂食台上 (图 1)。</li>
	<li>管理一个30µL 液滴的20% 蔗糖溶液或20% 蔗糖纳米微粒溶液与微的中心凹滑。在凹滑片上放置一个特定孔径的单张滤纸, 使滤纸的中心与纳米微粒溶液的液滴对齐。液滴与滤纸的接触导致溶液沿滤纸扩散, 填充孔隙 (图 1)。 注: 滤纸放在液滴的顶部, 而不是其他的周围, 以尽量减少在昆虫饲料的滤纸上的可能存在的纳米颗粒。</li>
	<li>将昆虫放在机械手的位置, 这样只有口器的远端区域才能接触到进料台上的湿润滤纸 (图 1)。使用昆虫引脚延长口器到滤纸上, 并允许昆虫在四十五年代进食。</li>
	<li>为了尽量减少在滤纸表面可能存在的液体膜上的昆虫, 请放置口器, 使它们与触及幻灯片平坦部分的滤纸的一部分 (i. e) 接触。, 而不是直接在幻灯片的凹部上方)。如果昆虫不表达对哺养的兴趣, 口器可以被拿着对滤纸与昆虫别针在哺养的时间期间。</li>
</ol>
3. 解剖
<ol>
	<li>将 PBS 溶液放在一个50毫米的手表玻璃上, 这样就有足够的解决方法来覆盖昆虫的身体。将手表玻璃置于立体和位置昆虫解剖设备 (弹簧微解剖剪刀, 昆虫针, 细点解剖钳) 旁边的立体。</li>
	<li>喂食后, 将昆虫从纸巾上取出, 并将其用翅膀封住。用弹簧微解剖剪刀将昆虫的头、腿和翅膀移开, 将昆虫放在手表玻璃的 PBS 溶液中 (图 2)。</li>
	<li>如果需要, 在解剖前麻醉昆虫。用钳子把昆虫的表皮靠近腹部的远端。用主手, 使用弹簧微解剖剪刀, 以削减前方向沿侧腹部的角质层, 从后端开始, 直到胸部达到。要特别小心, 确保只有角质层被切断, 并在昆虫的消化道没有损坏 (图 2)。</li>
	<li>用解剖剪刀做更多的角质层切开, 打开腹部, 露出消化道 (图 2)。去除腹部角质层, 脂肪体, 和其他结构的帮助下的昆虫引脚和搬迁他们的手表玻璃以外的后续处置, 只留下胸腔和消化道在手表玻璃。 注意: 解剖将揭示的作物, 这是一个囊状结构 (消化道的延伸) 位于靠近胸腔和腹部的交界处。</li>
	<li>如果作物没有暴露, 用剪刀在胸腔中附加切口直到作物显露出来。一旦作物是可见的, 切去剩下的部分昆虫只留下消化道与作物在手表玻璃 (图 2)。 注: 鳞翅目的作物几乎是透明的和玻璃纸一样的性质, 这可能难以识别, 如果它没有充满液体和扩大或如果它是在解剖切割。</li>
	<li>使用细点解剖钳将作物放置在片 (24 mm x 24 mm) 上进行后续成像 (图 2)。</li>
</ol>
4. 摄入纳米颗粒的测定
<ol>
	<li>将作物放在片上, 用细点解剖钳来防止作物破裂。使用 CY3 通道 (或相衬) 的倒置共焦显微镜成像20X 放大。在解剖后立即对作物进行成像, 以防止其干燥。</li>
	<li>在不控制显微镜操作阶段的手上, 手握一个磁力搅拌杆 (长度为41.3 毫米, 直径8毫米)。</li></ol>在作物附近来回挥动磁性搅拌棒 (距离裁剪大约 10 mm), 使每个来回运动大约需要一秒 (图 2)。
	<li>当磁性搅拌棒被挥动时, 检查庄稼为微粒。在近透明的作物中, 通过显微镜的眼球镜观察, 慢慢地来回移动操作阶段。如果纳米粒子存在于作物中, 表示正向摄食, 它们将响应并与磁性搅拌条 (图 2) 一致地波形。</li>

<ol>
	<li>Vijaysegaran, S., Walter, G. H., Drew, R. A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouthpart+structure,+feeding+mechanisms,+and+natural+food+sources+of+adult+Bactrocera+(Diptera:+Tephritidae).">Mouthpart structure, feeding mechanisms, and natural food sources of adult Bactrocera (Diptera: Tephritidae).</a> Ann Entomol Soc Am. 90, 184-201 (1997).</li><li>Lehnert, M. S., Monaenkova, D., Andrukh, T., Beard, C. E., Adler, P. H., Kornev, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic-hydrophilic+dichotomy+of+the+butterfly+proboscis.">Hydrophobic-hydrophilic dichotomy of the butterfly proboscis.</a> J R Soc Interface. 10, 1-10 (2013).</li><li>Zhao, J., Wu, J., Yan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erection+mechanism+of+glossal+hairs+during+honeybee+feeding.">Erection mechanism of glossal hairs during honeybee feeding.</a> J Theor biol. 386, 62-68 (2015).</li><li>Redak, R. A., Purcell, A. H., Lopes, J. R. S., Blua, M. J., Mizell, R. F., Andersen, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+xylem+fluid-feeding+insect+vectors+of+Xylella+fastidiosa+and+their+relation+to+disease+epidemiology.">The biology of xylem fluid-feeding insect vectors of Xylella fastidiosa and their relation to disease epidemiology.</a> Ann. Review Entomol. 49, 243-270 (2004).</li><li>B&#252;ttiker, W., Krenn, H. W., Putterill, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proboscis+of+eye-frequenting+and+piercing+Lepidoptera+(Insecta).">The proboscis of eye-frequenting and piercing Lepidoptera (Insecta).</a> Zoomorphology. 116, 77-83 (1996).</li><li>Light, J. E., Smith, V. S., Allen, J. M., Durden, L. A., Reed, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+history+of+mammalian+sucking+lice+(Phthiraptera:+Anoplura).">Evolutionary history of mammalian sucking lice (Phthiraptera: Anoplura).</a> BMC Evol Biol. 10, (2010).</li><li>Krenn, H. W., Aspock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form,+function+and+evolution+of+the+mouthparts+of+blood-feeding+Arthropoda.">Form, function and evolution of the mouthparts of blood-feeding Arthropoda.</a> Arthropod Struct Dev. 41, 101-118 (2012).</li><li>Lehnert, M. P., Pereira, R. M., Koehler, P. G., Walker, W., Lehnert, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Cimex+lectularius+using+heat+combined+with+dichlorvos+resin+strips.">Control of Cimex lectularius using heat combined with dichlorvos resin strips.</a> Med Vet Entomol. 25, 460-464 (2011).</li><li>Zaspel, J. M., Kononenko, V. S., Goldstein, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+blood+feeder?+Experimental+feeding+of+a+fruit-piercing+moth+species+on+human+blood+in+the+Primorye+Territory+of+far+eastern+Russia+(Lepidoptera:+Noctuidae:+Calpinae).">Another blood feeder? Experimental feeding of a fruit-piercing moth species on human blood in the Primorye Territory of far eastern Russia (Lepidoptera: Noctuidae: Calpinae).</a> J Insect Behav. 20, 437-451 (2007).</li><li>Barth, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insects and flowers: the biology of a partnership. , (1991).</li><li>Foil, L. D., Adams, W. V., McManus, J. M., Issel, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bloodmeal+residues+on+mouthparts+of+Tabanus+fuscicostatus+(Diptera:+Tabanidae)+and+the+potential+for+mechanical+transmission+of+pathogens.">Bloodmeal residues on mouthparts of Tabanus fuscicostatus (Diptera: Tabanidae) and the potential for mechanical transmission of pathogens.</a> J Med Entomol. 24, 613-616 (1987).</li><li>Monaenkova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butterfly+proboscis:+combining+a+drinking+straw+with+a+nanosponge+facilitated+diversification+of+feeding+habits.">Butterfly proboscis: combining a drinking straw with a nanosponge facilitated diversification of feeding habits.</a> J R Soc Interface. 9, 720-726 (2012).</li><li>Lehnert, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouthpart+conduit+sizes+of+fluid-feeding+insects+determine+the+ability+to+feed+from+pores.">Mouthpart conduit sizes of fluid-feeding insects determine the ability to feed from pores.</a> Proc. R. Soc. B. 284, (2017).</li><li>Grimaldi, D., Engel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evolution of the insects. , (2005).</li><li>Adler, P. H., Foottit, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect biodiversity: science and society. , (2009).</li><li>Tsai, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoporous+artificial+proboscis+for+probing+minute+amount+of+liquids.">Nanoporous artificial proboscis for probing minute amount of liquids.</a> Nanoscale. 3, (2011).</li><li>Krenn, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+sensilla+in+Vanessa+cardui+(Nympahlidae,+Lepidoptera):+Functional+morphology+and+significance+of+flower-probing.">Proboscis sensilla in Vanessa cardui (Nympahlidae, Lepidoptera): Functional morphology and significance of flower-probing.</a> Zoomorphology. 118, 23-30 (1998).</li><li>Plateau, J. A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+researches+on+the+figures+of+equilibrium+of+liquid+mass+withdrawn+from+the+action+of+gravity.+(Transl).">Experimental and theoretical researches on the figures of equilibrium of liquid mass withdrawn from the action of gravity. (Transl).</a> Annual Report of the Board Regents Smithsonian Institution. , 207-285 (1863).</li><li>Socha, J. J., Westneat, M. W., Harrison, J. F., Waters, J. S., Lee, W. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+phase-contrast+x-ray+imaging:+a+new+technique+for+the+study+of+animal+form+and+function.">Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function.</a> BMC Biol. 5, 6 (2007).</li><li>Westneat, M. W., Socha, J. J., Lee, W. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+biological+structure,+function+and+physiology+using+synchrotron+x-ray+imaging.">Advances in biological structure, function and physiology using synchrotron x-ray imaging.</a> Annu Rev Physiol. 70, 119-142 (2008).</li><li>Lee, W. -. K., Socha, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+hemolymph+flow+in+the+heart+of+a+grasshopper+(Schistocerca+americana).">Direct visualization of hemolymph flow in the heart of a grasshopper (Schistocerca americana).</a> BMC Physiology. 9, 2 (2009).</li><li>Lehnert, M. S., Mulvane, C. P., Brother, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouthpart+separation+does+not+impede+butterfly+feeding.">Mouthpart separation does not impede butterfly feeding.</a> Arthropod Struct Dev. 43, 97-102 (2014).</li><li>Lehnert, M. S., Beard, C. E., Gerard, P. D., Kornev, K. G., Adler, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+lepidopteran+proboscis+in+relation+to+feeding+guild.">Structure of the lepidopteran proboscis in relation to feeding guild.</a> J Morphol. 277, 167-182 (2016).</li><li>Yan, H., Sung, B., Kim, M. -. H., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+for+functionalizable+photoluminescent+magnetic+nanoparticles.">A novel strategy for functionalizable photoluminescent magnetic nanoparticles.</a> Mater. Res. Express. 1, 045032 (2014).</li><li>Kingsolver, J. G., Daniel, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanics+and+energetics+of+nectar+feeding+in+butterflies.">On the mechanics and energetics of nectar feeding in butterflies.</a> J Theor Biol. 76, 167-179 (1979).</li><li>Krenn, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+mechanisms+of+adult+Lepidoptera:+Structure,+function,+and+evolution+of+the+mouthparts.">Feeding mechanisms of adult Lepidoptera: Structure, function, and evolution of the mouthparts.</a> Ann Rev Entomol. 55, 307-327 (2010).</li><li>Tsai, C. -. C., Monaenkova, D., Beard, C. E., Adler, P. H., Kornev, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradox+of+the+drinking-straw+model+of+the+butterfly+proboscis.">Paradox of the drinking-straw model of the butterfly proboscis.</a> J Exp Biol. 217, 2130-2138 (2014).</li><li>Bauder, J. A. S., Handschuh, S., Metscher, B. D., Krenn, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+morphology+of+the+feeding+apparatus+and+evolution+of+proboscis+length+in+metalmark+butterflies+(Lepidoptera:+Riodinidae).">Functional morphology of the feeding apparatus and evolution of proboscis length in metalmark butterflies (Lepidoptera: Riodinidae).</a> Biol J Linn Soc. 110, 291-304 (2013).</li></ol>

作者没有什么可透露的。

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?...

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。

<table>
	<tbody>
		<tr>
			<td>20% sucrose solution</td>
			<td>Domino Sugar</td>
			<td></td>
			<td>Sugar needed to produce the sucrose solution with dH2O</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10X concentration diluted to 1X in dH2O for insect dissections</td>
		</tr>
		<tr>
			<td>Single depression concave slide</td>
			<td>AmScope</td>
			<td>BS-C6</td>
			<td>Slide is necessary for feeding stage setup</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY6004700 (60 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY4104700 (41 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY3004700 (30 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY2004700 (20 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>NY1104700 (11 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TCTP04700 (10 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TETP04700 (8 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>TMTP04700 (5 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>EMD Millipore</td>
			<td>RTTP04700 (1 &#181;m)</td>
			<td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td>
		</tr>
		<tr>
			<td>Iris microdissecting scissors</td>
			<td>Carolina Biological Supply Company</td>
			<td>623555</td>
			<td>Scissors used for dissections</td>
		</tr>
		<tr>
			<td>Insect pins (#1)</td>
			<td>Bioquip Products</td>
			<td>1208B1</td>
			<td>Pins used during dissections and feeding trials</td>
		</tr>
		<tr>
			<td>Extra-fine point dissecting forceps</td>
			<td>Carolina Biological Supply Company</td>
			<td>624684</td>
			<td>Dissecting equipment</td>
		</tr>
		<tr>
			<td>Leica M205 C Stereoscope</td>
			<td>Leica Microsystems</td>
			<td>M205 C</td>
			<td>Stereoscope used for dissections</td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td>Fluorescent microscope used to detect magnetic nanoparticles</td>
		</tr>
		<tr>
			<td>Fisherbrand PTFE Disposable Stir Bar</td>
			<td>Fisherscientific</td>
			<td>S68067</td>
			<td>Magnet used to detect nanoparticles</td>
		</tr>
		<tr>
			<td>Kimtech Science Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34155</td>
			<td>Tissues used to secure insects during feeding trials</td>
		</tr>
		<tr>
			<td>House fly (Musca domestica) pupae</td>
			<td>Mantisplace.com</td>
			<td></td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Blue bottle fly (Calliphora vomitoria) pupae</td>
			<td>Mantisplace.com</td>
			<td></td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Cabbage butterfly (Pieris rapae) larvae</td>
			<td>Carolina Biological Supply Company</td>
			<td>144102</td>
			<td>insects for experiments</td>
		</tr>
		<tr>
			<td>Finnpipette F1&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>4641080N</td>
			<td>micropipette for dispensing liquids</td>
		</tr>
		<tr>
			<td>Finntip 250 pipette tips</td>
			<td>ThermoFisher Scientific</td>
			<td>9400250</td>
			<td>micropipette tips</td>
		</tr>
		<tr>
			<td>Microscope Glass cover slides (=coverslips) (24 x 24 mm)</td>
			<td>AmScope</td>
			<td>CS-S24-100</td>
			<td>coverslips for viewing the insect&#39;s crop on confocal microscope</td>
		</tr>
	</tbody>
</table>

the ingestion of fluorescent, magnetic nanoparticles for determining fluid-uptake abilities in insects

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。

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?...

Watch this Scientific Journal Video about The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects at JoVE.com

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

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。

吸收荧光、磁性纳米微粒测定昆虫的吸液能力

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

the-ingestion-fluorescent-magnetic-nanoparticles-for-determining

Research

JoVE Journal

Biology

6.3K 次观看.  Kent State University at Stark. 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。

视频: The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.

Plant resistance to chewing insect herbivores can be tested in several ways. Here, we demonstrate how to set-up a choice and a no-choice experiment with the model plant Arabidopsis thaliana to identify resistance against the pest species Pieris rapae.

测试拟南芥耐咀嚼昆虫的选择和非选择性检测

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

科研

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

确定的能源系统，在运动的贡献

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

从白蛉提取内分泌物的方法<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

双正电子发射层析成像用 68 ga 芯掺杂氧化铁纳米粒子的合成/(t1) 磁共振成像

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

昆虫关键热极限的高通量测定

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...