Name: harvesting venom toxins from assassin bugs and other heteropteran insects
Uploaded: 2018-04-21T13:00:00.000+00:00
Duration: 9 min 45 s
Description: Watch this Scientific Journal Video about Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects at JoVE.com

我们承认澳大利亚研究理事会的财政支持 (赠款 DP130103813 和 LP140100832 G.F.K., DECRA 奖学金 DE160101142 EABU), 澳大利亚国家健康 & 医学研究理事会 (主要研究金APP1044414 到 G.F.K.) 和昆士兰大学 (博士后奖学金 A.A.W.)。

该议定书符合昆士兰大学在教学和研究中负责照料和使用动物的政策 (4.20.11) 以及国家卫生和医学研究理事会的澳大利亚保护和使用动物为科学目的(8th编辑 2013)。
注意: 在处理刺客 bug 时, 注意不要被 envenomated。在处理毒液防御的物种时, 要注意保护眼睛。注意不要伤害实验动物。这包括监测压力的限制, 如橡胶带和确保喙没有打破。
注: 有选择地, 麻醉动物通过接触 CO2为 0.5-2 分钟或冷却到4-10 °c 之前为目标1-3 的毒液收获促进安全转移和克制。Anaesthetization 不是严格的要求, 但可能有助于安全约束敏捷或强大的标本。然而, 动物必须是清醒的, 以允许毒液收割。在决定是否添加蛋白酶抑制剂时, 请牢记下游应用。
1. 刺激收集毒液毒素
<ol><li>获得活标本, 从中收获毒素。</li>
	<li>使用预先准备好的塑料镊子, 正负电极安装在任一尖端。将电气化镊子连接到 electrostimulator 或稳压电源, 以允许调整电压。<ol>
<li>对于小 (~ 10 毫米) 和大 (~ 25 毫米) 刺客臭虫, 使用峰值电压分别为15和 25 V。</li>
		<li>对于较大的 heteropterans, 如巨型水虫, 使用多达 40 v。</li>
	</ol>
</li>
	<li>通过在胸腔上使用橡皮筋将它们绑在平台上, 来抑制活虫。</li>
	<li>将喙尖放在适当的收集尖端。对于刺客臭虫, 使用 P200 吸管尖端。对于巨大的水虫, 切断 P200 尖端的肢体, 以增加光圈的大小。<ol>
<li>用闭合的清洁镊子轻轻举起喙, 将收集尖端的开口孔推到喙的末端。</li>
		<li>如果需要, 吸收5µL 超纯水之前, 把喙放入收集尖端。这减少了毒液残留在尖端的损失, 虽然收获的毒液将被稀释。</li>
	</ol>
</li>
	<li>应用刺激。将刺激电极浸在导电凝胶中, 如2.5 米 NaCl/50%glycerol。将电极应用于胸腔。对于 belostomatids, 将两个电极应用于头部后表面。</li>
	<li>贮存毒液以防止 autodegradation。在毒液被挤出后, 迅速将其转移到-20 摄氏度或-60 摄氏度的管子上, 或含有蛋白酶抑制剂鸡尾酒的管子。</li>
	<li>重复步骤1.5 和 1.6, 直到获得足够的毒液或没有进一步的毒液即将到来。</li>
</ol>2. 通过骚扰收集毒液毒素
<ol><li>准备动物的毒液收割和放置在收集前庭的喙尖, 如第1.1 和 1.3-1.4 所述。</li>
	<li>如果毒液自发地挤压, 请转到步骤2.3。如果没有, 用镊子轻轻抚摸它的腿, 腹部和触角, 直到毒液产生时, 骚扰动物。</li>
	<li>如果需要, 迅速将毒液转移到-20 摄氏度或-60 摄氏度的管子, 或含有蛋白酶抑制剂鸡尾酒的管子。</li>
</ol>3. 从毒液 "随地吐痰" 种的骚扰中获取毒液毒素
<ol><li>麻醉, 或部分麻醉, 在将昆虫从其外壳中除去之前, 防止任何过早的防御性吐痰。</li>
	<li>引发毒液吐痰行为。使用标准的 90 x 16 毫米培养皿的深盖子来控制和重新定位昆虫。把盖子稍后, 1-4 厘米以上的昆虫, 以防止飞行。大多数昆虫会随地吐痰几次, 往往是快速的连续。确保在盘子的底部收集所有毒液。</li>
	<li>用10µL 的超纯水冲洗培养皿底部的毒液。迅速将其转移到管在-20 °c 或-60 °c, 或一管含有蛋白酶抑制剂鸡尾酒。</li>
</ol>4. 通过腺体解剖收集毒液毒素
<ol><li>牺牲动物。严重麻醉或杀死动物使用 > 5 分钟暴露到 CO2。管道纯 CO2直接插入到动物外壳外壳的空气孔中。</li>
	<li>针昆虫到解剖托盘。对于刺客臭虫, 解剖通过腹面 (4.3)。对于巨大的水虫, 解剖通过背部表面 (4.4)。</li>
	<li>腹壁解剖<ol>
<li>插入三针到后腹部, 以保持昆虫下来, 不刺穿毒液腺体。</li>
		<li>用微型手术刀切开腹部腹面的短中线切口。使用微型剪刀将中线切口前方到头部, 注意切开外骨骼, 不损伤内部结构。</li>
		<li>为了暴露内部结构, 使多个横向切口延伸从中线切开到昆虫的侧面。然后, 把每个腹外骨骼的皮瓣钉回, 以揭示内部结构。</li>
		<li>对于大型刺客臭虫, 做四侧向切口, 在中腹部, 前腹部, 在第一和第二腿之间, 并且在第一条腿前面。</li>
	</ol>
</li>
	<li>背夹层<ol>
<li>移除底座附近的机翼。插入三针到后腹部, 以保持昆虫下来, 不刺穿毒液腺体。</li>
		<li>用微型剪刀和手术刀切开从头部到腹部的中线切口, 注意切开外骨骼, 而不是损伤内部结构。</li>
		<li>把昆虫的两半分开。沿昆虫的长度横向放置几个引脚, 使内腔暴露。</li>
		<li>使用镊子去除飞行肌肉。</li>
	</ol>
</li>
	<li>把解剖托盘给淹了。添加 PBS, 直到该 bug 被淹没, 以允许内部结构浮动和更容易可视化。</li>
	<li>使用镊子和微剪刀, 小心地去除结缔组织和神经组织和气管。毒液腺体呈拉长的、半透明的结构, 沿消化道的两侧延伸。<ol>
<li>以其特征外观识别主要腺体, 前后裂片和两个导管会在门。</li>
		<li>如果需要, 通过从门中追踪管道来识别副腺体。通过切割来自门的两个导管, 释放主腺体。</li>
	</ol>
</li>
	<li>收获所需的腺体流明。将腺体转移到含有30µL pbs 或 pbs 加蛋白酶抑制剂鸡尾酒的冰上的离心。用干净锋利的别针把腺体长矛。<ol>
<li>漩涡为十年代和离心机 (1 分钟, 5000 x g, 4 °c) 空的腺体流明。用镊子去除腺组织。</li>
	</ol>
</li>
	<li>澄清毒素提取物。离心机 (5 分钟, 1.7万 x 克, 4°c) 去除任何固体颗粒, 保留上清和丢弃颗粒。贮存在20°c 或-60 摄氏度, 以防止 autoproteolytic 降解。</li>
</ol>

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作者没有什么可透露的。

捕获刺客臭虫毒液最关键的一步是根据研究的目的选择合适的方法。三种收集 heteropteran 毒液的方法中, 每种都有优缺点, 这取决于下游的应用。
诱导臭虫从喙中排出毒液 (1-3 协议) 避免了腺组织对毒液的污染。此外, 这些方法是非致命的, 可以重复多次在一个 bug 的生活过程中。刺激通常提供最大数量的毒液, 并产生毒液具有强大的毒性对猎物昆虫根据几个研究5</s...

Heteropteran 毒液是完全生物活性物质1。例如, 血液喂养翅的毒液/唾液分泌物, 如接吻臭虫 (Triatominae) 和床虫 (Cimicidae) 通过扰乱止血2来促进喂养。这些毒液中的毒素靶向多个途径, 包括凝血、血小板聚集和血管收缩, 以及疼痛和瘙痒通路。大多数其他 heteropteran 物种的毒液都适于捕食, 而不是供血。他们的毒液导致瘫痪, 死亡和组织液化时, 注入无脊椎动物3,4。当注入脊椎动物时, 它们的毒液也可能产生剧烈的影响。例如, 从刺客 bug Holotrichius innesi中注入毒液会导致疼痛、肌肉麻痹和出血;由于呼吸麻痹5, envenomated 的老鼠很快就死了。
Transcriptomic 和蛋白质组学研究揭示了一些 heteropteran 毒液的蛋白质组成。捕食性天敌种类的毒液富含蛋白酶、其他酶, 以及未知结构和功能的多肽和蛋白质6, 7, 8.接吻臭虫毒液富含 triabin 蛋白家族, 其成员对凝血、血小板聚集和收缩29产生深刻影响。然而, 它不知道哪些毒素的基础上最具生物活性的毒液。例如, 接吻 bug Triatoma 疫病的毒液被报告为止痛药和抑制钠通道10, 但负责的组件仍有待阐明。同样, 还不知道刺客臭虫毒液的成分会导致麻痹或疼痛。确定对特定毒液生物活性负责的毒素, 以及表征新型毒液毒素的结构和功能的先决条件是获取毒液。
毒液已经从 heteropterans 获得了由刺激5,6,7,8,11,12,13, 挑衅防御响应4,8, 机械地压缩胸部12,14,15,16, 剖析毒液腺体8,17 、18、19、20、21、22和毒蕈乙酰胆碱受体的激动剂23的应用。判断任何方法的潜在优点和缺点是复杂的 heteropteran 毒液腺体的形态学, 它由一个主腺体与两个独立的流明, 前主腺体 (AMG) 和后主腺体 (普纳), 以及伴生的附属的腺体 (AG)。这些不同的腺体间隔产生不同的蛋白质分泌物, 这可能是专门用于不同的生物功能, 包括猎物捕获, 防御和额外口服消化8,17。在 peiratine 和 ectrichodiine 刺客臭虫, AMG 已经与猎物捕获和普纳的额外口服消化17。但是, 在 harpactorine bug Pristhesancus plagipennis中, 普纳专门用于捕获和消化, 而 AMG 则被假定为分泌防御毒液8。该 AG 已被描述为在刺客 bug8或作为一个主要的地方蛋白酶存储在巨型水臭虫23中的分泌功能很少。显然, 需要进一步的工作来澄清各个 heteropteran 子群之间各腺体间的功能, 并确定大多数毒液毒素的功能。在本报告中, 我们描述了从 heteropterans 到这个目标的收集毒液毒素的协议。

<table><tbody><tr><td>Electostimulator</td><td>Grass Technologies</td><td>S48 Square Pulse Stimulator</td><td>Electrostimulator allowing pulsed electrostimulation</td></tr><tr><td>Featherlight tweezers</td><td>Australian Entomological Supplies</td><td>E122B</td><td>For handling live venomous insects</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>4693124001</td><td>For preventing autoproteolytic digestion of venom</td></tr><tr><td>Dissection equipment</td><td>Australian Entomological Supplies</td><td>E152Micro</td><td>For fine dissections</td></tr><tr><td>Insect pins</td><td>Australian Entomological Supplies</td><td>E162</td><td>For fine dissections</td></tr></tbody></table>

harvesting venom toxins from assassin bugs and other heteropteran insects

Heteropteran 昆虫, 如刺客臭虫 (Reduviidae) 和巨大的水虫 (Belostomatidae) 从一个共同的捕食性天敌和恶毒的祖先后裔, 和大多数现存的 heteropterans 保留这一营养战略。一些 heteropterans 已经转变为喂养脊椎动物的血液 (如接吻臭虫, Triatominae; 和床虫, Cimicidae), 而另一些已经恢复到植物喂养 (大多数 Pentatomomorpha)。然而, 除了接吻虫子用来促进血液喂养的唾液之外, heteropteran 毒液与蜘蛛、蝎子和蛇的毒液相比, 很少被知晓。
heteropteran 毒液毒素特征的一个障碍是毒液/唇腺的结构和功能, 它们既形态复杂, 又具有多种生物作用 (防御、猎物捕获和超口服消化)。在本文中, 我们描述了三种方法, 我们已经成功地用于收集 heteropteran 毒液。首先, 我们提出刺激作为一种方便的方法收集毒液, 这往往是致命的, 当注射到猎物的动物, 并省却污染的腺组织。第二, 我们表明, 对动物的温柔骚扰足以产生毒液挤压从喙和/或毒液随地吐痰的一些群体的 heteropterans。第三, 我们描述的方法, 以收集毒液毒素的麻醉动物的解剖, 以获得毒液腺体。这种方法与其他方法相辅相成, 因为它可能允许从刺激和骚扰无效的分类群中收集毒素。这些协议将使研究人员能够从 heteropteran 昆虫身上获得毒素, 用于结构功能表征和在医药和农业中的可能应用。

某些 heteropteran 物种 (如 harpactorine P plagipennis和 reduviine Platymeris rhadamanthus) 可靠地产生大量 (5-20 µL) 的毒液, 以响应刺激 (表 1)。一般而言, 大多数 peiratine、reduviine 和 harpactorine bug 会产生毒液来响应这种方法。在 stenopodaine bug 中, 刺激从Oncocephalus sp 中引发毒液, 而不是Thodelmus sp。取样的 holoptiline 和 emesine bug 并没有产生显著的毒液 (?...

Watch this Scientific Journal Video about Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects at JoVE.com

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

虽然亚目翅 (昆虫纲: 半翅目) 中的许多昆虫是有毒的, 但它们的毒液成分和毒液毒素的作用大多是未知的。本议定书描述了利用刺激、骚扰和腺体解剖来收获 heteropteran 毒液以进一步表征的方法。

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

harvesting-venom-toxins-from-assassin-bugs-other-heteropteran

Universidad San Sebastian

Research

JoVE Journal

Environment

13.0K 次观看.  The University of Queensland. 虽然亚目翅 (昆虫纲: 半翅目) 中的许多昆虫是有毒的, 但它们的毒液成分和毒液毒素的作用大多是未知的。本议定书描述了利用刺激、骚扰和腺体解剖来收获 heteropteran 毒液以进一步表征的方法。

视频: Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Helminth Collection and Identification from Wildlife

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), &#34;thorny-headed worms&#34; (acanthocephalans), &#34;flukes&#34; (trematodes), and &#34;tapeworms&#34; (cestodes). The optimum method for collecting helminths is to examine a host that has been dead less than 4-6 hr since most helminths will still be alive. A thorough necropsy should be conducted and all major organs examined. Organs are washed over a 106 &#956;m sieve under running water and contents examined under a stereo microscope. All helminths are counted and a representative number are fixed (either in 70% ethanol, 10% buffered formalin, or alcohol-formalin-acetic acid). For species identification, helminths are either cleared in lactophenol (nematodes and small acanthocephalans) or stained (trematodes, cestodes, and large acanthocephalans) using Harris&#39; hematoxylin or Semichon&#39;s carmine. Helminths are keyed to species by examining different structures (e.g. male spicules in nematodes or the rostellum in cestodes). The protocols outlined here can be applied to any vertebrate animal. They require some expertise on recognizing the different organs and being able to differentiate helminths from other tissue debris or gut contents. Collection, preservation, and staining are straightforward techniques that require minimal equipment and reagents. Taxonomic identification, especially to species, can be very time consuming and might require the submission of specimens to an expert or DNA analysis.

Wild animals are commonly parasitized by a wide range of helminths.&#160; The four major types of helminths are &#8220;roundworms&#8221; (nematodes), &#8220;thorny-headed worms&#8221; (acanthocephalans), &#8220;flukes&#8221; (trematodes), and &#8220;tapeworms&#8221; (cestodes).&#160; Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically identified.&#160;

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), ...

科研

环境科学

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

重金属和微藻的产其他无机污染物的量化

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

测定掠夺取食行为<em&gt; Pristionchus</em&gt;及其他线虫

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

提取和表面活性剂的特性，从大气气溶胶

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Trichogramma dendrolimi 和毒液提取的人工宿主寄生

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

作者聚焦：Trichogramma 寄生黄蜂的高效毒液提取方法 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

生物化学

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

显微切割的黑寡妇蜘蛛丝绸生产腺体

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

生物学

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

摘要

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此视频中的章节

Helminth Collection and Identification from Wildlife

重金属和微藻的产其他无机污染物的量化

测定掠夺取食行为

从塑料颗粒中提取有机氯农药和塑料类型分析

提取和表面活性剂的特性，从大气气溶胶

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

使用人工宿主从寄生蜂 Trichogramma dendrolimi 中提取毒液

显微切割的黑寡妇蜘蛛丝绸生产腺体

提取毒液和毒腺Microdissections从蜘蛛的蛋白质组学和转录组分析