Name: a precise and quantifiable method for collecting hemolymph from small arthropods
Uploaded: 2023-04-28T12:00:00.000+00:00
Duration: 3 min 39 s
Description: Watch this Scientific Journal Video about A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods at JoVE.com

这项工作得到了国家重点研发计划（编号：2022YFD1401700）和国家自然科学基金（编号：32090013和32072385）的支持。

1. 昆虫饲养
<ol><li>提高本实验中使用的水稻幼苗（Oryza sativa cv. Japanbonbare）的SBPH。在培养箱（65mm x 200mm）中种植20棵水稻幼苗，并在16小时光照/ 8小时黑暗光周期下在25°C下生长。</li>
</ol>2. 解剖 SBPH 以收集血淋巴
<ol><li>将SBPH放入离心管中，并将它们置于冰浴中10-30分钟。 注意：不要将SBPH放入冰浴中少于10分钟，否则昆虫可能会复活。</li>
	<li>将冷冻的SBPH放在立体显微镜下的载玻片（见材料表）上，其腹部朝上。调整昆虫六条腿的焦点。</li>
	<li>准备两个带有超细尖端的高精度镊子（见 材料表）。用一把镊子压在昆虫的身体上以使其保持在原位，然后用另一把镊子小心地将一条腿从昆虫身上拉下来。 注意：对于硬壳昆虫，可以使用眼科手术刀切断其中一条腿。</li>
	<li>用镊子轻轻按压昆虫的胸部，使血淋巴通过伤口流出。 注意：血淋巴表现为透明飞沫，没有任何可见的白色絮状物。如果有任何白色絮凝物，则丢弃脂质，这代表脂肪体污染。</li>
</ol>3. 使用微量移液器收集血淋巴
<ol><li>准备微量移液器。将体积为1μL的毛细管（见材料表）放入移液器灯泡中（见材料表）。为了进行准确的测量，请确保刻度线可见。 注意：移液器灯泡顶部的一个小孔是必要的。</li>
	<li>收集血淋巴时，握住微量移液器的移液器灯泡，并将毛细管靠近昆虫伤口。通过简单地将毛细血管的尖端接触到血淋巴来收集从伤口渗出的血淋巴。</li>
	<li>当毛细管内的液体达到所需的刻度线时，用手指稍微堵住移液器灯泡顶部的小孔，以停止吸收过程。 注意：连续快速地收集一个 1 μL 血淋巴样本，以避免脂质污染和管堵塞。</li>
	<li>按下移液器灯泡，将收集的血淋巴液排出到 100 μL PBS 缓冲液中（137 毫摩尔/升 NaCI、2.7 毫摩尔/升 KCI、4.3 毫摩尔/升钠 2 HPO 4、1.4 毫摩尔/升KH2 PO 4，pH 7.2-7.4）。 注意：毛细管插入PBS缓冲液中。</li>
	<li>收集另外 1 μL 血淋巴样品时，更换为新的毛细管。</li>
</ol>4. 考马斯蓝染色
<ol><li>根据步骤3中描述的方案从3期幼虫收集3个相同体积（1μL）的血淋巴样品，并将收集的血淋巴排出到PBS缓冲液中，使总体积为100μL。</li>
	<li>将 25 μL 5x 蛋白质上样缓冲液（250 mmol/L Tris-HCI [pH 6.8]、10% W/V SDS、0.05% W/V 溴酚蓝、50% W/V 甘油、5% W/V β-巯基乙醇）加入到 100 μL 稀释的血淋巴样品中，加热使蛋白质变性。</li>
	<li>将 20 μL 煮沸的样品上样到 10% SDS-PAGE 蛋白质凝胶（参见 材料表）上以分离蛋白质。</li>
	<li>将凝胶染色在考马斯蓝溶液中（将1g考马斯亮蓝R-250与250mL异丙醇，100mL冰醋酸和650mLddH2O混合，并使用滤纸过滤掉任何颗粒）在室温下1小时，然后用脱色溶液（100mL乙酸， 400 mL 甲醇，500 mLddH 2O）。</li>
</ol>5. 蛋白质浓度测定
<ol><li>使用两倍稀释法将 10 mg/mL 牛血清白蛋白 （BSA） 稀释至 5 mg/mL、2.5 mg/mL、1.25 mg/mL、0.625 mg/mL 和 0.3125 mg/mL。</li>
	<li>在步骤5.1中吸出5μLPBS缓冲液和每种浓度的BSA溶液，加入1mL的1x布拉德福德染料试剂（参见 材料表），并在室温下孵育5分钟。使用布拉德福德染料试剂中的PBS缓冲液作为对照，OD = 595nm时为零。测量每种剩余蛋白质浓度的吸光度值，并制作标准曲线。</li>
	<li>将来自幼虫、雌性或雄性 SBPH 的 1 μL 血淋巴液加入 100 μL PBS 缓冲液中，然后在 4 °C 下以 1，000 x g 离心样品 10 分钟以除去血细胞。</li>
	<li>将90μL上清液吸入新的离心管中，并根据步骤5.2中的说明测量上清液的吸光度值。</li>
	<li>根据标准曲线的回归方程计算血淋巴样品的蛋白质浓度。在实验中包括三个生物学重复和三个技术重复。</li>
</ol>6. 显微检测
<ol><li>使用微量移液器收集不同发育阶段的10-15 SBPH的血淋巴。将收集的血淋巴液加入含有20μL4%多聚甲醛溶液的管中（参见 材料表）。将混合物在室温下孵育30分钟以固定血淋巴。</li>
	<li>将20μL固定的血细胞移液到涂有硅烷的载玻片的中心（参见 材料表）。</li>
	<li>在载玻片晾衣架中干燥液滴。 注意：避免过度干燥。</li>
	<li>用金抗淬灭试剂4'，6-二脒基-2-苯基吲哚（DAPI）覆盖样品（见材料表），并在倒置显微镜下检查载玻片（见材料表）。</li>
</ol>7. 细胞定量
<ol><li>用微量移液器从SBPH中收集1μL血淋巴，并将其溶解到20μLPBS缓冲液中。</li>
	<li>用PBS缓冲液将血淋巴样品稀释五倍。</li>
	<li>将20μL血淋巴溶液移液到细胞计数室的中心（参见 材料表）。用盖玻片盖住液滴（见 材料表）。</li>
	<li>在倒置显微镜下对细胞计数室（补充图1）上的五个方块中的细胞进行计数（参见 材料表）。在每个实验中包括三个生物学重复和三个技术重复。 细胞/μL = 5 个中间平方的总计数 × 5 × 稀释因子 × 104</li>
</ol>8. 统计分析
<ol><li>使用相应的软件进行统计分析（见 材料表）。创建一个新文件，选择列，导入数据，然后生成带有条形的散点图。</li>
	<li>使用列统计量计算每组数据的平均值和 SD，并使用单因子方差分析工具评估组间差异的显著性。通过三个独立实验从三个生物学重复中确定平均值和SD。</li>
</ol>

从小型节肢动物中收集血淋巴

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血淋巴是节肢动物循环系统的介质，虫媒病毒只有在恶劣的血淋巴环境中生存下来，才能侵入其他节肢动物组织。收集高质量的血淋巴样本是研究血淋巴中发生的载体-病毒相互作用的第一步。据报道，昆虫血淋巴可以从昆虫身体的几个部位获得，包括前腿上的伤口、头部区域的小切口或腹部的撕裂伤口26、27、28、<sup class="xref"...

动植物病毒都可以通过节肢动物传播，这些病毒对人类健康构成严重威胁，给农业造成巨大的经济损失1，2，3。重要的是，节肢动物血淋巴作为节肢动物的循环系统和免疫系统的重要组成部分，在调节虫媒病毒传播方面起着重要作用。通过节肢动物肠道获得的病毒只有在成功逃脱不良血淋巴环境后才会转运到其他组织4，5，6，7。节肢动物血淋巴中病毒的生命周期涉及病毒在血浆中存活、进入血细胞并转运到其他组织，血淋巴中发生各种病毒-载体相互作用机制8、9、10、11、12。例如，SBPH 对 RSV 的垂直传播取决于 SBPH 卵黄素蛋白和 RSV（米条纹病毒）衣壳蛋白13，14 之间的分子相互作用。一些病毒可能通过结合特定的载体因子15，16，17，18来逃避血淋巴的免疫反应。因此，研究节肢动物血淋巴中的载体-病毒相互作用对于更好地了解虫媒病毒传播非常重要。
一些小昆虫的血淋巴，如飞虱、叶蝉和一些蚊子，由于其体型的原因，很难收集。为了解决这个问题，已经开发了几种收集血淋巴的方法，包括将注射器针头直接插入昆虫体内以提取微量的血淋巴，用细尖镊子从伤口部位收集渗出物，以及直接离心。这些方法能够测量血淋巴内的相对基因表达水平和病毒滴度19，20，21。然而，对于这些小昆虫，目前还没有一种有效的血淋巴体积定量方法，这是血细胞计数、蛋白质定量和酶活性分析所必需的。
SBPH（小棕色飞虱）是一种体长约2-4毫米的小型昆虫媒介。SBPH能够传播多种植物病毒，包括RSV，玉米粗矮病毒和水稻黑条纹矮病毒22，23，24。在过去十年中，对SBPH和RSV之间的相互作用进行了深入研究。为了便于使用SBPH，我们开发了一种新颖而简单的血淋巴收集方法。这种方法基于毛细管力原理，使用带有刻度标记的毛细管以精确和可量化的方式获取昆虫的血淋巴。这使我们能够有效地从小昆虫中收集特定体积的血淋巴，并更详细地研究小载体的血淋巴环境。

<table><tbody><tr><td>10% SDS-PAGE protein gel</td><td>Bio-rad</td><td>4561035</td><td>Protein separation and detection</td></tr><tr><td>4% paraformaldehyde</td><td>Solarbio</td><td>P1110</td><td>For fixation of the cells or tissues&amp;nbsp;</td></tr><tr><td>Bradford dye reagent</td><td>Bio-rad</td><td>5000205</td><td>Protein concentration detection</td></tr><tr><td>Capillary</td><td>Hirschmann</td><td>9000101</td><td>For collecting hemolymph</td></tr><tr><td>Cell counting chamber</td><td>ACMEC</td><td>AYA0810</td><td>Hemocytes counting</td></tr><tr><td>Glass slide</td><td>Gitoglas</td><td>10127105A</td><td>For holding insects</td></tr><tr><td>Glass slide coated with silane</td><td>Sigma</td><td>S4651-72EA</td><td>For holding microscope samples</td></tr><tr><td>Gold antifade reagent with DAPI</td><td>Invitrogen</td><td>P36935</td><td>Nucleus staining</td></tr><tr><td>Microscope cover glass</td><td>Gitoglas</td><td>10212424C</td><td>For microscopic observation</td></tr><tr><td>Pipette bulb</td><td>Hirschmann</td><td>9000101</td><td>For collecting hemolymph</td></tr><tr><td>Prism 8.0 software</td><td>GraphPad Software</td><td>/</td><td>Statistical analyses</td></tr><tr><td>Stereomicroscope&amp;nbsp;</td><td>Motic</td><td>SMZ-168</td><td>For insect dissection</td></tr><tr><td>Tweezers</td><td>Tianld</td><td>P5622</td><td>For insect dissection</td></tr><tr><td>Zeiss inverted microscope</td><td>Zeiss</td><td>Observer Z1</td><td>Hemocytes observation</td></tr></tbody></table>

a precise and quantifiable method for collecting hemolymph from small arthropods

众所周知，节肢动物通过其血淋巴传播各种具有医学和农业重要性的病毒，这对于病毒传播至关重要。血淋巴采集是研究病毒-载体相互作用的基本技术。在这里，我们描述了一种新颖而简单的方法，用于定量收集小型节肢动物的血淋巴，使用 Laodelphax striatellus （小棕色飞虱，SBPH）作为研究模型，因为这种节肢动物是水稻条纹病毒（RSV）的主要载体。在该协议中，该过程首先用细尖镊子轻轻捏掉冷冻节肢动物的一条腿，然后将血淋巴从伤口中压出。然后，根据毛细管力原理，使用由毛细管和移液器灯泡组成的简单微量移液器从伤口收集漏出性血淋巴。最后，可以将收集的血淋巴溶解到特定的缓冲液中以供进一步研究。这种从小型节肢动物收集血淋巴的新方法是进一步研究虫媒病毒和载体-病毒相互作用的有用且有效的工具。

微量移液器模型和血淋巴收集 我们开发了一种简单的微量移液器，其作用基于毛细管的毛细管力。微量移液器由毛细管和移液器灯泡组成（图1A）。毛细管有不同的体积尺寸，范围从 1 μL 到 20 μL，毛细管体积可根据要求选择。不建议使用体积较小的毛细管，因为较小体积的毛细管的额外细孔会使吸收血淋巴等液体变得困难。移液器灯泡顶部有一个孔，在血淋...

Watch this Scientific Journal Video about A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods at JoVE.com

作者聚焦：从小型节肢动物中收集血淋巴液的精确且可量化的方法  

我们描述了一种从小型节肢动物中有效收集可量化血淋巴以进行后续分析的方法。

一种精确且可量化的小型节肢动物血淋巴收集方法

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus ...

我们的研究重点是虫媒病毒、昆虫和植物相互作用。从小型节肢动物中获取血淋巴样本对昆虫传播疾病的研究提出了技术挑战。该协议显示了解决此问题的精确且可量化的技术。虫媒病毒传播的最新研究进展发现，病毒可以操纵几个重要的媒介因素来突破宿主屏障。该方案是一种简单，具有成本效益且有价值的技术，可以精确定量血淋巴。这种方法还为启动病毒和载体之间的相互作用提供了有价值的技术。我们即将进行的研究将侧重于了解媒介昆虫传播病毒的分子机制，同时探索和增强新的操作技术。

a-precise-quantifiable-method-for-collecting-hemolymph-from-small

Research

JoVE Journal

Biology

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

1.5K 次观看.  Chinese Academy of Sciences. 我们的研究重点是虫媒病毒、昆虫和植物相互作用。从小型节肢动物中获取血淋巴样本对昆虫传播疾病的研究提出了技术挑战。该协议显示了解决此问题的精确且可量化的技术。虫媒病毒传播的最新研究进展发现，病毒可以操纵几个重要的媒介因素来突破宿主屏障。该方案是一种简单，具有成本效益且有价值的技术，可以精确定量血淋巴。这种方法还为启动病毒和载体?...

视频: 作者聚焦：从小型节肢动物中收集血淋巴液的精确且可量化的方法  

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

科研

发育生物学

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

免疫与感染

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

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, ...

环境科学

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

生物学

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

在无脊椎动物病理学研究中发现血淋巴收集和接种转移到大黄微加分中

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

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.

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

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www.jove.com/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
The Drosophila hemolymph serves as both blood and interstitial fluid, circulating throughout the body during the larva and adult stages. This video describes a protocol to collect larval hemolymph in order to quantify the circulating cells.

角质层破坏:一种从果蝇幼虫中收集血淋巴的方法

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
The Drosophila ...

一种精确且可量化的小型节肢动物血淋巴收集方法

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