Name: optimizing the rearing procedure of germ-free wasps
Uploaded: 2023-07-21T14:40:00.000+00:00
Duration: 5 min 39 s
Description: Watch this Scientific Journal Video about Optimizing the Rearing Procedure of Germ-Free Wasps at JoVE.com

资助：这项工作得到了中国国家自然科学基金（32270538）、国家重点研发计划（2022YFF0710603）、北京市自然科学基金（6222046）以及中科院通过CAS-CSIRO资助计划（152111KYSB20210011） 获得 的中科院战略资助。 作者贡献：所有作者都制定了综述的范围和重点，并为手稿的撰写做出了贡献。

1.无菌纳索尼亚饲养培养基的制备 
<ol><li>将市售的 绢毛蛹 （见 材料表）放在可以容纳所有蛹的表面上，例如托盘或一张纸。丢弃任何发育不全的幼虫、深色老蛹、空蛹壳、锯末或其他杂质。只保留棕红色的幼蛹，并将其转移到烧杯中（约3，000-4，000个蛹）。 注意：经过多次实验，发现由深色老蛹制成的培养基很容易变黑，并阻止了无菌黄蜂的发育。当使用幼棕红色蛹产生培养基时，不会发生这种现象。其原因尚不清楚，需要进一步调查以核实。</li>
	<li>向烧杯中加入足够的去离子水以覆盖蛹的整个表面。用锡纸或纱布包裹烧杯口，摇晃烧杯清洁蛹表面的杂质，然后倒出水。重复三到五次。</li>
	<li>将清洁的 绢毛蛹 放入大蒜压榨机的过滤罐中，用力挤压，并将 绢毛蛹 的组织液收集在 50 mL 无菌离心管中。然后，倒出蛹渣，将新蛹放入压蒜机的过滤罐中，直到全部被挤压。 注意：与Wang等人14使用的针状挤压装置相比，大蒜压榨机更方便，可以更彻底地分离组织液。这一进步为NRM的大规模生产和长期储存奠定了基础。</li>
	<li>将混合物在4°C（25，000× g）下离心10分钟。离心后，混合物将从下到上分成三层：沉淀层、蛋白质层和脂肪层（图 1）。</li>
	<li>为防止过滤过程中堵塞，请使用 18 G 无菌针吸出蛋白质层，并将其转移到新的 50 mL 锥形无菌聚丙烯离心管中。</li>
	<li>将商业液体 果蝇 培养基（见 材料表）以1：1的比例添加到蛋白质提取物中。 注意：与NRMv3相比，使用更方便的商业化 绢毛蚴作为 宿主和NRM原料。因此，在NRMv3的基础上，营养比例从1：2调整为1：1，更适合不育 Nasonia 的生长发育（图2）。</li>
	<li>使用具有不同孔径（8、1.2、0.8 和 0.45 μm;参见材料表）的真空过滤系统过滤混合培养基（果蝇培养基和绢毛蛹的蛋白质提取物）以去除不同大小的颗粒。为防止堵塞，请在流动减慢时更换滤纸。 注意：每次过滤都需要使用新的 50 mL 无菌离心管。</li>
	<li>再次将液体在4°C（15，000× g）下离心10分钟，并通过0.22μm注射器过滤器对上清液培养基进行灭菌。重复上述步骤一次。</li>
	<li>等分后将培养基储存在-20°C（图3A）以长期储存。 注意：为防止污染和反复冻融，建议在包装后使用培养基时仅打开每管NRM一次（图3A）。</li>
</ol>2. 无菌鸡蛋收集
<ol><li>为了确保后代中稳定的男女比例，由于它们的单倍体遗传特征（雄性从单倍体细胞发育而来，而雌性从受精卵产生的二倍体细胞发育而来），在蛹期以 50 雌性对 15 名雄性的比例将蛹放入果蝇小瓶中4，15。<ol><li>出襄成虫后，让雄性和雌配1.5天，然后将约40个 绢毛蛹 放入小瓶中。在寄生后12-24小时内，使用无菌解剖针在体视显微镜下小心地打开蛹壳的一端（图4）。</li>
		<li>用手握住另一端，找到黄蜂胚胎。使用解剖针将胚胎从 绢毛乳杆菌 组织表面转移到带有磷酸盐缓冲盐水（PBS）的无菌细胞过滤器14。 注意：较老的 绢毛蛹 应用于寄生，因为干燥的 绢毛蛹 组织更容易移植胚胎。为了保证卵从 绢毛蛹 组织表面顺利转移，尽量注意在解剖针打开蛹壳时不要刺穿组织，以免间质液流出。</li>
	</ol></li>
	<li>将 20-30 个胚胎放在细胞过滤器上，并用 1，000 μL 10% 商业次氯酸钠溶液均匀洗涤它们（参见 材料表），然后用 1，000 μL 1x 无菌 PBS 再次洗涤。然后，用 1，000 μL 70% 乙醇溶液洗涤一次，并用 1，000 μL 1x 无菌 PBS 洗涤三次。</li>
	<li>首先，将用1x PBS预润湿的5毫米直径聚丙烯网片（见 材料表）放入24孔板中。然后，使用无菌的小刷子，将细胞过滤器上的黄蜂胚胎轻轻刷到聚丙烯网片上。这可以对24孔板垂直柱中的所有四个孔进行。</li>
</ol>3. 无菌黄蜂饲养
<ol><li>向层流罩中的每个孔中加入 50 μL NRM。为了保持生长的潮湿环境，在 24 孔板中的每个孔之间加入 1 mL 无菌水。装有 30 mL 无菌水的小烧杯也可以放入带有 24 孔板的 5 L 无菌塑料盒中。<ol><li>在整个实验过程中，将无菌塑料盒和24孔板保持在25±2°C的恒温和恒定光照下的气候室中。</li>
	</ol></li>
	<li>在每天转移和添加NRM之前，在层流工作台上解冻冷冻的NRM。在无菌环境中，使用酒精消毒的镊子将带有幼虫的聚丙烯网从一个孔转移到另一个孔。最后，加入 50 μL 已平衡至室温的 NRM（图 3B）。每天重复上述操作，直到观察到蛹。 注意：随着黄蜂的发育，应适当增加NRM的用量，以确保无菌黄蜂有足够的营养。例如，向第四龄幼虫提供60至70μL的NRM。由于同一孔中可能共存不同的发育阶段，因此请使用无菌刷子转移蛹。向剩余的幼虫中添加少量NRM。使用无菌技术避免微生物入侵和污染。</li>
	<li>进食9至11天后，超过80%的幼虫发育成白色或黄色的蛹。此时，将过滤器移至干净的孔板并停止添加培养基以等待封闭。</li>
</ol>

饲养无菌 Nasonia 黄蜂

<ol>
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作者没有什么可透露的。

随着基因组学、代谢组学等高通量检测技术的应用，研究人员逐渐意识到肠道微生物群中存在巨大的遗传多样性和代谢复杂性16。这些共生细菌通过与宿主的复杂相互作用，与宿主营养代谢、肿瘤、免疫力、衰老等各种生理或病理状态密切相关17。然而，与宿主中微生物种群的组成和功能网络相关的研究非常困难，因为每个自然个体中微生物的组成并不完全相同...

无菌动物是指没有可检测到的活微生物和寄生虫的动物1.无菌胚胎可以通过在无菌条件下解剖母体来获得，随后在屏障系统中饲养2。这类动物可用于研究微生物对动物的影响，如对肠道微生物群、免疫系统和代谢的影响1。通过一定的技术手段，许多昆虫甚至哺乳动物都可以不育3，4。无菌动物具有独特的作用，并已广泛应用于微生物学研究的各个方面5.例如，使用无菌Nasonia黄蜂已经揭示了微生物可以帮助宿主在长期外源环境胁迫下适应新环境6，7。
Nasonia寄生蜂是小型寄生蜂，将卵注入苍蝇的蛹中4。Nasonia有四种已知的物种，包括Nasonia vitripennis，Nasonia longicornis，Nasonia giraulti和Nasonia oneida8。猪笼草可以在世界各地找到，而其他三个物种在北美的范围有限4. Nasonia寄生蜂因其易于栽培，繁殖周期短，基因组测序和长期滞育8，9等特点而被认为是理想的模式昆虫。它们可用于研究昆虫进化、遗传学、发育、行为和共生的各个方面10.此外，Nasonia寄生蜂还可以帮助控制农业中的有害苍蝇和疾病11。昆虫不育系统的成功建立涉及两个主要步骤：（1）胚胎绝育和（2）体外向幼虫提供无菌食品。为了获得无菌食品，布鲁克和博登斯坦12于2012年通过使用抗生素，漂白剂和胎牛血清等化学物质杀死细菌12，开发了Nasonia饲养培养基（NRMv1）。然而，化学灭菌方法导致猪笼草13的存活率和闭合率低。然后，在2016年，Shropshire等人通过使用过滤灭菌方法代替化学灭菌方法开发了NRMv2，以消除抗生素和其他物质的危害，并优化了育种过程13。不幸的是，这种方法仍然存在一些缺点，例如与制备和使用培养基相关的挑战，以及胚胎、幼虫和闭合蛹14 的溺水、喂养不足或脱水的风险。Wang和Brucker14最近改进了Nasonia饲养培养基版本3（NRMv3）和无菌饲养版本2（GFRv2）协议。这些改进降低了成本和介质消耗。然而，NRMv3的储存时间非常短，极易受到污染。
本研究基于NRMv3，优化了NRM制备工具的储存方法和养分比例。这种方法的改进有助于使用猪笼草作为微生物组研究的模型。与Wang等人开发的NRMv3相比14，与Wang等人使用的带底孔的60 mL注射器相比，NRM原料之一的改进型挤压大疱石棺蛹的工具大大提高了大疱沙棺组织液的生产效率14。我们调整了NRM的养分比例，使无菌Nasonia黄蜂的成活率有了一定的提高，而不影响其发育时间。此外，将NRM装入小容量离心管（1.5 mL）中，并在-20°C冰箱中冷冻以延长储存时间。值得注意的是，虽然我们使用家蝇露西莉亚绅雪菌作为NRM制备的宿主和来源，但该协议可能适用于实验室中可用的其他Nasonia宿主。

<table><tbody><tr><td>0.22 Sterile vacuum filter</td><td>NEST</td><td>331011</td><td></td></tr><tr><td>10% SodiumHypochlorite</td><td>LIRCON</td><td>XB-84BS-1</td><td></td></tr><tr><td>1x PBS solution</td><td>Solarbio</td><td>P1020</td><td></td></tr><tr><td>200 mesh nylon net</td><td>BIOBYING</td><td>BY-378Z</td><td></td></tr><tr><td>24 well-plate</td><td>NEST</td><td>702001</td><td></td></tr><tr><td>8, 1.2, 0.8, and 0.45 &amp;#181;m filters</td><td>Shanghai Xingya Purification Material Factory</td><td>HN-AA-JT-10079</td><td></td></tr><tr><td>Absolute ethyl alcohol</td><td>Macklin</td><td>E809057-500ml</td><td></td></tr><tr><td>Cell Strainer</td><td>BIOLOGIX</td><td>15-1100</td><td></td></tr><tr><td>Commercial Drosophila Medium</td><td>Boer</td><td>B645446-500ml</td><td></td></tr><tr><td>Dissecting needle</td><td>Bioroyee</td><td>17-9140</td><td></td></tr><tr><td>Garlic press</td><td>Taobao</td><td>No Catalog numbers</td><td>Purchase on Taobao</td></tr><tr><td>Lucillia sericata pupae</td><td>Hefei Dayuan Biotechnology Co., Ltd.</td><td>No Catalog numbers</td><td>Purchase on Taobao</td></tr><tr><td>Small writing brush</td><td>Cestidur</td><td>BL0508</td><td></td></tr><tr><td>Stereoscope</td><td>SOPTOP</td><td>RX50</td><td></td></tr><tr><td>Tweezers</td><td>SALMART</td><td>A109001-56</td><td></td></tr></tbody></table>

optimizing the rearing procedure of germ-free wasps

无菌饲养技术是一种在不育或几乎无菌的条件下培养昆虫的方法，可以有效消除外界微生物对昆虫菌群的影响，从而促进昆虫菌群研究的快速发展。Nasonia（黄蜂属）是一种寄生蜂昆虫，具有寿命短、遗传变异高、操作方便等优点，被广泛用作昆虫模型系统。与只能减少动物体内微生物数量的抗生素处理不同，无菌饲养技术可以控制动物体内微生物的组成和数量，进一步促进宿主-微生物相互作用的研究。然而，以前版本的Nasonia饲养培养基（NRM）存在一些缺陷和问题，例如制备过程复杂且耗时，容易被细菌或真菌污染以及储存时间短。因此，本研究通过优化NRM制备过程中使用的工具，储存条件和组分比例来解决这些问题。优化后的培养基可以在-20°C下储存至少3个月，并消除在喂食无菌黄蜂时NRM污染的可能性。这进一步提高了无菌Nasonia的存活率和健康水平，这对于使用Nasonia作为微生物研究的模型非常重要。

通过改进制备工具，大大提高了NRM的制备效率。此外，通过优化策略和保鲜方法，消除了饲喂过程中NRM污染的问题。同时，调整后的NRM对以 绢毛蚴为 寄主的无菌黄蜂的生长发育具有更适宜的营养配比。与使用GFRv2饲养的NRMv3无菌黄蜂相比，无菌黄蜂从幼虫到蛹的存活率显着提高（图2）。此外，无菌黄蜂和传统饲养的黄蜂之间的生成期没有差异（每代约14天）（<strong cl...

Watch this Scientific Journal Video about Optimizing the Rearing Procedure of Germ-Free Wasps at JoVE.com

作者聚焦：优化无菌黄蜂的饲养程序  

从蛹中解剖Nasonia蜂胚胎，寄生12-24 h后，用酒精和10%次氯酸钠溶液洗涤，得到无菌胚胎。在培育无菌胚胎并为其提供Nasonia饲养培养基进行体外生长和发育后，获得了无菌Nasonia成虫。

优化无菌黄蜂的饲养程序

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of ...

我们的研究重点是研究昆虫 - 微生物群相互作用，特别是调查这些共生细菌的影响，并探索宿主 - 微生物关系的动态。无菌Nasonia是进行这项研究的重要工具。目前的研究利用高通量测序、无菌昆虫饲养、转录组学、蛋白质组学和先进显微镜等技术。该工具能够对昆虫-微生物宿主相互作用进行全面分析和虚拟化，以促进我们对这一领域的理解。优化的Nasonia饲养培养基制备程序具有经济优势，并节省时间和精力。此外，它有效地降低了饲喂过程中NRM污染的风险，大大提高了无菌Nasonia的生产效率

optimizing-the-rearing-procedure-of-germ-free-wasps

Research

JoVE Journal

Biology

Optimizing the Rearing Procedure of Germ-Free Wasps

2.2K 次观看.  Chinese Academy of Sciences. 我们的研究重点是研究昆虫 - 微生物群相互作用，特别是调查这些共生细菌的影响，并探索宿主 - 微生物关系的动态。无菌Nasonia是进行这项研究的重要工具。目前的研究利用高通量测序、无菌昆虫饲养、转录组学、蛋白质组学和先进显微镜等技术。该工具能够对昆虫-微生物宿主相互作用进行全面分析和虚拟化，以促进我们对这一领域的理解。优化的Nasonia饲养培养基制?...

视频: 作者聚焦：优化无菌黄蜂的饲养程序  

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

科研

遗传学

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

免疫与感染

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

体外孤蜂饲养: 一种评估幼虫危险因素的工具

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

环境科学

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

生物工程

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

培养和遗传操纵昆虫致病线虫

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

维护伊蚊蚊蚊子感染沃尔巴克

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

生物学

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

Here we describe two protocols: first to propagate, extract, purify, and quantify large quantities of honey bee non-enveloped virus particles, including a method for removing honey bee pupae and second to test the effects of viral infection using a highly repeatable, high-throughput cage bioassay.

一种富含病毒的接种物用于蜜蜂口腔感染（Apis mellifera）的制备

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

自动化秀丽隐杆线虫的聚集定量

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, and parts of Africa over the last decade, becoming a devastating pest of various soft-skinned fruits in its invaded regions. Biological control, especially by means of self-perpetuating and specialized parasitoids, is expected to be a viable option for sustainable area-wide management of this highly mobile and polyphagous pest. Ganaspis brasiliensis Ihering (Hymenoptera: Figitidae) is a larval parasitoid that is widely distributed in East Asia, and has been found to be one of the most effective parasitoids of D. suzukii.
Following rigorous pre-introduction evaluations of its efficacy and potential non-target risks, one of the more host-specific genetic groups of this species (G1 G. brasiliensis) has been approved recently for introduction and field release in the United States and Italy. Another genetic group (G3 G. brasiliensis), which was also commonly found to attack D. suzukii in East Asia, may be considered for introduction in the near future. There is currently enormous interest in rearing G. brasiliensis for research or in mass-production for field release against D. suzukii. This protocol and associated video article describe effective rearing methods for this parasitoid, both on a small scale for research and a large scale for mass-production and field release. These methods may benefit further long-term research and use of this Asian-native parasitoid as a promising biological control agent for this global invasive pest.

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

Ganaspis brasiliensis-a larval parasitoid of Drosophila suzukii (a global invasive fruit crop pest)-has been approved or is considered for introduction into Europe and the United States for biological control of this pest. This article provides protocols for both small-scale and large-scale rearing of this parasitoid.

巴西寄生体加纳 斯菌的饲养方法，这是一种有前途的生物控制剂，用于侵入性 铃木果蝇

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, ...

优化无菌黄蜂的饲养程序

副本

摘要

探索更多视频

此视频中的章节

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

寄生蜂简介

体外孤蜂饲养: 一种评估幼虫危险因素的工具

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

培养和遗传操纵昆虫致病线虫

维护伊蚊蚊蚊子感染沃尔巴克

一种富含病毒的接种物用于蜜蜂口腔感染（Apis mellifera）的制备

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

自动化秀丽隐杆线虫的聚集定量

巴西寄生体加纳斯菌的饲养方法，这是一种有前途的生物控制剂，用于侵入性铃木果蝇

优化无菌黄蜂的饲养程序

副本

摘要

探索更多视频

此视频中的章节

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

寄生蜂简介

体外孤蜂饲养: 一种评估幼虫危险因素的工具

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

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