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>
	<li>Diviccaro, 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=Exploring+the+impact+of+the+microbiome+on+neuroactive+steroid+levels+in+germ-free+animals.">Exploring the impact of the microbiome on neuroactive steroid levels in germ-free animals.</a> International Journal of Molecular Sciences. 22 (22), 12551 (2021).</li><li>Pang, X., 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=Inter-species+transplantation+of+gut+microbiota+from+human+to+pigs.">Inter-species transplantation of gut microbiota from human to pigs.</a> The ISME Journal. 1 (2), 156-162 (2007).</li><li>Uzbay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-free+animal+experiments+in+the+gut+microbiota+studies.">Germ-free animal experiments in the gut microbiota studies.</a> Current Opinion in Pharmacology. 49, 6-10 (2019).</li><li>Zhu, Z., Liu, Y., Hu, H., Wang, G. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasonia-microbiome+associations:+a+model+for+evolutionary+hologenomics+research.">Nasonia-microbiome associations: a model for evolutionary hologenomics research.</a> Trends in Parasitology. 39 (2), 101-112 (2022).</li><li>Li, J., Wei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+efficient+germ-free+animal+system+to+support+functional+microbiome+research.">Establishment of an efficient germ-free animal system to support functional microbiome research.</a> Science China Life Sciences. 62 (10), 1400-1403 (2019).</li><li>Wang, G. H., 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=Changes+in+microbiome+confer+multigenerational+host+resistance+after+sub-toxic+pesticide+exposure.">Changes in microbiome confer multigenerational host resistance after sub-toxic pesticide exposure.</a> Cell Host Microbe. 27 (2), 213-224 (2020).</li><li>Wang, G. H., Dittmer, J., Douglas, B., Huang, L., Brucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coadaptation+between+host+genome+and+microbiome+under+long-term+xenobiotic-induced+selection.">Coadaptation between host genome and microbiome under long-term xenobiotic-induced selection.</a> Science Advances. 7 (19), (2021).</li><li>Dittmer, J., Brucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+your+host+shuts+down:+larval+diapause+impacts+host-microbiome+interactions+in+Nasonia+vitripennis.">When your host shuts down: larval diapause impacts host-microbiome interactions in Nasonia vitripennis.</a> Microbiome. 9 (1), 85 (2021).</li><li>Dittmer, J., 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=Disentangling+a+holobiont-recent+advances+and+perspectives+in+Nasonia+wasps.">Disentangling a holobiont-recent advances and perspectives in Nasonia wasps.</a> Frontiers in Microbiology. 7, 1478 (2016).</li><li>Brooks, A. W., Kohl, K. D., Brucker, R. M., van Opstal, E. J., Bordenstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylosymbiosis:+relationships+and+functional+effects+of+microbial+communities+across+host+evolutionary+history.">Phylosymbiosis: relationships and functional effects of microbial communities across host evolutionary history.</a> PLoS Biology. 14 (11), e2000225 (2016).</li><li>Heavner, M. E., 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=Partial+venom+gland+transcriptome+of+a+Drosophila+parasitoid+wasp,+Leptopilina+heterotoma,+reveals+novel+and+shared+bioactive+profiles+with+stinging+Hymenoptera.">Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera.</a> Gene. 526 (2), 195-204 (2013).</li><li>Brucker, R. M., Bordenstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cultivation+of+the+hymenoptera+genetic+model,+Nasonia.">In vitro cultivation of the hymenoptera genetic model, Nasonia.</a> PLoS One. 7 (12), e51269 (2012).</li><li>Shropshire, J. D., van Opstal, E. J., Bordenstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+approach+to+germ-free+rearing+in+the+jewel+wasp+Nasonia.">An optimized approach to germ-free rearing in the jewel wasp Nasonia.</a> PeerJ. 4, e2316 (2016).</li><li>Wang, G. H., Brucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+Nasonia+germ-free+rearing.">An optimized method for Nasonia germ-free rearing.</a> Scientific Reports. 12 (1), 219 (2022).</li><li>Brucker, R. M., Bordenstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hologenomic+basis+of+speciation:+gut+bacteria+cause+hybrid+lethality+in+the+genus+Nasonia.">The hologenomic basis of speciation: gut bacteria cause hybrid lethality in the genus Nasonia.</a> Science. 341 (6146), 667-669 (2013).</li><li>Fontaine, C. A., 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=How+free+of+germs+is+germ-free?+Detection+of+bacterial+contamination+in+a+germ+free+mouse+unit.">How free of germs is germ-free? Detection of bacterial contamination in a germ free mouse unit.</a> Gut Microbes. 6 (4), 225-233 (2015).</li><li>Mazmanian, S. K., Liu, C. H., Tzianabos, A. O., Kasper, 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=An+immunomodulatory+molecule+of+symbiotic+bacteria+directs+maturation+of+the+host+immune+system.">An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system.</a> Cell. 122 (1), 107-118 (2005).</li><li>Weersma, R. K., Zhernakova, A., Fu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+drugs+and+the+gut+microbiome.">Interaction between drugs and the gut microbiome.</a> Gut. 69 (8), 1510-1519 (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;well-plate</td>
			<td>NEST</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>8, 1.2, 0.8, and 0.45 &#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  

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

科研

生物学

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

小鼠肾移植程序

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

血红素加氧酶1表达上调在刮痧过程中的生物发光成像

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

一个优化的协议饲养 Fopius arisanus Tephritid果寄生蝇

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

试验样品为优化风暴超高分辨率显微镜

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

过程评价絮凝剂的效率的分散粒子的从植物提取物的去除

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Laboratory Rearing of Stable Flies and Other Muscoid Diptera

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in agronomic practices resulted in serious outbreaks of stable flies in several countries. These outbreaks disrupted livestock production and human recreation resulting in public demands for increasing research and management efforts for this pest. A simple and inexpensive procedure for rearing stable flies for laboratory studies is presented. The procedure uses locally available diet components, equipment and supplies. The procedure can be adapted for rearing other muscoid flies including face fly (Musca autumnalis), horn fly (Haematobia irritans), and house fly (Musca domestica). The procedure produces stable fly puparia averaging 12.5 mg and ~35% egg to adult survival. Approximately 3000 flies are produced in each pan.

A procedure for rearing stable flies (Stomoxys calcitrans) is presented. The procedure uses locally available materials for diet components, equipment and supplies.

稳定蝇及其他 Muscoid 双翅目的实验室饲养

Stable flies, Stomoxys calcitrans, are serious pests of livestock, humans, companion animals and wildlife worldwide. During the last 20+ years, changes in ...

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle&#160;(Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

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, ...

使用人工饲养方法提高狨猴的生存和发育

Hand-Rearing Method for Infant Marmosets

The common marmoset (Callithrix jacchus) is a small and highly social New World monkey with high reproduction rates, which has been proven to be a compelling non-human primate model for biomedical and neuroscience research. Some females give birth to triplets; however, the parents cannot raise all of them. To save these infants, we have developed a hand-rearing method for raising newborn marmosets. In this protocol, we describe the formula of the food, the time for feeding, the configuration of the temperature and humidity, as well as the adaptation of the hand-reared infants to the colony environment. This hand-rearing method significantly increases the survival rate of marmoset infants (without hand-rearing: 45%; with hand-rearing: 86%) and provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments. As the method is practical and easy to use, we anticipate that it could also be applied to other labs working with common marmosets.

作者聚焦：狨猴研究 - 范围和挑战 

Here, we describe a hand-rearing method for raising infant marmosets in an animal incubator. This method greatly increases the survival rate of marmoset infants, which provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments.