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。

Germ-free animals are animals that have no detectable living microorganisms and parasites1. Germ-free embryos can be obtained by dissecting the mother under aseptic conditions and subsequently raised in barrier systems2. Such animals can be used to study the effects of microorganisms on animals, such as on the intestinal microbiota, immune system, and metabolism1. With certain technical means, many insects and even mammals can be rendered sterile3,4. Germ-free animals have a unique role and have been widely used in various aspects of microbiology research5. For example, the use of germ-free Nasonia wasps has revealed that microorganisms can help hosts adapt to new environments under long-term exogenous environmental stress6,7.
Nasonia parasitoids are small parasitic wasps that inject their eggs into the pupae of flies4. There are four known species of Nasonia, including Nasonia vitripennis, Nasonia longicornis, Nasonia giraulti, and Nasonia oneida8. N. vitripennis can be found worldwide, while the other three species have limited ranges in North America4. Nasonia parasitoid wasps are regarded as ideal model insects because of their characteristics, such as easy cultivation, short reproduction cycle, sequenced genome, and long term diapause8,9. They can be used to study various aspects of insect evolution, genetics, development, behavior, and symbiosis10. Moreover, Nasonia parasitoid wasps can also help control harmful flies in agriculture and disease11. The successful establishment of a sterile insect system involves two major steps: (1) sterilization of the embryos and (2) provision of sterile food to the larvae in vitro. In order to obtain sterile food, Brucker and Bordenstein12 developed Nasonia rearing medium (NRMv1) in 2012 by using chemicals such as antibiotics, bleach, and fetal bovine serum to kill bacteria12. However, the chemical sterilization method resulted in low survival and eclosion rates of N. vitripennis13. Then, in 2016, Shropshire et al. developed NRMv2 by using a filter sterilization method instead of a chemical sterilization method to eliminate the hazards of antibiotics and other substances, and optimized the breeding process13. Unfortunately, this method still has some disadvantages, such as the challenges associated with preparing and using the medium, as well as the risks of drowning, underfeeding, or dehydration for the embryos, larvae, and closed pupae14. Wang and Brucker14 recently improved the Nasonia rearing media version 3 (NRMv3) and the germ-free rearing version 2 (GFRv2) protocols. These improvements reduced the cost and media consumption. However, the NRMv3 has a very short storage time and is highly susceptible to contamination.
Building on NRMv3, the NRM preparation tool storage method and nutrient ratio were optimized in this study. This methodological refinement facilitates using N. vitripennis as a model for microbiome studies. Compared with the NRMv3 developed by Wang et al.14, the improved tool for squeezing Sarcophaga bullata pupa, one of the NRM raw materials, greatly enhances the production efficiency of S. bullata pupa tissue fluid compared with the 60 mL syringe with a bottom hole used by Wang et al.14. We adjusted the nutrient ratio of NRM, which led to a certain increase in the survival rate of germ-free Nasonia wasps without affecting their development time. In addition, the NRM was packed into small-capacity centrifuge tubes (1.5 mL) and frozen in a -20 &#176;C refrigerator to extend the storage time. It is worth noting that while we used the housefly Lucilia sericata as the host and source for NRM preparation, this protocol can likely be adapted for other Nasonia hosts that are available in the laboratory.

作者聚焦：优化无菌黄蜂的饲养程序  

优化无菌黄蜂的饲养程序

Our research focuses on the study of insect-microbiota interactions, specifically investigating the impact of in those symbiotic bacteria and exploring the dynamics of host-microbe relationships. Germ-free Nasonia is an essential tool for conducting this research. Current research utilize technologies such as high-throughput sequencing, germ-free insect rearing, transcriptoptomics, proteomics, and advanced microscope.This tool enable comprehensive analysis and virtualization of insect-microbe host interaction for advancing our understanding of this field. The optimized the procedure for preparing the Nasonia Rearing Medium provides economic advantages and saves time and effort. Additionally, it effectively reduces the risk of NRM contamination during the feeding process, greatly improving the efficiency of producing aseptic Nasonia

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

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。

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

1.3K 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  

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of external microorganisms on insect microbiota and thus promote the rapid development of insect microbiota research. Nasonia (wasp genus) is a parasitic wasp insect that has many advantages, such as a short lifespan, high genetic variation, easy operation, etc., and is widely used as an insect model system. Unlike antibiotic treatment, which can only reduce the number of microorganisms in animals, aseptic rearing techniques can control both the composition and quantity of microorganisms in animals, further facilitating the study of host-microbe interactions. However, previous versions of Nasonia rearing medium (NRM) have some defects and problems, such as a complex and time-consuming preparation process, easy contamination by bacteria or fungi, and short storage time. Therefore, this study solves these problems by optimizing the tools used in the NRM preparation process, storage conditions, and component ratios. The optimized medium could allow storage at -20 &#176;C for at least 3 months and eliminate the possibility of NRM contamination during feeding sterile wasps. This further improves the survival rate and health level of aseptic Nasonia, which is important for using Nasonia as a model for microbial research.

Nasonia wasp embryos were dissected from Lucillia sericata pupae after parasitization for 12-24 h and washed with alcohol and 10% sodium hypochlorite solution to obtain germ-free embryos. After rearing the germ-free embryos&#160;and supplying them with Nasonia rearing medium to grow and develop in vitro, germ-free Nasonia adults were obtained.

科研

生物学

Preparation of Germ-Free Nasonia Rearing Medium (NRM)

Preparation of Germ-Free Nasonia Rearing Medium (NRM)  

To begin, place commercially available Lucillia sericata pupae on a sheet of paper. Discard any underdeveloped larvae, dark old pupae, empty pupil shells, sawdust, or other impurities. Transfer young brownish red colored pupae to a beaker and immerse the pupae in deionized water.Wrap the beaker with tinfoil or gauze and shake it to clean any surface impurities off the pupae. Then discard the water. Place the cleaned pupae in the filter tank of a garlic press, and squeeze hard.Then collect the pupae tissue fluid in a 50-millimeter sterile centrifuge tube, and centrifuge it at 25, 000 x g for 10 minutes at four degrees Celsius. Using a 22-gauge sterile needle, collect the protein layer into a new 50-millimeter polypropylene centrifuge tube, and add commercial liquid drosophila medium at an equal ratio to the protein extract. To remove the particles of different sizes from the mixture, filter it using a vacuum filtration system with filter paper of different pore sizes.To prevent clogging change the filter paper when the flow slows down. Once the filtrate is centrifuged, sterilize the supernatant through a 0.22 micrometer syringe filter, and store the prepared nasonia rearing medium, or NRM aliquots, at 20 degrees Celsius until further use.

无菌 纳索尼亚 饲养培养基（NRM）的制备

Germ-Free Wasp Embryo Collection and Rearing with Nasonia-Rearing Medium (NRM)

Germ-Free Wasp Embryo Collection and Rearing with Nasonia-Rearing Medium (NRM)  

To begin, parasitize about 40 Lucilia sericata pupae onto emerging adult male and female Nasonia wasps mated for 1.5 days. Within 12 to 24 hours after parasitization, carefully open one end of the pupal shell using a sterile dissecting needle. Hold the other end of the shell and locate the wasp embryo.Then transfer the embryo to a sterile cell strainer moistened with PBS. Once 20 to 30 embryos have been transferred, wash them with 1, 000 microliters of 10%sodium hypochlorite solution followed by sterile PBS and 70%ethanol. Finally, wash them three times with sterile PBS.Next, place a polypropylene mesh sheet pre-wetted with PBS into a 24 well plate. Using a sterilized small brush, gently transfer the wasp embryos from the cell strainer to the polypropylene mesh sheet. For the wasp rearing, add 50 microliters of prepared NRM to each well with a sheet.Then add one milliliter of sterile water between each well to maintain a humid environment for growth. The next day, transfer the polypropylene mesh containing the larva from one well to another well using alcohol disinfected tweezers, and add 50 microliters of equilibrated NRM. After nine to 11 days of feeding, over 80%of the larva should develop into white or yellow pupae.then transfer the mesh to a clean well plate. In the present study, the survival rate of the germ-free wasps from larva to pupae was significantly improved compared to rearing germ-free wasps with NRM version three. Additionally, there was no difference in the generation period between the germ-free and conventionally reared wasps.