本研究由国家自然科学基金项目（32172476,32102275）、农业科技创新计划项目（CAAS-ZDRW202203、CAAS-ZDRW202108）和中央指导地方科技发展基金（XZ202301YD0042C）资助。

注意：有关本协议中使用的所有材料、仪器、软件和试剂的详细信息 ，请参阅材料表 。
1. 昆虫培养物的收集和维护
<ol><li>使用 1：5 （v/v） 阿拉伯胶粉和水25,26 的溶液，将一组 ~5,000 个 Corcyra cephalonica （Stainton） 卵粘贴在 9 cm x 16 cm 的卡片上。 注意：避免在卡片上附加过多的宿主卵，因为过度拥挤会使后续的转移和解剖程序变得不切实际。</li>
	<li>为了防止宿主卵孵化，将宿主卵卡置于30分钟的紫外线（UV）照射下（图1-i）。随后，将装有灭活鸡蛋的纸切成厚度为1厘米，直径为8厘米的鸡蛋卡，每张包含约300-500个鸡蛋（图1-ii）25,26,27。</li>
	<li>将一组 80-120 只 T. denrolimi 黄蜂引入直径为 2 厘米、长度为 8 厘米的玻璃管中。用棉花密封管子。 注意：将黄蜂保持在25±1°C的温度下，光/暗循环为16小时，黑暗8小时，并保持约75%的相对湿度。</li>
	<li>向黄蜂出示寄主卵卡进行寄生（图1-ii）。让黄蜂将卵沉积在宿主卵中 6 小时，然后立即取出黄蜂。 注意：避免过度延长寄生期，因为这会导致寄主卵中 T. denrolimi 后代过度拥挤，导致后代黄蜂死亡率增加28,29。</li>
</ol>2. dsRNA的合成
<ol><li>设计含有 T7 启动子的引物组，用于合成双链 RNA （dsRNA）。从靶基因 （Ferhch） 中选择一个 300-400 bp 的片段进行 dsRNA 合成。</li>
	<li>获取商业合成的引物组，用于生产靶基因的dsRNA，以及用作阴性对照的dsGFP 。</li>
	<li>按照制造商的方案 9，使用参考试剂盒从 100 只 T. denrolimi 黄蜂中提取 RNA 含量。检查RNA含量的质量;如果 OD260/OD280 的值范围为 1.8 到 2.09，则继续。</li>
	<li>用 2 μL 5x 缓冲液 （1 U/50 μL）、1 μL DNAse （1 U/50 μL） 和 6.5 μL RNA （~100 ng/μL）、0.5 μL H2O 在 42 °C 下纯化 RNA 含量 2 分钟。</li>
	<li>进行逆转录酶聚合酶链反应 （RT-PCR） 以生成含有 10 μL 纯化 RNA （~100 ng/μL）、4 μL 5x 缓冲液 （1 U/50 μL）、1 μL 酶混合物 I （1 U/50 μL） 和 4 μL H2O 的 cDNA 模板。 使用以下设置进行RT-PCR： 37°C15分钟， 85°C5秒。将cDNA产物储存在-4°C直至使用。</li>
	<li>根据参考的预混液（10 μL Taq II [1 U/50 μL]、0.8 μL 正向引物 [0.4 μM]、0.8 μL 反向引物 [0.4 μM]、0.8 μL DNA 模板 [40 ng/μL] 和 7.6 μL H2O）进行聚合酶链反应 （PCR） 扩增 dsRNA 序列。使用以下设置进行PCR：94°C2分钟;35 次循环，其中 94 °C 30 秒，60 °C 30 秒，72 °C 30 秒;72°C2分钟。</li>
	<li>通过在2.0%琼脂糖凝胶5,9上电泳评估PCR产物的质量，并通过Sanger测序确认目标序列。</li>
	<li>根据制造商的指南9,23，使用凝胶提取试剂盒纯化PCR产物。</li>
	<li>利用RNAi系统用10μL 2x缓冲液（0.02 U / μL），8 μL DNA模板（75ng / μL）和2μL酶混合物（1U / 50μL）合成靶基因的dsRNA，设置如下：37°C30分钟，70°C10分钟和25°C20分钟。用 30 μL 无核酸酶水洗脱合成的 dsRNA。</li>
	<li>通过在 1.0% 琼脂糖凝胶 5,9 上观察 dsRNA 产物的质量来评估其质量。将 dsRNA 稀释至 7,000 ng/μL。 检查dsRNA产物的质量;如果 OD260/OD280 的值范围为 1.8 到 2.09，则继续。将dsRNA产物储存在-20°C直至使用。</li>
</ol>3. T. denrolimi 蛹的移植
<ol><li>在25±1°C下培养寄生的寄主卵约8天，保持16小时的光照/8小时的黑暗循环和~75%的相对湿度。寄生的宿主卵将在 5 天后变黑 30 （图 1-iv，v）。</li>
	<li>将宿主卵卡转移到解剖显微镜上。使用一对钨针（图1-vi）小心地从宿主卵中取出绒毛膜（图1-vii），并从内部取出蛹（图1-viii）5,17,18。 注意： 解剖针的尖端应小于 0.05 毫米。</li>
	<li>为基材准备一个干净的板。倒出 10-15 mL 的 15 g/L 琼脂溶液，使其自然冷却（图 2-i）。 注意：将琼脂溶液与0.5g链霉素混合，以抑制 体外污染物的生长。</li>
	<li>使用消毒刻刀蚀刻几个深度为 0.2-0.3 毫米、宽度为 0.4-0.8 毫米的凹槽（图 2-ii）。</li>
	<li>使用小刷子（图2-iii）将解剖的宿主卵中的蛹移植到琼脂基质上的一个凹槽中（图2-iv）。</li>
	<li>重复步骤3.4和3.5，将100-300个蛹逐个移植到凹槽中（图2-v）。</li>
</ol>4. 将 dsRNA 显微注射到 T. denrolimi 蛹中
<ol><li>使用玻璃针拔拔器拉动玻璃毛细管（图3-i，ii）。在拔针器上将热量设置为 280，将速度设置为 170，将延迟设置为 250，将拉力设置为 30（图 3-iii）。准备用于显微注射的多个毛细管玻璃针（图3-iv，v）。</li>
	<li>使用研磨板对玻璃针的尖端进行斜面处理（图3-vi）。 注意： 确保注射针的尖端保持锋利;否则，可能导致蛹死亡或阻碍试剂流过针头（图3-vii）。</li>
	<li>使用微进样泵将约2μL含有7,000ng / μL的dsRNA注射液加载到制备的注射针中（图3-vii）。</li>
	<li>将含有数百个蛹的琼脂底物转移到复合显微镜的平台上（图3-viii）。</li>
	<li>小心地将注射针插入 T. denrolimi 蛹的腹部，与晶状体下方的腹部成 ~30° 角（图 3-ix）。</li>
	<li>逐渐将~5nL的注射液（步骤4.3）尽可能轻柔地注入 T. denrolimi 蛹中。 注意：当注射dsRNA溶液时， T. denrolimi 蛹可能会略微膨胀。向蛹中注射过量的溶液或使用开口过大的针头可能会导致蛹过早死亡。当针头在注射几到几十个蛹后堵塞时更换针头。</li>
	<li>重复步骤4.5和4.6，将dsRNA逐个注射到600个蛹中。 注意：为了进行可靠的RT-qPCR分析，应从至少100个蛹中分离出RNA含量。在dsRNA处理中注射至少600个蛹进行3次重复，在 dsGFP 阴性对照中注射3次重复9。</li>
</ol>5. T. denrolimi 蛹的孵化
<ol><li>将含有注射蛹的底物转移到25±1°C的培养箱中，光/暗循环为16小时/8小时，RH为~75%。</li>
	<li>每次处理孵育约700 T. denrolimi 蛹24小时或48小时。从一组 100 个蛹中提取 RNA 内容，每次重复9. 注意：从样品中取出萎缩的蛹（死蛹）;否则，可能会导致基因表达检测出错。</li>
	<li>每次处理孵育~50 T. denrolimi 蛹，直到黄蜂出现（图3-x）。记录出现率和畸形率。</li>
</ol>6. 靶向基因表达检测
<ol><li>使用设计工具设计用于RT-qPCR的靶基因和参考基因的引物集。 注意：确保RT-qPCR引物的序列不与dsRNA的目标区域重叠。</li>
	<li>在RT-qPCR分析中采用基因 叉头盒O （FoxO）作为参考基因; FoxO 的引物之前已有报道 9.</li>
	<li>获得商业合成的引物组。使用 10 μL Taq II （1 U/50 μL）、0.8 μL 正向引物 （0.4 μM）、0.8 μL 反向引物 （0.4 μM）、0.8 μL 模板 （10 ng/μL） 和 7.6 μL H2O 进行 RT-qPCR。 使用以下设置进行 RT-qPCR：95°C 30 秒;35 次循环，其中 95 °C 持续 5 秒，57 °C 持续 30 秒，72 °C 持续 30 秒;95°C10秒;60°C5秒，95°C5秒。</li>
	<li>使用2-ΔΔCt方法计算目标基因的表达值9,25。</li>
	<li>采用Kruskal-Wallis检验分析不同治疗（dsFerhch、 dsGFP、非注射）影响下靶基因的表达。 注意：避免使用参数检验（例如 ，t 检验）进行事后比较，因为数据的异方差性和非正态性可能会导致错误的结论25.</li>
</ol>

推进树 突毛 虫 RNAi 技术用于基因功能分析

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Q., Zhao, Q., Dong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradual,+temperature-induced+change+of+secondary+sexual+characteristics+in+Trichogramma+pretiosum+infected+with+parthenogenesis-inducing+Wolbachia.">Gradual, temperature-induced change of secondary sexual characteristics in Trichogramma pretiosum infected with parthenogenesis-inducing Wolbachia.</a> PeerJ. 7, e7567 (2019).</li><li>Zhang, C., 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=Decreased+Wolbachia+titers+cause+gradual+change+in+masculinization+of+intersex+individuals+of+thelytokous+Trichogramma+dendrolimi.">Decreased Wolbachia titers cause gradual change in masculinization of intersex individuals of thelytokous Trichogramma dendrolimi.</a> Entomol Gen. 42 (5), 751-759 (2022).</li><li>Zhang, 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=Multi-parasitism:+A+promising+approach+to+simultaneously+produce+Trichogramma+chilonis+and+T.+dendrolimi+on+eggs+of+Antheraea+pernyi.">Multi-parasitism: A promising approach to simultaneously produce Trichogramma chilonis and T. dendrolimi on eggs of Antheraea pernyi.</a> Entomol Gen. 41 (6), 627-636 (2021).</li><li>Zhou, J. C., 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=Effects+of+Thelytokous+parthenogenesis-inducing+Wolbachia+on+the+fitness+of+Trichogramma+dendrolimi+Matsumura+(Hymenoptera:+Trichogrammatidae)+in+superparasitised+and+single-parasitised+hosts.">Effects of Thelytokous parthenogenesis-inducing Wolbachia on the fitness of Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) in superparasitised and single-parasitised hosts.</a> Front Ecol Evol. 9, 730664 (2021).</li><li>Flanders, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notes+on+the+life+history+and+anatomy+of+Trichogramma.">Notes on the life history and anatomy of Trichogramma.</a> Ann Entomol Soc Am. 30 (2), 304-308 (1937).</li></ol>

提交人声明他们没有利益冲突。

Trichogramma黄蜂被认为是有效的生物防治剂，专门针对农业和林业中的一系列鳞翅目害虫1。这些体型较小的黄蜂在宿主卵中经历其未成熟阶段，这一特征在进行 RNAi 实验时提出了挑战 5,18。本研究为在T. denrolimi中进行RNAi实验提供了全面的视觉指南。鉴于毛滴虫物种之间共有的生物学特征，该程序可以很容易地适用于?...

毛滴虫属是一组卵类寄生虫，已被广泛用作高效生物防治剂，可对抗全球农业和森林生态系统中的各种鳞翅目害虫1,2,3,4。大规模饲养的Trichogramma的应用为害虫的可持续管理提供了一种环境友好的方法5,6,7。通过研究基因调控和基因组编辑的方法10，了解毛滴虫的分子生物学，为提高这些生物防治剂的批量饲养效率和田间性能提供了宝贵的见解8,9。
自 1998 年在秀丽隐杆线虫中发现双链 RNA （dsRNA） 介导的特异性遗传干扰以来，RNA 干扰 （RNAi） 方法已发展成为通过抑制靶基因表达来探索生物体调控机制的重要遗传工具包11。RNAi实验已成为一种标准方法，广泛应用于研究多种昆虫物种的基因功能12,13。然而，RNAi的操作在许多寄生物种中提出了巨大的挑战，特别是在属于内寄生的Chalcidoidea家族的物种中14,15,16。RNAi方法已在至少13种寄生虫物种14,15,16,17,18,19中记录。其中，RNAi方法已在Nasonia黄蜂中全面进行，并适用于整个发育阶段，包括胚胎、幼虫、蛹和成虫14,15,16。值得注意的是，Nasonia 黄蜂是体外寄生虫，它们的后代在宿主蛹和蛹之间的间隙中发育，使它们能够在体外培养并使其耐受某些治疗，例如显微注射。与 Nasonia 黄蜂不同，Trichogramma 个体在宿主卵内经历整个胚胎、幼虫和蛹发育。胚胎和幼虫阶段的层（可能阻碍dsRNA的通透性）、易受损害以及体外存活困难是巨大的障碍20,21,22。此外，Trichogramma个体的体型很小，成年或蛹的长度约为~0.5毫米，这使得它们非常复杂，无法操纵20,21,22。
在本研究中，我们概述了在 Trichogramma denrolimi Matsumura 中进行 RNA 干扰 （RNAi） 实验的综合程序。该程序包括以下程序：（1）双链RNA（dsRNA）的设计和合成，（2）丹罗利米蛹的显微注射，（3）这些蛹的移植和体外培养，以及（4）通过RT-qPCR分析检测靶基因敲低。为RNAi实验选择的靶基因是铁蛋白重链同源性（Ferhch）。FerHCH是一种铁结合蛋白，含有具有抗氧化能力的铁氧化酶中心，促进Fe2+氧化为Fe3+。它通过维持氧化还原平衡和铁稳态，在各种生物的生长发育中起着不可或缺的作用。氟乙烯氯环己烷的消耗可导致铁的过度积累，导致不可逆转的组织损伤，并经常导致显著的表型改变，包括生长缺陷、畸形和死亡23,24。本研究为在T. denrolimi中进行RNAi提供了分步指南，这对于在更广泛的Trichogramma黄蜂背景下研究基因功能非常宝贵。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2x ES Taq MasterMix (Dye)</td>
			<td>Cowin Biotech, China</td>
			<td>CW0690H</td>
			<td>To amplify the dsRNA sequences</td>
		</tr>
		<tr>
			<td>20x PBS Buffer, DEPC treated (7.2-7.6)</td>
			<td>Sangon Biotech, China</td>
			<td>B540627-0500</td>
			<td>To dilute dsRNA</td>
		</tr>
		<tr>
			<td>Agar strip</td>
			<td>Shishi Globe Agar Industries Co.,Ltd, China</td>
			<td>n/a</td>
			<td>To make culture medium</td>
		</tr>
		<tr>
			<td>Ampicillin sodium</td>
			<td>Sangon Biotech, China</td>
			<td>A610028</td>
			<td>To make culture medium</td>
		</tr>
		<tr>
			<td>Bioer Constant temperature metal bath&#160;</td>
			<td>BIOER, China</td>
			<td>MB-102</td>
			<td>To synthesis dsRNA</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary&#160;</td>
			<td>WPI, USA</td>
			<td>1B100-4</td>
			<td>To pull capillary glass needle</td>
		</tr>
		<tr>
			<td>Clean bench&#160;</td>
			<td>Airtech, China</td>
			<td>SW-CJ-1FD</td>
			<td>To extract RNA</td>
		</tr>
		<tr>
			<td>Double distilled water</td>
			<td>Sangon Biotech, China</td>
			<td>A500197-0500</td>
			<td>To dilute cDNA</td>
		</tr>
		<tr>
			<td>Environmental Testing chamber&#160;</td>
			<td>Panasonic, Japan</td>
			<td>MLR-352H-PC</td>
			<td>To culture T. denrolimi</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge&#160;</td>
			<td>Eppendrof, Germany</td>
			<td>5418R</td>
			<td>To store RNA content</td>
		</tr>
		<tr>
			<td>Eppendorf FemtoJet 4i</td>
			<td>Eppendrof, Germany</td>
			<td>FemtoJet 4i</td>
			<td>To inject T. denrolimi</td>
		</tr>
		<tr>
			<td>Eppendorf Refrigerated Centrifuge&#160;</td>
			<td>Eppendrof, Germany</td>
			<td>5810R</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Ethanol solution (75%, RNase-free)</td>
			<td>Aladdin, China</td>
			<td>M052131-500ml</td>
			<td>To extract RNA</td>
		</tr>
		<tr>
			<td>Gel Extraction Kit</td>
			<td>Omega, USA</td>
			<td>D25000-02</td>
			<td>To extract cDNA</td>
		</tr>
		<tr>
			<td>GUM Arabic</td>
			<td>Solarbio, China</td>
			<td>CG5991-500g</td>
			<td>To make egg card</td>
		</tr>
		<tr>
			<td>Isopropyl alchohol</td>
			<td>Aladdin, China</td>
			<td>80109218</td>
			<td>To extract RNA</td>
		</tr>
		<tr>
			<td>Laser-Based Micropipette Puller&#160;</td>
			<td>SUTTER, USA</td>
			<td>P-2000</td>
			<td>To pull capillary glass needle</td>
		</tr>
		<tr>
			<td>Microloader&#160;</td>
			<td>Eppendrof, Germany</td>
			<td>20 &#181;L</td>
			<td>To load dsRNA</td>
		</tr>
		<tr>
			<td>Multi-sample tissue grinder&#160;</td>
			<td>LICHEN, China</td>
			<td>LC-TG-24</td>
			<td>To grind T. denrolimi</td>
		</tr>
		<tr>
			<td>Needle Grinder&#160;</td>
			<td>SUTTER, USA</td>
			<td>BV-10-E</td>
			<td>To grind capillary glass needle</td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Sangon Biotech, China</td>
			<td></td>
			<td>To dilute RNA</td>
		</tr>
		<tr>
			<td>OLYMPUS Microscope</td>
			<td>OLYMPUS, Japan</td>
			<td>XZX16</td>
			<td>To observe T. denrolimi</td>
		</tr>
		<tr>
			<td>PCR machine&#160;</td>
			<td>Bio-rad, USA</td>
			<td>S-1000</td>
			<td>For DNA amplification</td>
		</tr>
		<tr>
			<td>PowerPac Basic</td>
			<td>Bio-rad, USA</td>
			<td>PowerPacTM Basic</td>
			<td>To detect the quality of dsRNA&#160;</td>
		</tr>
		<tr>
			<td>Primer of dsGFP (Forward)</td>
			<td></td>
			<td></td>
			<td>[TAATACGACTCACTATAGGG] 
			ACAAACCAAGGCAAGTAATA</td>
		</tr>
		<tr>
			<td>Primer of dsGFP (Reverse)</td>
			<td></td>
			<td></td>
			<td>[TAATACGACTCACTATAGGG] 
			CAGAGGCATCTTCAACG</td>
		</tr>
		<tr>
			<td>Primer of Ferhch for qPCR (Forward)</td>
			<td></td>
			<td></td>
			<td>TGAAGAGATTCTGCGTTCTGCT</td>
		</tr>
		<tr>
			<td>Primer of Ferhch for qPCR (Reverse)</td>
			<td></td>
			<td></td>
			<td>CTGTAGGAACATCAGCAGGCTT</td>
		</tr>
		<tr>
			<td>Primer of Ferhch for RNAi (Reverse)</td>
			<td></td>
			<td></td>
			<td>[TAATACGACTCACTATAGGG]AG 
			TAGCCATCATCTTTCC</td>
		</tr>
		<tr>
			<td>Primer of Ferhch for RNAi(Forward)</td>
			<td></td>
			<td></td>
			<td>[TAATACGACTCACTATAGGG] 
			ACACTGTCAATCGTCCTG</td>
		</tr>
		<tr>
			<td>Primer of FoxO for qPCR (Forward)</td>
			<td></td>
			<td></td>
			<td>CTACGCCGATCTCATAACGC</td>
		</tr>
		<tr>
			<td>Primer of FoxO for qPCR (Reverse)</td>
			<td></td>
			<td></td>
			<td>TGCTGTCGCCCTTGTCCT</td>
		</tr>
		<tr>
			<td>PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time)</td>
			<td>TaKaRa, Japan</td>
			<td>RR047A</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantitative Real-time PCR&#160;</td>
			<td>Bio-rad, USA</td>
			<td>CFX 96 Touch</td>
			<td>To perform reverse transcriptase polymerase chain reaction (RT-PCR)&#160;</td>
		</tr>
		<tr>
			<td>Real-time PCR (TaqMan) Primer and Probes Design Tool</td>
			<td></td>
			<td></td>
			<td>https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool/</td>
		</tr>
		<tr>
			<td>T7 RiBoMAX Express RNAi System</td>
			<td>Promega, USA</td>
			<td>P1700</td>
			<td>To synthesis dsRNA &#160;in vitro&#160;</td>
		</tr>
		<tr>
			<td>TB Green Premix Ex TaqTM <img alt="Equation 1" src="/files/ftp_upload/66250/66250eq01.jpg" style="margin: auto;" /> (Til RnaseH Plus)</td>
			<td>TaKaRa, Japan</td>
			<td>RR820A</td>
			<td>To perform RT-qPCR</td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>KESHI, China</td>
			<td>GB/T682-2002</td>
			<td>To extract RNA</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Ambion, USA</td>
			<td>15596018</td>
			<td>To extract total RNA content from samples</td>
		</tr>
		<tr>
			<td>Ultra-low Temperature Freezer&#160;</td>
			<td>Thermo, USA</td>
			<td>Forma 911</td>
			<td></td>
		</tr>
	</tbody>
</table>

rna interference in the egg parasitoid, trichogramma dendrolimi matsumura

卵寄生虫（Trichogramma spp）被认为是针对农业和森林中各种鳞翅目害虫的有效生物防治剂。Trichogramma后代的未成熟阶段在宿主卵中发育，表现出显着的矮小（成体长度约为0.5毫米）。RNA干扰（RNAi）方法已成为阐明众多生物体基因功能的重要工具。然而，在某些小型寄生虫物种（如Trichogramma）中操纵RNAi通常带来了重大挑战。在这项研究中，我们提出了一种有效的 RNAi 方法。概述的程序包括从宿主卵中获取和分离单个 T. dendrolimi 标本、双链 RNA （dsRNA） 的设计和合成、T. dendrolimi 蛹的体外移植和培养、dsRNA 的显微注射，以及随后通过 RT-qPCR 分析评估靶基因敲低。本研究为在树状锥虫中进行RNAi实验提供了一个全面、视觉上详细的程序，从而使研究人员能够研究该物种的基因调控。此外，该方法适用于其他毛滴虫物种的RNAi研究或显微注射，只需稍作调整，使其成为在其他内寄生物种中进行RNAi实验的宝贵参考。

Trichogramma spp. are a group of egg parasitoids that have been extensively utilized as highly efficient biological control agents against a wide spectrum of lepidopteran pests in agricultural and forest ecosystems worldwide1,2,3,4. The application of mass-reared Trichogramma provides an environmentally friendly approach for the sustainable management of pests5,6,7. Understanding the molecular biology of Trichogramma wasps provides valuable insights into enhancing the mass-rearing efficiency and field performance of these biological control agents8,9by investigating the methodology of gene regulation and genome editing10.
Since the discovery of double-stranded RNA (dsRNA)-mediated specific genetic interference in Caenorhabditis elegans in 1998, the RNA-interference (RNAi) method has evolved into a vital genetic toolkit for exploring the regulatory mechanisms of organisms by suppressing the expression of target genes11. RNAi experiments have become a standard methodology widely applied to study gene function in numerous insect species12,13. Nevertheless, the manipulation of RNAi presents a formidable challenge in many parasitoid species, particularly among those belonging to the endoparasitic Chalcidoidea family14,15,16. The RNAi method has been documented in at least 13 parasitoid species14,15,16,17,18,19. Among these, the RNAi approach has been comprehensively conducted in Nasonia wasps and is applicable throughout the developmental stages, including embryos, larvae, pupae, and adults14,15,16. It is noteworthy that Nasonia wasps are ectoparasitoids, with their offspring developing in the interstitial space between the host pupa and the puparium, enabling their cultivation in vitro and making them tolerate certain treatments, such as micro-injection. Unlike Nasonia wasps, Trichogramma individuals undergo their entire embryonic, larval, and pupal development inner the host egg. The layer at embryo and larva stages (which may impede dsRNA permeability), vulnerability to damage, and the difficulty in surviving in vitro present formidable obstacles20,21,22. Additionally, the diminutive size of Trichogramma individuals, approximately ~0.5 mm in adult or pupal length, renders them exceedingly intricate to manipulate20,21,22.
In the present study, we outline a comprehensive procedure for conducting RNA interference (RNAi) experiments in Trichogramma denrolimi Matsumura. This procedure encompasses the following procedures: (1) the design and synthesis of double-stranded RNA (dsRNA), (2) microinjection of T. denrolimi pupae, (3) the transplantation and in vitro incubation of these pupae, and (4) the detection of target gene knockdown through RT-qPCR analysis. The target gene selected for the RNAi experiment is the ferritin heavy chain homology (Ferhch). FerHCH, an iron-binding protein, contains a ferroxidase center endowed with antioxidant capabilities, facilitating the oxidation of Fe2+ to Fe3+. It plays an indispensable role in the growth and development of various organisms by maintaining redox equilibrium and iron homeostasis. Depletion of FerHCH can result in the overaccumulation of iron, leading to irreversible tissue damage, and often culminating in significant phenotypic alterations, including growth defects, deformities, and mortality23,24. This study offers a step-by-step guide for conducting RNAi in T. denrolimi, which will be invaluable for investigating the gene functions within the broader context of Trichogramma wasps.

作者聚焦：研究 Wolbachia 诱导的 Thelytokous 孤雌生殖和通过 RNA 干扰的遗传工具包开发

卵寄生虫中的RNA干扰， Trichogramma dendrolimi Matsumura

Trichogramma wasps are a group of egg parasitoids used to control lepidopteran pests in agriculture. The intracellular bacteria Wolbachia induces thelytokous parthenogenesis in some Trichogramma wasps. We are trying to answer the mechanism of Wolbachia and sex-determination systems in Trichogramma wasps.We have developed the RNA interference method in Trichogramma wasps. This method is a vital genetic toolkit for investigating the gene functions of Trichogramma. The immature stages of Trichogramma offspring develop within the host egg.The body size of Trichogramma is extremely small and is approximately 0.5 millimeter in adult length. The manipulation of genetic tools such as RNA interference and genome editing presents a formidable challenge for these tiny parasitoid wasps. For decades, gene functions within the entire Trichogramma genus have been rarely explored.The present methodology provides a robust model for investigating gene regulation during developmental and physiological activities in Trichogramma wasps.

注射dsFerhch的T. denrolimi蛹的出现率显著低于注射dsGFP或未注射的蛹（表1）。在新出现的黄蜂中，51.85%的T. denrolimi黄蜂经受dsFerhch后发育出畸形的小翅膀。在注射dsGFP或未注射任何疫苗的黄蜂中未观察到变形的黄蜂（表1）。此外，注射dsFerhch的T. denrolimi蛹的腹部表现出黑色素，这是铁代谢异常的表型23。?...

Watch this Scientific Journal Video about RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura at JoVE.com

RNA干扰（RNAi）的操纵在许多体型较小的寄生虫物种（如 黄蜂毛 滴虫）中提出了巨大的挑战。本研究描述了一种有效的 RNAi 方法。本方法为研究 毛滴 虫黄蜂的基因调控提供了一个稳健的模型。

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and ...

Trichogramma 黄蜂是一组用于控制农业中鳞翅目害虫的卵寄生虫。细胞内细菌沃尔巴克氏体在一些毛滴虫黄蜂中诱导孤雌生殖。我们试图回答沃尔巴克氏体和Trichogramma黄蜂性别决定系统的机制。我们开发了Trichogramma黄蜂的RNA干扰方法。该方法是研究毛滴虫基因功能的重要遗传工具包。Trichogramma后代的未成熟阶段在宿主卵中发育。Trichogramma的体型非常小，成年长度约为0.5毫米。RNA干扰和基因组编辑等遗传工具的操纵为这些微小的寄生蜂带来了巨大的挑战。几十年来，整个Trichogramma属的基因功能很少被探索。本方法为研究毛滴虫黄蜂发育和生理活动期间的基因调控提供了一个稳健的模型。

rna-interference-egg-parasitoid-trichogramma-dendrolimi

Research

JoVE Journal

Biology

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

973 次观看.  Shenyang Agricultural University. Trichogramma 黄蜂是一组用于控制农业中鳞翅目害虫的卵寄生虫。细胞内细菌沃尔巴克氏体在一些毛滴虫黄蜂中诱导孤雌生殖。我们试图回答沃尔巴克氏体和Trichogramma黄蜂性别决定系统的机制。我们开发了Trichogramma黄蜂的RNA干扰方法。该方法是研究毛滴虫基因功能的重要遗传工具包。Trichogramma后代的未成熟阶段在宿主卵中发育。Trichogramma的体型非常小，成年长度约为0....