本研究得到了重大科技创新项目（2020CXGC010802）的支持。

注意：在诱捕野生型迁徙昆虫之前要注意安全措施，建议穿戴安全装备（手套、长袖衬衫和护目镜）。此外，不使用时请关闭疏水阀，以免出现其他安全隐患和灯过热。解剖前遵循安全规程很重要，例如戴手套、护目镜和实验室外套，以防止接触体液和化学物质。
1. 迁徙昆虫的诱捕
<ol><li>通过使用探照灯诱捕昆虫来开始此协议。在本协议中，测试昆虫来源为中国山东省济南市济阳区（北纬36.977088°，东经116.982747°）。</li>
	<li>使用探照灯灯的主体，即不生锈的钢，盒子，这是一个矩形的主体，GT75型卤素大灯，功率为1000W，将前照灯放在中间作为光源。</li>
	<li>在灯的内部和底部放置一个漏斗状的昆虫收集通道，放置一个直径为5厘米的昆虫收集箱，然后放置一个60阶的昆虫收集网袋（0.5 m x 0.5 m），用于收集被光困住的昆虫。已知的光投射距离地面约500 m。</li>
	<li>该协议强调野生型雌性的迁徙动态和卵巢发育;因此，收集不同种类的昆虫（这里的收集是在 2021 年至 2022 年期间的 4 月至 8 月进行的）。避免收集小型和受伤的昆虫，选择类似大小的大型害虫进行此实验。</li>
</ol>2.昆虫的制备
<ol><li>将所有收集的昆虫从网袋（0.5 m）转移到网笼（30 cm x 30 cm），然后提供含有10%蜂蜜水灭菌溶液的培养皿（喂食可选）。将笼子置于27±2°C，65%±12%相对湿度下，并在黑暗中保持8-12小时。</li>
	<li>选择同一天飞入笼子内的野生型雌性，小心地将它们转移到单独的小瓶管中，并用棉盖关闭每个管子。避免直接处理，否则可能会因压力过大而损坏或伤害害虫。 注意：所有野生型雌性均在夜间捕获，并在白天进行解剖。因此，每个实验都在一天内进行。</li>
</ol>3.昆虫麻痹法的制备方法（图1）
<ol><li>将选定的雌性害虫单独放入中间的苍蝇瓶管中，并通过握住吹枪针使用CO2 气体麻醉雌性害虫，以引起轻度麻痹。要确认害虫是否瘫痪，请使用软刷轻轻推或触摸害虫。对软刺激和不动没有反应表明瘫痪成功。 注意：低温（-20°C）也可用作麻痹昆虫的替代技术。</li>
</ol>4. 昆虫解剖
<ol><li>将新鲜瘫痪的雌性放入含有无水乙醇（10mL）的解剖培养皿中。为避免解剖过程中鳞片毛和翅膀粉末的影响，用无水酒精浸入活的或瘫痪的昆虫中，并用清水冲洗。</li>
	<li>使用两对镊子将背翅与胸部和腹部身体的交界处分开。</li>
	<li>将腹部转移到新的一次性培养皿中，装有适量的水（2-5mm深），并使用解剖镊子沿着背腹线从尖嘴到尾巴轻轻剥离腹部外骨骼。在另一侧重复相同的步骤，然后将其放入清水中以分散完整的组织。</li>
	<li>用镊子小心地剥离表皮脂肪组织，轻轻拉动并释放卵巢。</li>
	<li>使用解剖镊轻轻去除卵巢周围的脂肪颗粒和其他器官。一般害虫卵巢多在腹部两侧向内折叠，在展开卵巢的同时尝试在液体环境中操作，从中间慢慢剥离交配囊，挑出附着在卵巢管上的脂肪颗粒。</li>
	<li>从垂直的后端轻轻抓住卵巢和交配囊，然后小心地向下展开。为避免在展开过程中损坏，将卵巢转移到装有水的新培养皿或干净的培养皿中。握住卵巢尖端，向内展开卵巢;小心执行此步骤以避免卵泡受损。</li>
</ol>5. 分析卵巢组织解剖学数据
<ol><li>在此步骤中，根据卵的颜色和大小评估每种昆虫的卵发育，以判断其成熟度。然后，根据卵子发育情况判断卵巢等级。 注意：卵巢发育水平的划分主要分为卵子前或卵黄沉淀水平的卵发育。为了更清楚起见，昆虫的卵粒发育进一步分为几个阶段，如卵黄发生阶段、卵黄成熟阶段和卵黄死亡阶段。蛋粒的成熟度取决于鸡蛋的饱满度、颜色和大小来判断其成熟度。</li>
	<li>解剖后，确保将雌配囊组织与完整的卵巢分开，并观察形态以区分物种，因为大多数交配囊的解剖结构因物种而异。因此，使用交配囊来区分物种。</li>
	<li>在此步骤中，评估卵巢解剖结构，并分析卵巢发育分级。将卵巢组织分为五个等级（1级至5级）。<ol><li>寻找以下组织的变化和结构：一级（1）是发育的早期阶段（乳白色透明），腹部饱满，柔软，脂肪体蓬松，颜色浅，难以剥离。二级（2）是卵黄沉积阶段，在观察更长和更厚的卵巢管后，如果需要，可以单独对卵巢进行分级。三年级（3）的脂肪体较少，只有少数颗粒附着在子房上。四年级（4）看起来弹性较差，容易折断，子宫管中间可能存在一些卵子。五年级 （5） 很容易识别，身体脂肪较少或没有脂肪，卵巢萎缩和脆弱。</li>
	</ol></li>
	<li>根据实验需要使用数码相机拍摄图像。</li>
</ol>

通过卵巢生殖分析鉴定迁徙昆虫的解剖

作者没有利益冲突需要声明。

卵巢分析方法通常用于植物保护，以阐明昆虫飞行和种群的运动，以预测19,20,21，并详细阐述昆虫的生理变化。人们注意到，棉铃虫、粘虫、芋头毛虫、甜菜蛾等常见农业害虫独特的迁徙和快速扩散能力，使其他地区难以预测。一些研究认为 Athetis lepigone 是迁徙性害虫22,23，但...

昆虫的迁徙在全球昆虫分布的种群动态中起着至关重要的作用，这些昆虫如棉铃虫、东方粘虫、草地贪夜蛾（芋头毛虫）、甜菜粘虫等昆虫的种群动态，这些昆虫在中国被报告为严重害虫 1,2,3,4.迁徙距离长、季节变化大、迁徙性强、生态因素等因素给这些害虫的预测、预报和防控带来了很大的困难5。需要对有害生物迁移进行监测，以揭示根据气候变化或周期促进迁移性有害生物的适应性和行为变化6.为了维持它们的生长、繁殖和生存，昆虫在进化过程中获得了连续的适应性;这一系列的适应性生活使生殖系统发生了许多变化，例如迁徙策略导致在漫长的迁徙过程中控制卵巢发育。
卵巢发育在迁徙性害虫中很常见，这会影响其种群的增长7.因此，卵巢发育长期以来一直是迁徙性害虫研究的热点。一系列研究导致了几个卵巢发育指标和分类策略。到目前为止，已经使用了几种方法来分析卵巢发育，例如，Loxostege sticticalis - 草甸蛾 - 卵巢发育，包括初始羽化阶段、早期产卵期、产卵期和产卵期结束 8.一些研究人员根据迁徙性鳞翅目害虫的蛋黄颜色发育来划分卵巢水平，例如甜菜粘虫 S. exigua、Pseudaletia unipuncta - 真正的粘虫和 Cnaphalocrocis medinalis（水稻叶文件夹等）9,10,11,12.以往研究将棉铃虫、水稻卷叶虫等害虫的卵巢发育水平分为卵黄沉积期、卵粒成熟期、成熟等待期、卵源高峰期和产卵期结束13,14。欧洲玉米螟的卵巢发育分为6个发育阶段：卵黄沉积期、卵成熟期、卵前处置期、产卵高峰期和末产卵期15。
此外，同一属昆虫的发育阶段不同，草地贪夜蛾（草地贪夜蛾）的卵巢发育水平分为四个层次：卵黄沉积期、成熟等待分娩期、卵阳性高峰期和产卵期结束16。另一方面，甜菜蛾（甜菜蛾）的卵巢发育有五个层次：透明、卵生、卵成熟、产卵和晚期产卵水平17。
以前的研究只能使用卵黄的颜色成熟度、产卵和卵子发育来对单个卵巢发育水平到多个卵巢发育水平进行分类，但不能根据生殖系统的解剖结构进行分类。基于形态发生解剖学的卵巢发育是一个研究较少的领域。本文采用解剖方法，利用两种卵巢组织类型预测种群中的迁徙雌性，基于卵巢发育阶段和交配囊的解剖形态发生，阐述其生殖动力学，为区分迁徙野生型雌性提供直接证据。
一些研究发现，洄游 夜蛾科 昆虫物种经常被探照灯捕获18。大多数洄游 夜蛾科 昆虫的子房在迁徙初期处于发育的早期阶段，卵巢水平随着迁徙的进行而增加。本研究描述了卵巢发育等级的解剖方法，研究了探照灯捕获的不同雌性种群害虫的两种生殖组织。该方法不仅推进了对迁徙动态的研究，而且为昆虫分类、昆虫生理学研究、害虫预测和雌性害虫物种的预测提供了便利。

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			<td>Digital camera</td>
			<td>Canon ( China ) co., LTD</td>
			<td>EOS 800D</td>
			<td></td>
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			<td>Dropper</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td></td>
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			<td>Ethanol absolute (99.7%)</td>
			<td>Shanghai Hushi Laboratory Equipmentco., LTD</td>
			<td></td>
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			<td>Forceps</td>
			<td>&#160;Vetus Tools co., LTD</td>
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			<td>GT75 type halogen headlamp (1000 W)</td>
			<td>Shanghai Yadeng Industry co., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Helicoverpa armigera, Mythimna separate, Spodoptera litura, Spodoptera exigua</td>
			<td>Jiyang district, Jinan city, Shandong province, China</td>
			<td></td>
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			<td>Measuring cylinder, beaker, flask</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td></td>
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			<td>Net bag&#160;</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td>0.5 m&#160;</td>
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			<td>Net cages&#160;</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td>30 cm x 30 cm</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td>&#160;60 mm diameter</td>
		</tr>
	</tbody>
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dissection and grading of ovarian development in wild-type female insects

迁徙性害虫对全世界的粮食生产和安全构成严重挑战。可以使用探照灯陷阱监测和捕获迁徙性害虫。迁徙性有害生物预测的最重要技术之一是识别迁徙物种。但是，在大多数情况下，仅凭外观很难获得信息。因此，利用通过对雌性生殖系统进行系统分析获得的知识，有助于了解探照灯陷阱捕获的野生型迁徙昆虫的卵巢交配囊和卵巢发育分级的综合解剖形态。为证明该方法的适用性，直接评估了 蛤蜊、蛤蜊、 苔 藓荠蛾和 苔 藓蛾的卵巢发育状况和卵粒发育阶段，并研究了卵巢交配囊 Agrotis ipsilon、Spaelotis valida、Helicoverpa armigera、Athetis lepigone、Mythimna separata、Spodoptera litura、Mamestra brassicae 和 Spodoptera exigua，探索他们的关系。本工作展示了预测野生型迁徙昆虫的具体解剖方法，比较了不同迁徙昆虫的独特生殖系统。然后，进一步研究了两个组织，即卵巢和交配囊。该方法有助于预测野生型雌性迁徙昆虫生殖系统的动态和结构发育。

Migration of insects plays a vital role in population dynamics of global insect distribution for insects like Helicoverpa armigera - the cotton bollworm, Mythimna separate - the oriental armyworm, Spodoptera litura - the taro caterpillar, Spodoptera exigua - the beet armyworm, that have been reported as serious pests in China1,2,3,4. The long travel distances, seasonal movements, high fecundity of migratory pests, and ecological factors have brought great difficulties in the prediction, forecast, and control of these pests5. Pest migration monitoring is required to reveal the adaptability and behavioral changes that facilitate migratory pests according to climate changes or cycles6. To sustain their growth, reproduction, and survival, insects have acquired sequential adaptability during evolution; this series of adaptive life has generated many changes in the reproductive system, such as migratory strategy leading to control of ovarian development in the long migratory process.
Ovarian development is common in migratory pests, which affects the growth of their population7. Therefore, ovarian development has been a hot topic of migratory pest research for a long time. A series of studies have led to several ovarian development indicators and classification strategies. Until now, several methods have been used to analyze ovary development, e.g., Loxostege sticticalis - the meadow moth- ovary development which includes the initial feathering stage, the early spawning period, the spawning period, and the end of oviposition8. Some researchers divide ovarian levels on the bases of yolk color development in migratory Lepidopteran pests, such as S. exigua - the beet armyworm, Pseudaletia unipuncta - the true armyworm, and Cnaphalocrocis medinalis- the rice leaf-folder, etc.9,10,11,12. In previous studies, the ovarian development levels for pests, such as cotton bollworm and rice leaf roller, were divided into five stages: yolk deposition stage, egg grain maturity stage, mature waiting for birth, peak ovogenesis period, and end spawning stage13,14. The ovarian development of the European corn borer was divided into six developmental stages: yolk deposition stage, egg maturation, pre-eggs dispositioning, peak spawning stage, and end-spawning stage15.
Moreover, insects of the same genus have different stages of development, such as ovarian development levels of Spodoptera frugiperda - the fall armyworm - falls into four levels: yolk deposition stage, mature waiting for delivery, peak ovi-positivity, and end spawning stage16. On the other hand, ovarian development in Spodoptera exigua - the beet moth - has five levels: transparent, vitellogenesis, maturation of eggs, egg release, and late egg-laying levels17.
Former studies can only classify development from single to multiple ovarian development levels using color maturity of yolk, oviposition, and egg developments, but classification cannot be done based on anatomy of the reproductive system. The development of an ovary based on the morphogenesis anatomy is a less studied area. Here, the dissection method was designed to predict migratory females in the population using two ovarian tissue types, to elaborate their reproductive dynamics based on the anatomical morphogenesis of -ovarian development stage and mating sac- providing direct evidence to distinguish migratory wild-type females.
Some studies have found that, migratory Noctuidae insect species were frequently captured by searchlights18. The ovary of most migratory Noctuidae insect species is in the early stages of development during the initial stage of migration and the ovarian level increases with the migratory progress. In this study, the dissection method for ovarian development grades is described, to study the two reproductive tissues of different female population pests, captured by search light. This method not only advances the research to understand the migratory dynamics, but also facilities in insect classification, insect physiology study, pest prediction, and forecasting of female pest species.

作者聚焦：分析迁徙昆虫的卵巢解剖结构以克服实验挑战 

野生型雌性昆虫卵巢发育的解剖和分级

卵子的发育 上述方案用于分析卵巢中卵子的发育。为此，首先将卵分为四个阶段，以区分所有物种（如棉铃虫、粘虫、芋头毛虫和甜菜蛾）的卵发育早期和成熟阶段。在这里，观察到羽化的早期阶段（乳白色透明阶段）。 图2A 显示卵巢尚未开始发育，卵巢导管呈丝状，弹性好。由于鸡蛋的美味，形成的蛋粒在日光下无法看到，并且通过拉伸卵巢管可以看到透...

Watch this Scientific Journal Video about Analysis of Ovarian Anatomy in Migratory Insects to Overcome Experimental Challenges at JoVE.com

该协议展示了一种简单易行的解剖方法，适用于用探照灯陷阱捕获的野生型迁徙雌性昆虫。该技术可以通过比较两种生殖组织（即野生型雌性昆虫的交配囊和卵巢发育）来显着阐明同一物种。

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using ...

我们的目标是将这种简单和最新的方法应用于迁徙性害虫，以描述迁徙雌性的卵巢解剖结构。这些信息将有助于分析相似的单个物种。预测迁徙的雌性昆虫是相当具有挑战性的。因此，卵巢解剖结构的可视化非常容易且非常有用。

dissection-grading-ovarian-development-wild-type-female

Research

JoVE Journal

Behavior

Dissection and Grading of Ovarian Development in Wild-Type Female Insects

990 次观看.  Shandong Academy of Agricultural Sciences. 我们的目标是将这种简单和最新的方法应用于迁徙性害虫，以描述迁徙雌性的卵巢解剖结构。这些信息将有助于分析相似的单个物种。预测迁徙的雌性昆虫是相当具有挑战性的。因此，卵巢解剖结构的可视化非常容易且非常有用。

视频: 作者聚焦：分析迁徙昆虫的卵巢解剖结构以克服实验挑战 

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using searchlight traps. One of the most important techniques for migratory pest forecasting is to identify the migratory species. However, in most cases, it is difficult to get the information just by appearance. Therefore, using knowledge acquired by systematic analysis of the female reproductive system can help to understand the combined anatomic morphology of the ovarian mating sac and ovary developmental grading of wild-type migratory insects captured with searchlight traps. To demonstrate the applicability of this method, ovarian development status and egg grain development stages were directly assessed in Helicoverpa armigera, Mythimna separata, Spodoptera litura, and Spodoptera exigua for the ovarian anatomy, and the ovarian mating sacs were studied in Agrotis ipsilon, Spaelotis valida, Helicoverpa armigera, Athetis lepigone, Mythimna separata, Spodoptera litura, Mamestra brassicae, and Spodoptera exigua, to explore their relationships. This work shows the specific dissection method to predict wild-type migratory insects, comparing the unique reproductive system of different migratory insects. Then, both tissues, namely, the ovary and mating sacs, were further investigated. This method helps to predict the dynamics and the structural development of reproductive systems in wild-type female migratory insects.

The protocol demonstrates a simple and easy dissection method, suitable for wild-type migratory female insects captured with searchlight traps. This technique can significantly clarify the same species by comparing both reproductive tissues, namely the mating sac and ovarian development of wild-type female insects.