野生型雌性昆虫卵巢发育的解剖和分级
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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?…
Representative Results
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…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了重大科技创新项目（2020CXGC010802）的支持。
Materials
| Digital camera | Canon ( China ) co., LTD | EOS 800D | |
| Dropper | Qingdao jindian biochemical equipment co., LTD | ||
| Ethanol absolute (99.7%) | Shanghai Hushi Laboratory Equipmentco., LTD | ||
| Forceps | Vetus Tools co., LTD | ST-14 | |
| GT75 type halogen headlamp (1000 W) | Shanghai Yadeng Industry co., LTD | ||
| Helicoverpa armigera, Mythimna separate, Spodoptera litura, Spodoptera exigua | Jiyang district, Jinan city, Shandong province, China | ||
| Measuring cylinder, beaker, flask | Qingdao jindian biochemical equipment co., LTD | ||
| Net bag | Qingdao jindian biochemical equipment co., LTD | 0.5 m | |
| Net cages | Qingdao jindian biochemical equipment co., LTD | 30 cm x 30 cm | |
| Petri dishes | Qingdao jindian biochemical equipment co., LTD | 60 mm diameter |
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Cite This Article
Sindhu, L., Guo, S., Song, Y., Li, L., Cui, H., Guo, W., Lv, S., Yu, Y., Men, X. Dissection and Grading of Ovarian Development in Wild-Type Female Insects. J. Vis. Exp. (197), e65644, doi:10.3791/65644 (2023).