This study was supported by the major scientific and technological innovation project (2020CXGC010802).

NOTE: Pay attention to safety measurements before trapping wild-type migratory insects, it is suggested to wear safety gear (gloves, long-sleeved shirts, and goggles). Also, turn off the trap when not in use to avoid other safety hazards and overheating the light. It is important to follow safety protocols before dissection, such as wearing gloves, goggles, and lab coat to prevent exposure to body fluids and chemicals.
1. Trapping of migrant insects
<ol>
	<li>Begin this protocol by trapping insects using the searchlight lamp. In this protocol, test insect source is Jiyang district, Jinan city, Shandong province, China (36.977088&#176; N, 116.982747&#176; E).</li>
	<li>Use the main body of the searchlight lamp, that is made of non-rusted steel, the box, which is a rectangular body, and the GT75 type halogen headlamp, with a power of 1000 W. Place the headlamp in the middle as the light source.</li>
	<li>Place a funnel-shaped insect-collecting channel inside and at the bottom of the light, place a box for insect-collection with a diameter of 5 cm, followed by a 60-order insect-collecting net bag (0.5 m x 0.5 m), which is used to collect insects trapped by light. The known projection of the light is about 500 m above the ground.</li>
	<li>This protocol emphasizes on the immigratory dynamics of wild-type females and ovary development; therefore, collect different species of insects (here collection was done from April to August, during 2021 to 2022). Avoid collecting small sized and injured insects, select similarly sized large pests for this experiment.</li>
</ol>
2. Preparation of insects
<ol>
	<li>Transfer all collected insects from net bag (0.5 m) to the net cage (30 cm x 30 cm), and then provide a Petri dish containing sterilized solution of 10% honey water (feeding is optional). Place the cage at 27 &#177; 2 &#176;C, 65% &#177; 12 % relative humidity, and a maintain in dark for 8-12 h.</li>
	<li>Select wild-type females that have flown inside the cage on the same day, carefully transfer them into the individual vial tubes, and close each tube with a cotton lid. Avoid direct handling, that could damage or injure the pest due to excessive pressure. 
	&#8203;NOTE: All wild-type females were captured in the night-time and dissection was performed in daytime. Thus, each experiment was performed within a day.</li>
</ol>
3. Preparation for insect paralyzing method (Figure 1)
<ol>
	<li>Place selected female pest individually into a fly vial tube in the middle and anesthetize female using CO2 gas by holding blow gun needle to cause mild paralysis. To confirm whether the pest is paralyzed or not, gently nudge or touch the pest using a soft brush. No response to soft stimuli and immobility indicates a successful paralysis. 
	&#8203;NOTE: Low temperature (-20 &#176;C) can also be used as an alternative technique to paralyze insects.</li>
</ol>
4. Dissection of insects
<ol>
	<li>Place freshly paralyzed female into the dissecting Petri dish containing absolute ethanol (10 mL). To avoid the influence of scale hairs and powder of wings during dissection, infiltrate the living or paralyzed insect with absolute alcohol and rinse in clean water.</li>
	<li>Separate the dorsal wings from the junction of the chest and abdomen body, using two pairs of forceps.</li>
	<li>Transfer the abdomen into a new disposable Petri dish, containing an appropriate amount of water (2-5 mm deep), and gently peel the abdominal exoskeleton along the dorsal ventral line from the pointed mouth to the tail, using dissecting forceps. Repeat the same steps on the other side, and then put it into clean water to disperse the intact tissues.</li>
	<li>Carefully peel off the epidermis fat tissues using forceps, and gently pull and release the ovaries.</li>
	<li>Use dissecting forceps to gently remove fat particles and other organs around the ovaries. Generally, the pest ovaries are mostly folded inward on both sides of the abdomen, try to operate in a liquid environment while unfolding the ovaries, and slowly peel off the mating sac from the middle, and pick out the fat particles attached to the ovarian tubes.</li>
	<li>Gently hold on to the ovary and mating sac from the vertical posterior end and unfold it carefully downwards. To avoid damage during unfolding, transfer the ovary into a new or clean Petri dish containing water. Hold the ovary tip and unfold the ovary inwardly; carefully perform this step to avoid damage of the ovarioles.</li>
</ol>
5. Analyzing data for ovarian tissues anatomy
<ol>
	<li>At this step assess the eggs development for each insect, following the color and size of the eggs, to judge its maturity. Then, judge the ovarian grade according to egg development. 
	NOTE: The division of various ovarian development levels is mainly divided, either before egg laying or egg development with yolk precipitation level. Insects egg grain development are further divided into stages for more clarity such as, yolk occurrence stage, yolk maturity stage, and yolk demise stage. The maturity of egg grain depends on the fullness, color, and size of the egg to judge its maturity.</li>
	<li>After dissection, ensure to separate the female mating sacs tissues from the intact ovaries, and observe the morphology to distinguish the species because most mating sacs anatomy varies from species to species. Therefore, use mating sacs to distinguish between species.</li>
	<li>At this step, evaluate the ovarian anatomy, and analyze the ovarian development grading. Divide the ovarian tissues into five grades (grade 1 to grade 5).
	<ol>
		<li>Look for the following changes and structure to organize the tissue: first grade (1) is an early stage of development (milky transparent), full abdomen, soft, fat body fluffy, light color, difficult to peel. Second grade (2) is the yolk deposition stage and grade the ovaries separately if needed after observing the longer and thicker ovarian canal. Third grade (3) has fewer fat bodies and only a few granules attached to the ovary. Fourth grade (4) appears less elastic and easy to break, and some eggs can be present in the middle of uterine tube. Fifth grade (5) is easy to identify with less or no fat bodies, atrophic and fragile ovaries.</li>
	</ol>
	</li>
	<li>Capture images using a digital camera as per experimental need.</li>
</ol>

Dissection for Identifying Migratory Insects via Ovarian Reproductive Analysis

The authors have no conflicts of interest to declare.

Ovarian analysis methods are routinely used in plant protection, to elucidate the movement of insect flight and population for forecasting19,20,21 and to elaborate on the physiological variations in insects. It has been noticed that the unique migration and rapid dispersion ability of common agricultural pests, such as bollworm, armyworm, taro caterpillar,&#160;and beet moth, make it difficult for prediction from other regions. ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digital camera</td>
			<td>Canon ( China ) co., LTD</td>
			<td>EOS 800D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dropper</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute (99.7%)</td>
			<td>Shanghai Hushi Laboratory Equipmentco., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>&#160;Vetus Tools co., LTD</td>
			<td>ST-14</td>
			<td></td>
		</tr>
		<tr>
			<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>
			<td></td>
		</tr>
		<tr>
			<td>Measuring cylinder, beaker, flask</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Net bag&#160;</td>
			<td>Qingdao jindian biochemical equipment co., LTD</td>
			<td></td>
			<td>0.5 m&#160;</td>
		</tr>
		<tr>
			<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>
</table>

dissection and grading of ovarian development in wild-type female insects

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.

Author Spotlight: Analysis of Ovarian Anatomy in Migratory Insects to Overcome Experimental Challenges 

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

We aim to apply easy and this newest method for migratory pest, in describing the ovarian anatomy of migratory females. This information will be useful to analyze similar individual species. To predict migratory female insects is pretty challenging.Therefore, visualization of ovarian anatomy is quite easy and very useful.

Development of the eggs 
The above protocol was applied to analyze the development of eggs in the ovary. For this purpose, firstly, eggs were classified generally into four stages to distinguish early and mature stage of egg development among all species e.g., bollworm, armyworm, taro caterpillar, and beet moth. Here, the early stage of feathering (milky white transparent stage) was observed. Figure 2A shows that ovaries have not yet begun to develop, the ovarian duct i...

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

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.

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

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

Research

JoVE Journal

Behavior

Noctuidae Insect Preparation, Dissection, and Ovarian Tissue Analysis

Noctuidae Insect Preparation, Dissection, and Ovarian Tissue Analysis  

Begin by transferring all the collected Noctuidae insects from the net bag to a net cage. Next, provide a Petri dish containing a sterilized 10%honey water solution. Set the cage in a stable environment.Choose wild type female insects that have ventured inside the cage that same day. Carefully transfer these insects into individual vial tubes, ensuring each tube is sealed with a cotton lid. For insect dissection, first place a freshly paralyzed female insect into a dissecting Petri dish containing absolute ethanol.To ensure a clean dissection, infiltrate the insect with absolute alcohol and rinse it in clean water. Next, using forceps, separate the dorsal wings at the junction of the chest and abdomen. Transfer the abdomen into a new disposable dish filled with two to five millimeters of water.Using dissecting forceps, gently peel the abdominal exoskeleton, starting from the pointed mouth dorsoventrally to the tail. Replicate these steps on the other side and place it in clean water to disperse the intact tissues. Now delicately peel off the epidermis fat tissues using forceps and gently pull and release the ovaries.With a pair of dissecting forceps, gently remove fat particles and any surrounding organs from the ovaries. Gradually peel off the mating sac from the middle and eliminate fat particles attached to the ovarian tubes. Gently hold onto the ovary and mating sac from the vertical posterior end, then slowly unfold it downwards.To prevent damage, transfer the ovary into a new or clean Petri dish filled with water. Hold the ovarian tip and unfold the ovary inwardly. Assess the egg maturity for the insect using color and size indicators.Then judge the ovarian grade according to the egg development. After dissection, make sure to separate the female mating sac tissues from the intact ovaries. Examine the morphology as mating sac anatomical features differ from species to species.Next, analyze the ovarian anatomy by dividing the ovarian tissues into five distinct grades. Lastly, capture images of the ovary using a digital camera as per the requirements of the experiment. Four different stages of ovarian development were observed, including the milky white transparent stage with underdeveloped ovaries, the yolk deposition stage with large slightly immature eggs, the ripening stage with bright yellow bead-shaped eggs and the end spawning stage with few or no egg grains.The mating sac of a wild type insect Helicoverpa armigera had a columnar right helix with an inner lumen. Mythimna separata had a G-shaped mating sac with dark brown bands. The mating sac of Agrotis ipsilon was linear with large coils outer back cysts.Spaelotis valida had a slightly brownish thin J-shaped mating sac, while Spodoptera exigua showed a white translucent dumbbell-shaped sac with a bubble cyst in the middle. The mating sac of Spodoptera litura was red with an upper milky white transparent mouth and a faintly visible internal cystic cavity. Pseudoptera lepigone mating sac was small and coiled with closed outer cysts.Mamestra brassicae had olive colored double coiled sacs with outer cysts. Old worm ovarian grading showed transparent and elastic ovarian ducts with grade one visible oocytes. At grade two, the ovarian duct became transparent with tender yellow eggs.At grade three, large fat bodies were absent with the duct being thickest at the ovipositor. Grades four and five showed clusters and fewer eggs in the ovaries with pale yellow and greenish color. All five ovarian grades were also observed in the armyworm, in the tarot caterpillar, and in the bollworm.

933 Views.  Shandong Academy of Agricultural Sciences.  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.

Dissection for Identifying Migratory Insects via Ovarian Reproductive Analysis

Summary