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.

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

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

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 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 ...

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.

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

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992 Views.  Shandong Academy of Agricultural Sciences. 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.

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

Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice

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Disease, head injury, genetic modifications, and treatment of mice with drugs can have profound effects on behavior. Utilizing well-characterized and validated approaches such as marble burying and nestlet shredding, compulsive-like behaviors can be documented accurately in mice as models of human obsessive-compulsive disorder and autism spectrum disorder.

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a ...

A Method for Investigating Age-related Differences in the Functional Connectivity of Cognitive Control Networks Associated with Dimensional Change Card Sort Performance

The ability to adjust behavior to sudden changes in the environment develops gradually in childhood and adolescence. For example, in the Dimensional Change Card Sort task, participants switch from sorting cards one way, such as shape, to sorting them a different way, such as color. Adjusting behavior in this way exacts a small performance cost, or switch cost, such that responses are typically slower and more error-prone on switch trials in which the sorting rule changes as compared to repeat trials in which the sorting rule remains the same. The ability to flexibly adjust behavior is often said to develop gradually, in part because behavioral costs such as switch costs typically decrease with increasing age. Why aspects of higher-order cognition, such as behavioral flexibility, develop so gradually remains an open question. One hypothesis is that these changes occur in association with functional changes in broad-scale cognitive control networks. On this view, complex mental operations, such as switching, involve rapid interactions between several distributed brain regions, including those that update and maintain task rules, re-orient attention, and select behaviors. With development, functional connections between these regions strengthen, leading to faster and more efficient switching operations. The current video describes a method of testing this hypothesis through the collection and multivariate analysis of fMRI data from participants of different ages.

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Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

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Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

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An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

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The Drosophila model has been invaluable for the study of neurological function and for understanding the molecular and cellular mechanisms that underlie ...