Method for observing external genitals of female adult Gynura without dissection

Abstract

The invention discloses a method for observing external genitals of female adult Gynura without dissection, and belongs to the technical field of insect identification. The invention discloses a method for observing external genitals of female adult Gynura without dissection. The method comprises the following steps: firstly, preparing a 600-fold diluent of 40% chlorantraniliprole thiamethoxam water dispersible granules with water; immersing a living female adult Gynura into the liquid medicine for 1-3 minutes, then taking out the female adult, and standing for 0-4 hours until the external genitals of the female adult are completely exposed; then transferring the female adults with exposed external genitals into a centrifugal tube filled with absolute ethyl alcohol to be soaked for 2 minutes; and finally, taking out the imago, and observing the exposed external genitalia structure under a microscope. The method disclosed by the invention is simple and convenient to operate, an identification technology, namely genital microdissection, which can be mastered by only a few professionals is avoided, and an almost perfect female adult external genital form is obtained, so that an identification threshold is remarkably reduced.

Description

Technical Field

[0001] This invention relates to the field of insect identification technology, and more specifically to a method for observing the genus *Gnaphalium* without dissection. Carpophilus Methods for examining the external genitalia of adult females. Background Technology

[0004] The macadamia nut beetle is a significant pest of macadamia nuts. The term "nitidulidae" refers to insects in the family Nitidulidae of the order Coleoptera. These are a group of polyphagous insects distributed worldwide, with approximately 4,500 species recorded globally, and about 150 species recorded in my country. They can damage both field crops and stored goods, including field crops, fruits, vegetables, forests, medicinal herbs, and dried fruits. Some nitidulidae feed on sap, fungi, and fermenting, decaying plant and animal tissues; some are predatory; some can spread pathogens, causing damage to crops and trees, and have even caused large-scale outbreaks of oak wilt disease; damaged grains become moldy, rotten, and produce toxins harmful to humans and animals, significantly reducing yield and quality; furthermore, some can act as natural pollinators, increasing fruit set and yield in fruit trees. Therefore, the nitidulidae has extremely important economic significance.

[0005] In macadamia nut producing areas of Yunnan, the thoracic breech-end beak has been discovered. Carpophilus dimidiatus (Fabricius)], pseudospora brevis (Fabricius) Carpophilus mutilatus Erichson), large armpit tail nail ( Carpophilus marginellus Species such as *Motschulsky* damage macadamia nut kernels; these species all belong to the genus *Motschulsky*. Carpophilus Stephens (1829). This genus contains 195 known species worldwide, with 18 species recorded in China.

[0006] The external genitalia (i.e., ovipositor) of adult females are an important identifying feature of insects in the family Asparagidae, and play a key role, especially in the identification of similar species.

[0007] Previously, the external genitalia of female adults were mainly obtained through dissection. As documented in the literature by Chen Xiaoxiao, Liu Meike, Chen Ying, Hui Ziyan, Wang Guoli, and Natalia Reales, this was achieved through dissection. The specific method was as follows: Fresh specimens were dissected directly. The fresh specimen was placed ventrally upwards between the index finger and the thumb, pressing down on the cephalothorax to fix it, exposing the abdomen. A very thin, scalpel-like needle was used to repeatedly puncture the dorsal membrane connecting the anal plate and the terminal sternum. A No. 0 needle (with a self-made hook) fixed to a thin wooden stick or pen refill was inserted as deeply as possible into the abdomen. When the external genitalia, which were covered by muscle, were felt, the needle was hooked and slowly removed (to prevent tearing the muscle and damaging the external genitalia structure). A drop of glycerin was placed on a concave slide beforehand. The removed external genitalia with muscle tissue was placed in the glycerin, and a small amount of muscle outside the external genitalia was gently removed with a No. 0 or No. 00 insect needle. If the muscle was difficult to remove directly, it needed to be dissolved in 10% NaOH. After processing, the specimen can be directly observed, identified, photographed, and drawn. If it is a dry specimen, use a small brush to apply a small amount of water to the joint between the specimen and the cardboard to soften the glue and allow the specimen to separate naturally. Soak it in warm water for 6 hours until it softens. Place the softened specimen on the tip of your left index finger, ventral side up, and add a drop of water to prevent it from becoming brittle under the light of a stereomicroscope. Use your left thumb to fix the insect's cephalothorax, exposing the abdomen. Use a homemade dissecting needle (a No. 0 insect needle fixed to the tip of a pen refill or disposable chopstick with a homemade hook at the tip) or dissecting forceps to gently insert into the dorsal membrane between the fifth abdominal segment and the anal plate. Pry open the end of the abdomen and gently insert the dissecting needle or dissecting forceps into the insect's abdomen to hook out the genitals. Place them on a concave slide pre-dried with glycerin to remove excess muscle. If the muscle is difficult to remove, soak it in a 10% NaOH solution for 12 hours or boil it for 5 minutes to remove excess muscle. Rinse the treated genital tissue several times with water and add glycerin for observation. After observation, the genitals were placed in a 0.2 ml centrifuge tube containing glycerol and inserted below the adult specimen for preservation.

[0008] Obtaining the external genitalia through dissection is a highly specialized and difficult task, requiring skilled professionals and involving potential damage to the insect. This is because the beetle itself is relatively small (most are only 1-3 mm in length), and the female adult's external genitalia are even smaller (most are only 0.5 mm). Only those proficient in microscopic dissection techniques can obtain the female adult's external genitalia intact. This undoubtedly raises the bar for species identification using this characteristic, making it difficult for the average person to perform. However, to perfectly present the identification feature, researchers have had to use hand-drawn illustrations to compensate for potential damage during dissection. This is why early identification atlases often used hand-drawn illustrations (such as...). Figure 1 One of the important reasons is that...

[0009] Furthermore, since the insect specimens used for dissection are mostly dried specimens after death, the external genitalia of these specimens often lose their original extended shape due to twisting, shriveling, etc., making it impossible to clearly display their natural characteristics, thus causing distortion of identification information. For example, Figure 2 The external genitalia of the female adult *Ceratophora melilotus* shown in the image exhibit this problem, affecting the accurate identification of the species (correspondingly, Figure 8 (This demonstrates the natural characteristics of the external genitalia of adult female *Ceratophora gracilis*). Based on this, the field of morphological identification urgently needs to develop a method that can easily obtain the external genitalia of adult female *Ceratophora* while preserving their natural, perfect state.

[0010] Therefore, providing a method for observing the external genitalia of adult female *Gnaphalium* without dissection is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0011] In view of this, the present invention provides a method for observing the external genitalia of female adult beetles of the genus *Gnaphalium* without dissection.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: A method for observing the external genitalia of adult female *Gnaphalium* beetles without dissection includes the following steps: (1) Preparation of chlorantraniliprole·thiamethoxam dilution: 40% chlorantraniliprole·thiamethoxam water-dispersible granules were diluted with water at a mass ratio of 1:599 to obtain a 600-fold diluted solution of chlorantraniliprole·thiamethoxam. (2) Infiltrating insects: Live female adult beetles of the genus *Gnaphalium* were transferred into a 50-100 mesh sieve bag. The entire sieve bag was then immersed in a 600-fold diluted solution of chlorantraniliprole and thiamethoxam for 1-3 minutes. After removal, the beetles were left to stand for 0-4 hours until their external genitalia were fully exposed. Different species have varying sensitivities, so the time to reach maximum exposure varies (for example, the large axillary tract has strong resistance and takes 4 hours). Even within the same species, the time it takes for the external genitalia to be fully exposed does not always coincide for different individuals.

[0013] (3) Fixed: Female adult worms with fully exposed external genitalia were immersed in centrifuge tubes containing anhydrous ethanol for 2 minutes to fix their exposed external genitalia. (4) Observation: The morphology of the exposed external genitalia was observed under a stereomicroscope, and the characteristics were recorded by microscopic photography.

[0014] As can be seen from the above technical solution, compared with the prior art, this invention discloses a method for observing the external genitalia of female adult *Gnaphalium* without dissection. By utilizing the action of chlorantraniliprole-thiamethoxam solution, the female adult *Gnaphalium* is induced to actively expose its external genitalia. This method is simple to operate, avoids the identification technique of genital microdissection mastered only by a few professionals, and overcomes the defect of easily deformed female adult external genitalia in desiccated specimens, obtaining near-perfect morphology of female adult external genitalia, thus significantly lowering the identification threshold. It is particularly important to note that the female adult *Gnaphalium* used in this method are live. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 A hand-drawn illustration of the female adult's external genitalia (Ovipositor); in which (a) the thoracic breech beak is exposed. Carpophilus dimidiatus (Fabricius, 1792)];(b) Exposed tail armor (likely a type of thoracic armor) Carpophilus mutilatus (Erichson, 1843); derived from Dobson, 1954.

[0017] Figure 2 Anatomical diagram of the external genitalia of a female adult of the large-axillary beetle.

[0018] Figure 3 After implementing the method of the present invention, the thoracic spine and tail carapace are exposed. Carpophilus dimidiatus The female adult's external genitalia are exposed.

[0019] Figure 4 This is a magnified view of the external genitalia of a female adult spinothoracic beetle after implementation of the method of the present invention.

[0020] Figure 5 The method of this invention is used to expose the tail carapace (spinal thoracic shell) after implementation. Carpophilus mutilatus The female adult's external genitalia are exposed.

[0021] Figure 6 This is a magnified view of the external genitalia of a female adult *Pterygota tectorum* after implementation of the method of this invention.

[0022] Figure 7 After the implementation of the method of the present invention, the tailbone is exposed in the axilla ( Carpophilus marginellus The female adult's external genitalia are exposed.

[0023] Figure 8 This is a magnified view of the external genitalia of a female adult beetle with exposed tail after the implementation of the method of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 A method for observing the external genitalia of adult female *Gnaphalium* beetles without dissection includes the following steps: (1) Preparation of chlorantraniliprole·thiamethoxam dilution: 40% chlorantraniliprole·thiamethoxam water-dispersible granules were diluted with water at a mass ratio of 1:599 to obtain a 600-fold diluted solution of chlorantraniliprole·thiamethoxam. (2) Infiltrating insects: Five live female adult beetles (3 groups in total) were placed in a 100-mesh sieve bag. The entire sieve bag was then immersed in a 600-fold diluted solution of chlorantraniliprole and thiamethoxam for 1 minute. After removal, the beetles were left to stand for 0-2 hours until their external genitalia were fully exposed.

[0026] The quantity and proportion of exposed items are shown in Table 1.

[0027] The results of screening tests for different drugs are shown in Table 2.

[0028] (3) Fixed: Female adult worms with fully exposed external genitalia were immersed in centrifuge tubes containing anhydrous ethanol for 2 minutes to fix their exposed external genitalia.

[0029] (4) Observation: The morphology of the exposed external genitalia was observed under a stereomicroscope, and microscopic photographs were taken to record the characteristics. Results are shown below. Figure 3 and Figure 4 .

[0030] Table 1. Proportion of female adult beetles with exposed genitalia within 2 hours of exposure.

[0031] Table 2. Proportion of female adult spinoceretta beetles with exposed genitalia after treatment with different agents (4 hours)

[0032] Note: Different lowercase letters in the same column indicate significant differences (p=0.05); all agents were diluted 600 times with water; for 40% chlorantraniliprole·thiamethoxam water-dispersible granules, the exposure rate at 4 hours in Table 2 was the same as the exposure rate at 2 hours in Table 1, indicating that the values ​​did not change between 2 and 4 hours.

[0033] Table 2 shows that the pesticide with the highest exposure rate is 40% chlorantraniliprole·thiamethoxam water dispersible granules.

[0034] Example 2 A method for observing the external genitalia of adult female *Gnaphalium* beetles without dissection includes the following steps: (1) Preparation of chlorantraniliprole·thiamethoxam dilution: 40% chlorantraniliprole·thiamethoxam water-dispersible granules were diluted with water at a mass ratio of 1:599 to obtain a 600-fold diluted solution of chlorantraniliprole·thiamethoxam. (2) Infiltrating insects: Five live female adult beetles were placed in a group of three (total of three groups) into an 80-mesh sieve bag. The entire sieve bag was then immersed in a 600-fold dilution of chlorantraniliprole and thiamethoxam for 2 minutes. After removal, the beetles were left to stand for 0–3 hours until their external genitalia were fully exposed. The number and proportion of exposed beetles are shown in Table 3.

[0035] (3) Fixed: Female adult worms with fully exposed external genitalia were immersed in centrifuge tubes containing anhydrous ethanol for 2 minutes to fix their exposed external genitalia.

[0036] (4) Observation: The morphology of the exposed external genitalia was observed under a stereomicroscope, and microscopic photographs were taken to record the characteristics. Results are shown below. Figure 5 and Figure 6 .

[0037] Table 3. Proportion of female adult *Pterygota tectorum* with exposed genitalia within 3 hours.

[0038] Example 3 A method for observing the external genitalia of adult female *Gnaphalium* beetles without dissection includes the following steps: (1) Preparation of chlorantraniliprole·thiamethoxam dilution: 40% chlorantraniliprole·thiamethoxam water-dispersible granules were diluted with water at a mass ratio of 1:599 to obtain a 600-fold diluted solution of chlorantraniliprole·thiamethoxam. (2) Infiltrating insects: Five live adult female beetles were placed in a group of three (total of three groups) into a 50-mesh sieve bag. The entire sieve bag was then immersed in a 600-fold diluted solution of chlorantraniliprole and thiamethoxam for 2 minutes. After removal, the beetles were left to stand for 0–4 hours until their external genitalia were fully exposed. The number and proportion of exposed beetles are shown in Table 4.

[0039] (3) Fixed: Female adult worms with fully exposed external genitalia were immersed in centrifuge tubes containing anhydrous ethanol for 2 minutes to fix their exposed external genitalia.

[0040] (4) Observation: The morphology of the exposed external genitalia was observed under a stereomicroscope, and microscopic photographs were taken to record the characteristics. Results are shown below. Figure 7 and Figure 8 .

[0041] Table 4. Proportion of female adult beetles with exposed genitalia within 4 hours

[0042] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for observing the external genitalia of female adult *Gnaphalium* beetles without dissection, characterized in that, Includes the following steps: (1) Preparation of chlorantraniliprole·thiamethoxam dilution: 40% chlorantraniliprole·thiamethoxam water-dispersible granules were diluted with water at a mass ratio of 1:599 to obtain a 600-fold diluted solution of chlorantraniliprole·thiamethoxam. (2) Infiltrating insects: Live female adult beetles of the genus *Gnaphalium* were transferred into a 50-100 mesh sieve bag. The entire sieve bag was then immersed in a 600-fold diluted solution of chlorantraniliprole and thiamethoxam for 1-3 minutes. After removal, the beetles were left to stand for 0-4 hours until their external genitalia were fully exposed. (3) Fixed: Female adult worms with fully exposed external genitalia were immersed in centrifuge tubes containing anhydrous ethanol for 2 minutes to fix their exposed external genitalia. (4) Observation: The morphology of the exposed external genitalia was observed under a stereomicroscope, and the characteristics were recorded by microscopic photography.

Images