Microinjection method of scleroderma guani eggs

Abstract

The invention provides a microinjection method of scleroderma guani eggs. The method mainly comprises the following steps: firstly, inducing Scleroderma guani to lay eggs and collecting the Scleroderma guani; long-horned beetle larvae are provided for female scleroderma guani which is mated after eclosion, and eggs on the surfaces of the long-horned beetle larvae are collected after the female scleroderma guani spawns. Secondly, the collected scleroderma guani eggs are arranged on the edge of the cover glass, and the rear ends of the eggs are uniformly arranged in the direction where the injection needle is located; then, the injection needle is drawn, microinjection is conducted on the egg granules, and the injection position is the rear ends of the egg granules; and finally, transferring the injected egg granules to the surface of the frozen long-horned beetle larva for culturing. The method is reasonable and efficient in design, conforms to the egg growth characteristics of the scleroderma guani, and provides a brand new way for gene function research of the scleroderma guani.

Description

Technical Field

[0001] This invention relates to the field of entomology, specifically to a microinjection method for eggs of the tube-shaped parasitic wasp. Background Technology

[0002] *Symplocos guani* is an ectoparasitic wasp that paralyzes its host with venom and lays eggs on the host's surface. The hatched larvae feed on the host's hemolymph, causing death. *Symplocos guani* is widely used to control borer pests such as longhorn beetle larvae and has high economic value. However, current methods for optimizing the parasitism efficiency of *Symplocos guani* face bottlenecks, with research at the gene function level lagging behind due to a lack of methods for studying its gene function. Summary of the Invention

[0003] To overcome the aforementioned technical deficiencies, this invention proposes a microinjection method for *S. guanis* eggs, enabling the delivery of gene function research tools into *S. guanis* eggs. This method is rationally designed and highly efficient, laying the foundation for gene function research in *S. guanis* and can be used for bee breed improvement based on genetic engineering. Specifically, it includes the following steps: 1. Inducing oviposition by *Symplocos guani*: Soak 0.4g-0.6g of longhorn beetle larvae in a 10% ethanol aqueous solution for 1-2 minutes for disinfection. After rinsing the larvae with clean water, provide the larvae to 8-10 female *Symplocos guani* wasps that have mated 5-10 days after emergence. 2. Egg Collection and Arrangement: After the female wasp lays eggs, collect the eggs on the surface of the longhorn beetle larvae every hour, and immediately perform the following arrangement and injection procedures. Fix an 18mm×18mm coverslip onto a 25.4mm×76.2mm slide with a small amount of 502 glue. Then arrange the eggs of *Triplophysa tubi* along the edge of the fixed coverslip, with the longitudinal axis of the eggs parallel to the long side of the slide, and the posterior ends of the eggs all facing the direction of the injection needle. 3. Injection needle pulling: The needle was pulled using a NARISHIGE PC-100 vertical injection needle pulling instrument. The borosilicate capillary was a Sutter BF100-50-10 with two light counterweights. The needle pulling parameters were: Two-stage pull mode, LV1: 65 degrees Celsius, LV2: 65 degrees Celsius. 4. Microinjection of oocytes: Microinjection was performed using an Eppendorf Femtojet 4i pneumatic injection system. The system parameters were: Manual mode, pc=1000-2500hPa. The injection needle was positioned at a 15-25 degree acute angle to the longitudinal bearing of the oocyte, and the injection site was the posterior end of the oocyte. 5. Egg culture after injection: Longhorn beetle larvae weighing 0.4g-0.6g were stored in a freezer at -20 degrees Celsius for 24-48 hours in advance. After thawing at room temperature, the injected eggs were placed on the surface of the longhorn beetle larvae for culture. The culture conditions were 24-26 degrees Celsius and 40%-60% humidity. Attached Figure Description

[0004] Figure 1 This is a diagram showing the arrangement of the eggs on the glass slide. Figure 2 This is a diagram showing the injection site. Figures 1-2 In the diagram, the names of each structure are: coverslip 1, slide 2, egg 3, and injection needle 4. Detailed Implementation

[0005] Example 1: The specific injection procedure is as follows: (1) Step 1. Preparation before injection: Store 1-2 longhorn beetle larvae weighing 0.4g-0.6g in a freezer at -20 degrees Celsius for 24-48 hours in advance, and thaw them before injection; (2) Step 2. Preparation before injection: such as Figure 1 Fix the 18mm×18mm coverslip to the 25.4mm×76.2mm slide with a small amount of 502 glue; (3) Step 3. Inducing oviposition by *Symplocos guani*: Soak 0.4g-0.6g of longhorn beetle larvae in a 10% ethanol solution for 1-2 minutes for disinfection. Wash the larvae with clean water and place them in a 5-10mL test tube. Add 8-10 mated female *Symplocos guani* wasps that have emerged 5-10 days ago to the test tube and plug the test tube opening with a cotton ball. 10-20 longhorn beetle larvae can be prepared at the same time to ensure that enough eggs can be collected; (4) Step 4. Egg collection: Usually, the tube-shaped parasitic wasp needs to stay with the longhorn beetle larvae in the test tube for 3-5 days before it gradually begins to lay eggs. When eggs are found on the surface of the longhorn beetle larvae, collect the eggs with tweezers once every 1 hour. (5) Step 5. Egg arrangement: such as Figure 1 The collected eggs are placed on the glass slide prepared in step 2 using tweezers and arranged along the edge of the coverslip. The longitudinal axis of the eggs is parallel to the long side of the glass slide, and the rear ends of the eggs are all arranged in the direction of the injection needle. (6) Step 6. Injection needle pulling: Use Chengmao PC-100 vertical injection needle pulling instrument to pull the needle. Install the borosilicate capillary on the pulling instrument, adjust the pulling parameters, and press the "START" button. (7) Step 7. Place the slide prepared in step 5 under an optical microscope. Use the Eppendorf microloader pipette tip to draw up the liquid to be injected and add it into the injection needle. Connect the injection needle to the air pump injection system. Click the "clean" button on the air pump injection system and observe the liquid flowing out of the injection needle tip to confirm that the injection needle tip has an opening. Connect the air pump injection system to the injection needle, as follows: Figure 2 The injection needle tip is at a 15-25 degree acute angle to the longitudinal bearing of the egg. The injection needle tip is inserted into the rear end of the egg for injection. The parameters of the air pump injection system are: Manual mode, pc=1000-2500hPa. (8) Step 8. Transfer the injected eggs to the surface of the thawed longhorn beetle larvae in step 1 using tweezers for culture. The culture conditions are a temperature of 24-26 degrees Celsius and a humidity of 40%-60%.

Claims

1. A method for injecting *Triplophysa kusnezoffii* eggs, wherein the microinjection of *Triplophysa kusnezoffii* eggs is performed using a micro-injection needle on a microscopic platform, the microscopic platform comprising an optical microscope, an air pump injection system, a micromanipulator, and a vertical injection needle puller, characterized in that: The microinjection of bee eggs into the swollen leg of the tube includes the following steps: (1) Inducing and collecting eggs from *Symplocos guanis*: Soak 0.4g-0.6g of longhorn beetle larvae in 10% ethanol aqueous solution for 1-2 minutes for disinfection. After rinsing the longhorn beetle larvae with clean water, provide the longhorn beetle larvae to 8-10 female *Symplocos guanis* that have mated 5-10 days after emergence. Collect the eggs on the surface of the longhorn beetle larvae every hour after the female beetle lays eggs, and immediately perform the following injection operation. (2) Arrange the eggs of the worm: Fix an 18mm×18mm coverslip to a 25.4mm×76.2mm slide with a small amount of 502 glue, and then arrange the eggs of the worm on the edge of the fixed coverslip. The longitudinal axis of the eggs is parallel to the long side of the slide, and the rear end of the eggs are uniformly arranged in the direction of the injection needle. (3) Injection needle pulling: The needle was pulled using a NARISHIGE PC-100 vertical injection needle pulling instrument. The borosilicate capillary was a Sutter BF100-50-10 with two light counterweights. The needle pulling parameters were: Two-stage pull mode, LV1: 65 degrees Celsius, LV2: 65 degrees Celsius. (4) Microinjection: Microinjection was performed using an Eppendorf Femtojet 4i air pump injection system. The system parameters were: Manual mode, pc=1000-2500hPa. The injection needle was positioned at a 15-25 degree acute angle to the longitudinal bearing of the egg, and the injection site was the posterior end of the egg. (5) Egg culture after injection: Longhorn beetle larvae weighing 0.4g-0.6g were stored in a freezer at -20 degrees Celsius for 24-48 hours in advance. After thawing at room temperature, the injected eggs were placed on the surface of the longhorn beetle larvae for culture. The culture conditions were 24-26 degrees Celsius and 40%-60% humidity.

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