Female parent RNA interference method based on specific development window period of parasitic wasps

Abstract

The invention discloses a female parent RNA interference method based on a parasitic wasp specific developmental window period, and relates to the technical field of insect functional genomics and genetic manipulation, and the method comprises the following steps: systematically comparing microinjection effects of a pre-pupal period and an adult period, determining an efficient and safe window of parasitic wasp female parent RNA interference in the adult period, the method comprises the following steps: selecting a conservative transform gene in an insect sex determination pathway as a target gene to prepare specific double-stranded RNA, and performing targeted microinjection on the belly of a female bee in an adult stage, so that efficient silencing of the target gene is realized on the premise of ensuring high survival rate and normal reproductive capacity of a female parent. According to the method, by defining an efficient and safe operation window of the parasitic wasp female parent RNAi in the adult stage, the technical problems of high death rate, developmental disorder, reproductive power decline and the like caused by injection in the pre-pupal stage are thoroughly solved, the cumulative survival rate of the female parent within 20 days is not lower than 50%, the operation repeatability is high, and a stable and reliable technical normal form is provided for research on the gene function of the parasitic wasp.

Description

Technical Field

[0001] This invention relates to the field of insect functional genomics and genetic manipulation technology, specifically to a method for maternal RNA interference based on a specific developmental window of parasitic wasps. Background Technology

[0002] Parasitic wasps, as core natural enemies in the biological control of agricultural and forestry pests, rely heavily on the precise regulation of key biological processes such as reproduction and development for large-scale breeding and genetic improvement. However, there is currently a severe lack of efficient, safe, and universally applicable techniques for gene function research on parasitic wasps, especially many small oviparous parasitic wasps with significant application value. Due to their small size and unique life cycle, conventional genetic manipulation techniques (such as microinjection) face significant bottlenecks in application, including low delivery efficiency, unclear manipulation windows, and high toxicity to host development, which greatly limits in-depth analysis of the growth, development, and reproductive regulation of parasitic wasps.

[0003] RNA interference (RNA) technology offers the possibility of directly regulating sex at the gene level. Maternal RNA interference is known to be a powerful tool for studying the function of early developmental genes in various insects; however, its effectiveness in parasitic wasps is highly dependent on establishing a technical system that minimizes the impact on the mother while ensuring efficient gene silencing in offspring. Existing attempts often overlook the decisive influence of developmental stage selection on the success rate of the technique. For example, microinjection during the sensitive prepupal stage, while potentially achieving gene knockdown, easily leads to extremely high host mortality or reproductive decline, complicating the interpretation of experimental data and severely hindering the reliability and reproducibility of the technique. Therefore, developing a universal method that can clearly define the optimal operating window, minimize physiological interference with the mother, and stably achieve maternal gene function research in various parasitic wasps has become an urgent technical need in this field.

[0004] This methodological breakthrough will directly drive the development of multiple applications. One key application is the precise control of the sex ratio in parasitic wasps. Whether for parthenogenetic or hermaphroditic strains, controlling the sex of offspring by intervening in specific maternal genes is of significant value for the genetic breeding of natural enemy insects, population structure optimization, and improving the efficiency of biological control. However, current methods relying on long-term antibiotic treatment to alter reproductive patterns and obtain males suffer from problems such as long cycles and irreversible changes in population characteristics, failing to achieve flexible, on-demand sex control. Therefore, a new technological approach based on direct genetic intervention, which is simple to operate and does not alter the basic reproductive patterns of the population, is urgently needed.

[0005] Therefore, developing a method to establish an efficient and safe maternal RNAi method in parasitic wasps by scientifically comparing and determining the optimal developmental window is of great theoretical value and urgent application need. Summary of the Invention

[0006] The purpose of this invention is to provide a method for maternal RNA interference based on a specific developmental window period of parasitic wasps, in order to solve the problems mentioned in the background art.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for maternal RNA interference based on a specific developmental window in parasitic wasps includes the following steps:

[0009] (a) Determination and selection of key developmental windows: Through parallel comparative experiments, the optimal developmental stage for microinjection and RNA interference of the target parasitic wasp was determined to be the adult stage, and the prepupal stage was excluded;

[0010] (b) Target gene selection and dsRNA preparation: The conserved transformer gene in the insect sex determination pathway was selected as the RNA interference target, and the green fluorescent protein gene sequence was used as the control. Specific double-stranded RNA targeting this gene was designed and synthesized.

[0011] (c) Recipient preparation: Select healthy female parasitic wasps in the optimal developmental window period determined in step (a) – the adult stage – as recipients;

[0012] (d) Targeted injection during adult stage: Using a microinjection system, the double-stranded RNA solution prepared in step (b) is precisely injected into the abdominal cavity of the female adult selected in step (c);

[0013] (e) Phenotypic assessment: After injection, the female bees were raised and parasitized under standard conditions. The sex ratio of their offspring was recorded, and the survival rate of the female bees, the sex ratio of their offspring, and the male morphology and behavior were assessed.

[0014] A further improvement of the technical solution of the present invention is that the parallel comparison experiment in step (a) specifically includes: setting up an untreated group (prepupal stage uninjected group and adult stage uninjected group) and a treated group (prepupal stage injected group and adult stage injected group); the untreated group is not injected, and the treated group is injected with an equal amount of dsRNA, and the differences between the two groups in adult lifespan, number of parasites in adults within 10 days, progeny emergence rate, male ratio and tra gene silencing efficiency are systematically compared; the prepupal stage uninjected group will die during pupation and adulthood, the prepupal stage injected group shows an unacceptably high mortality rate and developmental impairment, while the adult stage uninjected group and the injected group show high survival rate and high efficiency.

[0015] A further improvement of the technical solution of the present invention is that: the prepupal stage refers to a specific stage in which the larvae are fully developed, have stopped feeding, but have not yet entered the formal pupal stage; the adult stage refers to the stage in which the female adult has completed molting and whose sexual organs are mature.

[0016] A further improvement of the technical solution of the present invention is that: the injection site for the adult bee in step (d) is the junction of the ovipositor and the body on the front abdomen of the female bee.

[0017] A further improvement of the technical solution of the present invention is that: in step (b), the dsRNA is synthesized using plasmids of the tra gene and the GFP gene as DNA templates, and after PCR amplification and gel purification, it is synthesized and purified using a transcription kit and an RNA purification kit.

[0018] A further improvement of the technical solution of the present invention is that: in step (d), the injection dose of dsRNA via microinjection is 150 ng / female bee, and the injection volume is calculated and determined based on the dsRNA concentration.

[0019] A further improvement of the technical solution of the present invention is that: the standard feeding conditions in step (e) are a temperature of (24±1)℃, a humidity of (70±5)%, and a light-dark ratio of 14:10. During the feeding process, the female bees are provided with 30% honey water or 1mol / L fructose water as food.

[0020] A further improvement of the technical solution of the present invention is that: the proportion of male offspring of adult female bees treated by the method reaches more than 95%, and the cumulative survival rate of the female bees after 20 days is not less than 50%, which is significantly higher than that of the pre-pupal injection group.

[0021] The present invention also proposes a parasitic wasp offspring population with a directionally reversed sex ratio obtained by the above method. This population is produced by parthenogenetic female wasps treated with the adult tra gene maternal RNAi, in which male individuals are absolutely dominant and male individuals have complete courtship and mating behaviors.

[0022] Due to the adoption of the above technical solution, the technical progress achieved by this invention compared to the prior art is as follows:

[0023] 1. This invention provides a method for maternal RNA interference based on a specific developmental window period of parasitic wasps. By clearly defining the adult stage as the efficient and safe operating window for maternal RNAi in parasitic wasps, it completely solves the technical pain points such as high mortality, developmental disorders and reproductive decline caused by injection during the prepupal stage. The cumulative survival rate of the maternal parent after 20 days is not less than 50%, and the operation is highly reproducible, providing a stable and reliable technical paradigm for the study of gene function in parasitic wasps.

[0024] 2. This invention provides a method for maternal RNA interference based on a specific developmental window of parasitic wasps, which targets and silences the core sex-determining gene tra, achieving a sex ratio of ≥95% in offspring and a directional reversal of female-to-male conversion. The regulatory precision far exceeds that of traditional methods, and it does not change the basic reproductive pattern of the parasitic wasp population, thus meeting the core need for flexible and on-demand regulation of sex structure in the large-scale breeding of natural enemy insects.

[0025] 3. This invention provides a method for maternal RNA interference based on a specific developmental window of parasitic wasps. The induced male parasitic wasps are morphologically normal and have complete courtship and mating behaviors. They can be directly used as paternal parents for genetic improvement and strain hybridization of parasitic wasps, providing renewable and high-quality male resources for the breeding of natural enemy insects. This invention breaks through the technical bottleneck of obtaining effective paternal parents for parthenogenetic female-producing strains.

[0026] 4. This invention provides a method for maternal RNA interference based on a specific developmental window period of parasitic wasps. This method can be applied to gene function analysis and sex regulation of various parasitic wasps. It not only provides a key tool for the study of sex determination pathways in non-model parasitic wasps, but can also be directly transformed into productivity in the biological control of agricultural and forestry pests. By optimizing the parasitic wasp population structure, it can improve control efficiency. Attached Figure Description

[0027] Figure 1 This is a statistical analysis of mortality in the prepupae stage non-injection group and the injection group in this embodiment of the invention;

[0028] Figure 2 This is a comparison chart of the 20-day cumulative survival curves of the non-injected group and the injected group during the prepupal and adult stages in this embodiment of the invention.

[0029] Figure 3 This is a comparison chart of the silencing efficiency between the non-injected group and the injected group during the prepupal and adult stages in this invention embodiment;

[0030] Figure 4 This is a bar chart comparing the number of parasites, offspring emergence rate, and male ratio in the prepupal and adult stages of the present invention, between the uninjected and injected groups.

[0031] Figure 5 The RNAi male bee morphology produced after the preferred scheme (injection during the adult stage) of this invention;

[0032] Figure 6 This is a diagram illustrating the mating behavior of RNAi male bees according to the present invention. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to embodiments:

[0034] Example 1

[0035] Comparison of the RNAi window effect between the prepupal and adult stages and application of the optimal scheme

[0036] 1. Test Insects: The parasitic wasps collected from the wild were first morphologically and molecularly identified as *Triplophysa gansuensis*. Adult wasps were collected using a fine brush into plastic jars (12.5 × 6.5 cm in size). The jar openings were covered with appropriately sized pieces of white cloth and secured with rubber bands. Each jar contained 20 female wasps. A 30% honey solution was brushed onto the jar walls (care should be taken not to stick the wasps to the ground). The jars were then placed in a cultivation greenhouse under constant conditions of (24±1)℃, (70±5)% humidity, and a light-dark ratio of 14:10. After approximately 5 days, 300 *Tetracentron sinense* eggs were introduced for parasitism to allow the next generation to reproduce.

[0037] 2. RNA extraction:

[0038] (1) Collect 5 adult Gansu flat-bellied wasps into 1.5 mL enzyme-free centrifuge tubes, add sterilized magnetic beads (about 5 beads) to the Gansu flat-bellied wasp sample, and put the centrifuge tube into a multi-sample tissue grinder to grind thoroughly for 2-3 min;

[0039] (2) After grinding, add 1 mL of pre-cooled Trizol to the centrifuge tube and centrifuge at 4℃ and 12000 rpm for 10 min to separate the RNA from the tissue sample;

[0040] (3) Add 200 μL of pre-cooled chloroform, shake for 30 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4℃ and 12000 rpm for 20 minutes to separate RNA from protein and other components.

[0041] (4) Take the supernatant and add 200 μL of pre-cooled isopropanol. Mix well and let stand on ice for 10 min to precipitate RNA. Centrifuge at 4℃ and 12000 rpm for 20 min to precipitate RNA.

[0042] (5) Discard the supernatant, add 300 μL of pre-cooled 75% enzyme-free ethanol solution, and wash the RNA by centrifugation at 7500 rpm for 5 min at 4℃.

[0043] (6) Remove the ethanol and add 20-50 μL of Nuclease-Free Water to dissolve the RNA. Use a nucleic acid protein analyzer to determine the concentration of RNA and the ratio of OD260 / OD280, and record the concentration value (the ratio of OD260 / OD280 should be between 1.8 and 2.0).

[0044] 3. cDNA Acquisition: Total RNA samples from *Gansu flat-bellied wasp* were processed using PrimeScript. RTThe kit is used to synthesize the first strand of cDNA. The specific reaction steps are as follows: Add 2 μL of 5×gDNA Eraser Buffer, 1 μL of gDNA Eraser, and 1 μg of RNA to the PCR tube in sequence, and finally add RNase-Free ddH2O to make up to 10 μL. Incubate at 42℃ for 2 min to remove DNA from the total RNA. Add the premixed solution of PrimeScript RT Enzyme Mix I, RT Primer Mix, 5×PrimeScript Buffer II, and Nuclease-Free Water to the PCR tube, mix well, and incubate in a PCR instrument at 37℃ for 15 min, followed by an incubation at 85℃ for 5 s. Store at -20℃.

[0045] 4. dsRNA synthesis: Primers were designed based on the green fluorescent protein (GFP) gene sequence and the Gansu flat-bellied wasp transformer sequence (Agtra). A T7 promoter sequence was added to the 5' end of all primers. Plasmids containing both genes were used as DNA templates for dsRNA synthesis, and TakaRa Taq was employed. TM PCR amplification was performed using Version 2.0 plus dye DNA polymerase. After gel recovery and purification of the DNA fragments, dsRNA was synthesized and purified according to the instructions of Thermo Scientific TranscriptAid T7 High-Yield Transcription Kit and GeneJET™ RNA Purification Kit.

[0046] 5. Window period comparison experiment design: (1) Prepupal stage without injection group: carefully dissect the late-stage mature larvae that have stopped feeding and have white body color, and place them in a transparent petri dish lined with filter paper. The petri dish is placed in an incubator (25±1℃, 70%±5% RH, 14:10hL:D photoperiod) until emergence. (2) Adult stage without injection group: select healthy female adults that have just emerged (<6h), feed them 1mol / L fructose water every day and give them 30 tussah silkworm eggs for parasitism. (3) Prepupal stage injection group: carefully dissect the late-stage mature larvae that have stopped feeding and have white body color (n = 50). Under stereomicroscopy, microinjection was performed on the lateral abdomen. The treatment group (n = 50) was injected with a solution containing 150 ng dsAgtra per insect, while the control group (n = 50) was injected with a solution containing 150 ng dsGFP. (4) Adult injection group: Healthy female adults that had just emerged (< 6 h) were selected. After brief hypothermic anesthesia, microinjection was performed at the 2nd-3rd abdominal intersegmental membrane at the ovipositor connection suture on the front of the abdomen. The treatment group (n = 30) was injected with a solution containing 150 ng dsAgtra per insect, while the control group (n = 30) was injected with a solution containing 150 ng dsGFP.

[0047] 6. Specific injection procedure:

[0048] (1) Insert a borosilicate glass capillary tube into the needle puller and pull out the needle according to the operating requirements of the P-2000 needle puller (the parameters used by the P-2000 needle puller are HEAT:300; FIL:4; VEL:60; DEL:80; PULL:99).

[0049] (2) Load 1 μL of dsRNA into the needle using an Eppendorf microsampler;

[0050] (3) Open the needle tip and install the pulled-out needle at a 30° angle on the needle grinding instrument BV-10-E (SUTTER, USA) to grind the needle opening; or slide the needle tip onto the surface made of two overlapping sliders; or you can use an ophthalmic scalpel to open the needle tip to form a sharp edge;

[0051] (4) Place the arranged Gansu flat-bellied wasp prepupae or adult wasps under a dissecting microscope, insert the needle into the injection tube of the micromanipulator and tighten the handle;

[0052] (5) Observe the needle under the microscope, turn on the micromanipulator and rotate the knob to the appropriate height, set the parameters, use 150ng as the fixed injection mass, and calculate the injection volume according to the concentration of each dsRNA.

[0053] (6) Carefully insert the needle into the abdomen of the Gansu flat-bellied wasp using the control lever, at a vertical angle of about 30°; after reaching the fixed injection volume, stop the injection; slowly pull out the needle and carefully move it to the next pupa or wasp, repeating these steps;

[0054] (7) Place the injected pupae in a petri dish with clean filter paper at the bottom, cover it, spray with an appropriate amount of sterile water to maintain humidity, and place it in an incubator (25 ± 1 ℃, 70% ± 5% RH, 14:10hL:D photoperiod) until the Gansu flat-bellied wasp emerges; after injection, place the adult wasps in a plastic jar (12.5 × 6.5 cm in size), cover the jar opening with a piece of white cloth cut to the appropriate size, and secure it with a rubber band. Each jar contains approximately 20 wasps, and use a small brush to brush 1 mol / L fructose water onto the jar wall (be careful not to stick the parasitic wasps to the wall). Place the jar in the same incubation temperature. The uninjected group is operated the same as the injected group except that no microinjection is performed.

[0055] 7. Tracking evaluation and data collection:

[0056] (1) Survival and development tracking: After each operation and treatment, all individuals were raised individually under standard conditions, and observations were recorded daily. For the prepupal group, it was necessary to record whether pupation was successfully completed (body wall hardening, pupal shell formation), whether the pupal stage was dead, whether the larvae were successfully molted, and the daily survival status after molting. For the adult group, it was necessary to record the survival rate within 20 days after injection and the subsequent daily survival status. The mortality time of all pupae after molting into adults was recorded, and survival curves were plotted.

[0057] (2) Reproduction and offspring evaluation: All female wasps that successfully survived to sexual maturity (surviving individuals in the adult stage group and individuals that emerged from the prepupa stage group) were provided with 30 fresh host eggs per day for parasitism. The parasitized eggs were placed in a climate chamber (25 ± 1℃, 70% ± 5%RH, 14:10hL:D photoperiod) until they emerged, and the sex ratio of the offspring (F1) was recorded.

[0058] Example 2

[0059] Comparison results

[0060] 1. Survival rate and developmental cost (see...) Figure 1 , Figure 2 ):

[0061] (1) Prepupal stage without injection group: 24 hours after the larvae were removed from the host eggs, 36% of all dead individuals failed to pupate successfully; after successful pupation, another 18% died during the pupal stage; and 45% of the individuals died after emergence (control group). There was no significant difference in lifespan between the adult bee and the adult stage without injection group after emergence.

[0062] (2) Adults who were not injected: 100% survival rate 24 hours after injection.

[0063] (3) Prepupal injection group: Within 24 hours after the operation, about half of the dead individuals in the dsGFP and dsAgtra injection groups failed to pupate successfully; after successful pupation, about 17% (dsGFP) and 9% (dsAgtra) died during the pupal stage; the number of individuals that died after emergence accounted for about 31% (dsGFP) and 39% (dsAgtra). Moreover, the adults that emerged from the dsAgtra group were very weak, with an average lifespan of less than 50% of that of the dsGFP group.

[0064] (4) Adult injection group: Survival rate >95% 24 hours after injection. The subsequent survival curve was not significantly different from that of the uninjected adult group, and the lifespan of the emerging adults was normal.

[0065] 2. Comparison of gene silencing efficiency (see...) Figure 3 The target gene was silenced in the injection groups during the prepupal and adult stages, with a silencing efficiency of over 70% on day 3 and over 85% on day 5.

[0066] 3. Comparison of gender reversal efficiency (see...) Figure 4 ):

[0067] (1) The average male ratio of adults that successfully emerged from the prepupal group and the adult group that was not injected was extremely low, about 2%–5%.

[0068] (2) Adults that successfully emerged from the prepupal injection group failed to successfully parasitize the silkworm eggs, and correspondingly, no offspring emerged.

[0069] (3) The sex reversal efficiency of the adult injection group was extremely high and stable, with an average male ratio of 99.9%, which was significantly different from the uninjected group and the dsGFP injection group (2%-3%).

[0070] Example 3

[0071] Conclusions and Optimal Solution Establishment

[0072] The above comparative experiments clearly demonstrate that: under no-injection conditions, simply dissecting the insect body results in a high mortality rate among pupating adults during the prepupal stage; under the same injection conditions, prepupal injection causes unacceptable developmental damage to the host, making it technically infeasible; while adult injection achieves extremely high sex regulation efficiency with very low host cost (compared to the uninjected adult group). Therefore, the adult stage is the only recommended window for implementing this technology.

[0073] Example 4

[0074] Validation of the Optimal Solution Product

[0075] Phenotypic analysis was performed on the large number of RNAi drones produced in the adult injection group (see...). Figure 5 These male bees are identical to normal male bees in terms of body size, antennae, and external genitalia. Figure 5 B, a, and b represent normal male testes; c and d represent testes treated with dsAgtra. Behavioral observations showed that over 85% of individuals (20 pairs) exhibited complete courtship behavior towards female bees, and 30% of individuals (20 pairs) exhibited complete mating behavior (see...). Figure 6 This indicates that the technology can successfully induce individuals with complete male behavior and normal external sexual characteristics, thus verifying its effectiveness and potential in precisely regulating sex determination pathways.

[0076] The above embodiments fully illustrate the complete process and significant advantages of the present invention in determining the optimal developmental window through comparative studies and establishing an efficient and safe sex regulation method based on this.

[0077] The present invention has been described in detail above. However, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. A method for maternal RNA interference based on a specific developmental window period in parasitic wasps, characterized in that: Includes the following steps: (a) Determination and selection of key developmental windows: Through parallel comparative experiments, the optimal developmental stage for microinjection and RNA interference of the target parasitic wasp was determined to be the adult stage, and the prepupal stage was excluded; (b) Target gene selection and dsRNA preparation: The conserved transformer gene (tra) in the insect sex determination pathway was selected as the RNA interference target, and the green fluorescent protein gene sequence was used as the control. Specific double-stranded RNA targeting this gene was designed and synthesized. (c) Recipient preparation: Select healthy female parasitic wasps in the optimal developmental window period determined in step (a) – the adult stage – as recipients; (d) Targeted injection during adult stage: Using a microinjection system, the double-stranded RNA solution prepared in step (b) is precisely injected into the abdominal cavity of the female adult selected in step (c); (e) Phenotypic assessment: After injection, the female bees were raised and parasitized under standard conditions. The sex ratio of their offspring was recorded, and the survival rate of the female bees, the emergence rate of the offspring, the sex ratio, the male morphology and behavior were assessed.

2. The method for maternal RNA interference based on a specific developmental window period of parasitic wasps according to claim 1, characterized in that: The parallel comparison experiment in step (a) specifically includes: setting up an untreated group—prepupal stage uninjected group and adult stage uninjected group, and a treated group—prepupal stage injected group and adult stage injected group. The untreated group was not injected. After the treated group was injected with an equal amount of dsRNA, the differences between the two groups in adult lifespan, number of parasites in adults within 10 days, progeny emergence rate, male ratio and tra gene silencing efficiency were systematically compared. The prepupal stage uninjected group showed mortality during pupation and adulthood. The prepupal stage injected group showed unacceptably high mortality and developmental disorders. The adult stage uninjected group and the injected group showed high survival rate and high efficiency.

3. The method for maternal RNA interference based on a specific developmental window period of parasitic wasps according to claim 1, characterized in that: The prepupal stage refers to the specific stage where the larvae are fully developed, have stopped feeding, but have not yet entered the formal pupal stage; the adult stage refers to the stage where the female adult has completed molting and whose sexual organs are mature.

4. The method for maternal RNA interference based on a specific developmental window period of parasitic wasps according to claim 1, characterized in that: The injection site for the adult bee in step (d) is the junction between the ovipositor and the body on the front abdomen of the female bee.

5. The method for maternal RNA interference based on a specific developmental window period of parasitic wasps according to claim 1, characterized in that: In step (b), the dsRNA was synthesized using plasmids of the tra gene and GFP gene as DNA templates. After PCR amplification and gel purification, the dsRNA was synthesized and purified using a transcription kit and an RNA purification kit.

6. The method for maternal RNA interference based on a specific developmental window period of parasitic wasps according to claim 1, characterized in that: In step (d), the dsRNA injection dose via microinjection is 150 ng / female bee, and the injection volume is calculated based on the dsRNA concentration.

7. The method for maternal RNA interference based on a specific developmental window period of parasitic wasps according to claim 1, characterized in that: The standard rearing conditions in step (e) are a temperature of (24±1)℃, a humidity of (70±5)%, and a light-to-dark ratio of 14:

10. During the rearing process, the female bees are provided with 30% honey water or 1mol / L fructose water as food.

8. The method for maternal RNA interference based on a specific developmental window period of parasitic wasps according to claim 1, characterized in that: The adult female bees treated by this method have a male offspring ratio of over 95%, and the cumulative survival rate of the female bees after 20 days is not less than 50%, which is significantly higher than that of the pre-pupal injection group.

9. A parasitic wasp population with a directionally reversed sex ratio obtained by the method of any one of claims 1-8, characterized in that, This population was produced by parthenogenetic female wasps treated with the adult tra gene maternal RNAi, with males being absolutely dominant and exhibiting complete courtship and mating behaviors.

10. The application of the method according to any one of claims 1-7 in comparing and verifying the applicability of gene manipulation techniques to parasitic wasps at different developmental stages.

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