A method for breeding of montezumia spinifrons and application in prevention and treatment of monochamus alternatus

Abstract

The application discloses a method for artificially breeding Sirex gregarivora Cresson and applying the Sirex gregarivora Cresson to biological prevention and control of Anoplophora solenitica. The application determines optimal host size, bee connection ratio, breeding environment and other conditions when the Sirex gregarivora Cresson is artificially bred, can significantly improve parasitization rate of artificial bee breeding and reproduce a large number of parasitic bees in a short period. Meanwhile, conditions suitable for storing adult Sirex gregarivora Cresson are also determined, so that the shelf life of the adult Sirex gregarivora Cresson can be extended to 2 months. The Sirex gregarivora Cresson reproduced through the application is applied to biological prevention and control of Anoplophora solenitica larvae in the wild, and optimal prevention and control effect can be obtained according to equal ratio release of bees and worms, and low cost investment is also considered.

Description

Technical Field

[0001] This invention belongs to the field of biological control, specifically relating to the technical field of artificial breeding and biological control of natural enemy insects, and more specifically to a method for breeding the parasitic natural enemy Braconid wasp *Stylos glabripennis*, and a method for controlling *Stylos glabripennis* using this natural enemy. Background Technology

[0002] The Asian longhorn beetle (Anoplophor alabripennis) (Coleoptera: Cerambycidae) is a major forest pest in my country. Its larvae primarily bore into the trunk and main branches of the host tree, disrupting nutrient and water transport and potentially causing the entire tree to die. The Asian longhorn beetle hosts trees from over 20 genera and 18 families, but in China, it mainly damages trees in the genera *Populus*, *Salix*, *Acer*, and *Ulmus*. In the "Three-North" shelterbelt project, the Asian longhorn beetle almost destroyed the first phase of the poplar-dominated shelterbelt project, and its damage remains severe in this region. Furthermore, the large-scale planting of willows and maples in many Chinese cities has made this pest a serious urban landscaping pest in recent years.

[0003] The Asian longhorn beetle (Anoplophora glabripennis) hides under the bark and within the xylem throughout its entire larval stage, making its control difficult due to its long development period. Biological control is considered the most effective method for controlling longhorn beetle-like borers. Currently, *Sclerodermusguani* and *Dastarcus helophoroides* are mainly used for the biological control of *Anoplophora glabripennis*. *Sclerodermusguani* can parasitize young larvae of *Anoplophora glabripennis*. However, *Sclerodermusguani* has poor host specialization, and the female adults lack wings and cannot fly, resulting in weak dispersal ability in the wild and insufficient control efficacy. *Dastarcus helophoroides* mainly parasitizes mature larvae and pupae of *Anoplophora glabripennis*, exhibiting strong parasitic control. However, by the time *Anoplophora glabripennis* develops into a mature larva or pupa, its borer damage to trees is already severe, and even with effective parasitism, the affected trees often fail to survive. Therefore, developing and utilizing natural enemies of the early larval stages of the Asian longhorn beetle, especially those with high host specialization and strong dispersal capabilities, is key to effectively controlling the damage caused by the Asian longhorn beetle in its early life cycle, reducing the population of this pest, and saving affected trees.

[0004] For a long time, apart from the parasitic wasp *Spathius anoplophora*, no other natural enemies have been available for the biological control of the larval stage of *A. anoplophora*. In recent years, we have conducted natural enemy attraction experiments in various locations across China where *A. anoplophora* is present, by placing wooden sticks infested with *A. anoplophora* larvae on various sticks. We discovered a parasitic wasp that parasitizes the young larvae of *A. anoplophora*—*Spathius anoplophorae* (Hymenoptera: Braconidae)—in Beijing, Shanghai, Zunyi, Kunming, and other locations. The attraction experiments showed that this parasitic wasp has an average natural parasitism rate of 20% on *A. anoplophora* larvae, demonstrating a strong control effect. The distribution of this parasitic wasp in many cities throughout my country indicates a wide suitable habitat area, suggesting that it could be developed into a natural enemy product for control in multiple locations. Furthermore, this parasitic wasp exhibits strong host specificity towards *A. anoplophora*, and its adults are winged with strong flight dispersal capabilities, making it a superior natural enemy for the biological control of *A. anoplophora*. Currently, there are no reports on the application of braconid wasps as natural enemies in the biological control of the Asian longhorn beetle in my country. As a new natural enemy, solving its artificial breeding techniques, achieving large-scale propagation, and exploring its application in forests are fundamental to its application in biological control. Against this backdrop, we have developed a method for breeding the braconid wasp *Pterocarya stenoptera* (a type of wasp) and its application in the control of the Asian longhorn beetle. Summary of the Invention

[0005] The purpose of this invention is to provide a method for artificially breeding the *Braconchae pedunculata*, a type of longhorn beetle. Another purpose of this invention is to provide a method for controlling the longhorn beetle using *Braconchae pedunculata*.

[0006] As a first aspect of the present invention, the present invention provides a method for artificially breeding *Aegilops glabripennis*, characterized by comprising the following steps:

[0007] (1) Using the second to fourth instar larvae of the longhorn beetle as hosts, they were inoculated onto willow tree segments to obtain sample trees with hosts;

[0008] (2) Place the sample wood with the host in a mesh insect rearing cage, preferably with a mesh size of 100;

[0009] (3) The mated female Braconid wasps of the Asian longhorn beetle were introduced into the above-mentioned insect rearing cage and placed together in an artificial climate chamber for cultivation. The ratio of female Braconid wasps of the Asian longhorn beetle to the host Asian longhorn beetle larvae was 2:1 to 1:6.

[0010] (4) After the female bee completes parasitism, the female bee is removed, and the insect rearing cage and sample wood are kept in the artificial climate chamber until the young bees develop to adulthood.

[0011] (5) Collect newly emerged male and female bees and store them in a low-temperature incubator for use in forest pest control.

[0012] In a specific implementation, in step (1), the larvae of the longhorn beetle are 2 to 3 instars, preferably 3 instars.

[0013] In a specific implementation, the method for preparing the host sample wood in step (1) is as follows: cut a 2cm diameter willow wood into 15cm long sections; make a 1cm×1.5cm oblique cut into the willow wood section with a knife, remove the xylem of the cut but retain the bark; place one larva of the longhorn beetle into one cut and cover it with bark, then fix the bark of the cut with Parafilm; attach 12 host plants to each 15cm long willow wood section.

[0014] In a specific implementation, in step (2), in order to facilitate large-scale inoculation and breeding, the insect rearing cage is 20cm×20cm×20cm in size, and 25 host sample trees are inserted into each insect rearing cage.

[0015] In a specific embodiment, in step (3), the ratio of the number of female Braconid wasps of the longhorn beetle to the number of host longhorn beetle larvae is 1:1 to 1:4, preferably 1:4.

[0016] In a specific implementation, in step (3), the artificial climate chamber conditions are set to a temperature of 26-30°C, a photoperiod of L:D = 14-16:10-8h, and a relative humidity of RH = 50-70%; preferably 30°C, L:D = 14:10h, and RH = 70%.

[0017] In a specific implementation, in step (4), the female wasp completes the parasitism cycle in 2 to 4 days, preferably 2 days.

[0018] In a specific embodiment, in step (5), the conditions of the low-temperature incubator are set to a temperature of 8-12°C, a relative humidity of RH = 80-90%, and no light; preferably 8°C, RH = 90%, and no light.

[0019] As a second aspect of the present invention, the present invention also provides a method for using the *Begonia sclerotiorum* bred according to the above method in the control of the *Begonia sclerotiorum*. Preferably, it includes the following steps:

[0020] (1) During the early larval stage of the Asian longhorn beetle, the population density of the Asian longhorn beetle in the forest was investigated.

[0021] (2) Based on the population density of the longhorn beetle, female parasitic wasps of the longhorn beetle were taken and placed at the base of the trees damaged by the longhorn beetle.

[0022] Optionally, the following steps may also be included:

[0023] (3) Fifteen days after releasing the bees, dissect the bark around the grooves of the longhorn beetle on the tree to investigate the parasitism of the longhorn beetle larvae by the longhorn beetle parasitoid wasp and evaluate the effectiveness of biological control.

[0024] In a specific implementation, in step (1), the population density of the longhorn beetle is investigated by random sampling, specifically by using the average number of grooves per beetle to represent the average larval density per beetle.

[0025] In a specific implementation, in step (2), the female wasp of the stalked-belly braconid beetle is placed in a plastic centrifuge tube, the centrifuge tube is placed at the base of the affected tree with the tube opening facing upwards, and the tube cap is removed.

[0026] In a specific implementation, in step (2), the ratio of female *Begonia scleroderma* wasps is 3:1 to 1:1, preferably 1:1.

[0027] This invention, by testing the parasitism preference of *Begonia sclerotioides* larvae of different sizes, clarifies the optimal age of the host for artificial breeding, thus improving the parasitism rate during artificial breeding. Research on the optimal inoculation ratio revealed that a parasitism rate of over 90% can still be achieved at a ratio of 1:4, saving on the number of female wasps required while maintaining a high parasitism rate. The female *Begonia sclerotioides* larvae identified in this invention can complete sufficient parasitism within 2 days. Based on this, the female wasps can be transferred for re-inoculation, fully utilizing their reproductive potential, which is significant for improving breeding efficiency and reducing inoculation costs. Furthermore, under the larval breeding environment provided by this invention, *Begonia sclerotioides* completes a generation in only about 12 days, significantly shortening the breeding cycle and making it possible to mass-produce this parasitoid wasp in a short period. More importantly, because the inoculation and breeding environment provided by this invention allows for a shorter time for both host parasitism and the completion of larval development, it eliminates the need for moisturizing measures such as floral soil or hydroponics to maintain the freshness of the willow logs. The logs can be directly stored in batches within insect rearing cages to complete the bee breeding process, greatly simplifying the operation and improving efficiency. Under the adult bee storage environment specified in this invention, the average lifespan of female *Braconchae stenoptera* (a parasitic wasp) can reach over two months, significantly extending the shelf life of this parasitic wasp. This invention also clarifies the optimal release ratio of *Braconchae stenoptera* for controlling *Braconchae stenoptera*. When released in equal proportions, not only is the optimal control effect ensured, but the amount of female wasps released is also minimized, reducing the cost of biological control. This invention provides a new natural enemy product for the biological control of *Braconchae stenoptera*, enriching the technical aspects and resources for integrated pest management of *Braconchae stenoptera*. Furthermore, it supports reducing the use of chemical pesticides in the control of *Braconchae stenoptera*, providing technical assurance for protecting the ecological environment. Attached Figure Description

[0028] Figure 1 Method for preparing a willow log with a host. Left: Cutting; Middle: Removing the xylem while retaining the phloem; Right: Secured with sealing film after grafting the host.

[0029] Figure 2 The cocoon left behind by the molting beetle *Begonia glabripennis* after its emergence.

[0030] Figure 3 The young larvae of the *Anoplophora glabripennis* parasitize the body surface of the larvae of the *Anoplophora glabripennis*.

[0031] Figure 4 The cocoon of the young wasp parasitizing the larvae of the longhorn beetle in the wild. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below:

[0033] Example 1

[0034] (1) Parallel experimental setup: Larvae of the Asian longhorn beetle of different instars were selected as hosts for bee breeding, including treatment 1:1 instar; treatment 2:2 instar; treatment 3:3 instar; treatment 4:4 instar; and treatment 5:5 instar. Willow logs with a diameter of 2 cm were cut into 15 cm long segments. A 1 cm × 1.5 cm incision was made obliquely into each segment, removing the xylem but retaining the bark. One Asian longhorn beetle larva was placed into each incision and covered with bark. The bark was then fixed with Parafilm. Twelve larvae of the same instar were inoculated onto each sample log. As an example, Figure 1 The method of making the willow wood with the host plant is shown.

[0035] (2) Place 25 sample logs inoculated with larvae of the same age of the longhorn beetle in a 100-mesh mesh cage with dimensions of 20cm×20cm×20cm.

[0036] (3) 75 mated female Brachiosa stylipoda were introduced into each insect rearing cage and placed in an artificial climate chamber at 30℃, L:D=14:10h, RH=70% for cultivation. This was recorded as one replicate.

[0037] (4) Two days after receiving the bees, the female bees are removed, and the insect rearing cage and sample wood are kept in the artificial climate chamber until the young bees develop and emerge.

[0038] (5) After the offspring emerge, dissect the sample wood. Statistical analysis of the parasitism rate of *Begonia glabripennis* on hosts of different ages was performed, with successful parasitism defined as the presence of a parasitoid cocoon inside the cut surface. As an example, Figure 2The image shows the cocoon left by the molted parasitic wasp *Braconchis stenoptera* from the *A. stenoptera*. The number of *Braconchis stenoptera* parasitic wasps that could reproduce using *A. stenoptera* larvae of different instars as hosts was counted.

[0039] Each treatment was repeated 3 times, and the results are shown in Table 1:

[0040] Table 1. Parasitism rate and number of offspring of *A. sclerotium* (a type of longhorn beetle) using *A. sclerotium* of different ages as the host.

[0041]

[0042]

[0043] Table 1 shows that the parasitism rate of *Blastophyrus stenoptera* on *A. stenoptera* larvae of different instars differed significantly, initially increasing and then decreasing with increasing host instar (df = 4, 10, F = 894.04, P < 0.001). Treatments 2 and 3, i.e., when the *A. stenoptera* larvae were 2nd or 3rd instar, showed the highest parasitism rate, exceeding 90%. The average parasitism rate of *Blastophyrus stenoptera* on 1st instar larvae of *A. stenoptera* was only 3.67%. As the host instar increased (i.e., the size of the host increased, such as 4th or 5th instar larvae), the parasitism rate of *Blastophyrus stenoptera* on the host decreased. This is because when the host is too large, its resistance to parasitic wasp attacks is stronger, and the female wasp cannot adequately paralyze and subdue the host, thus reducing the parasitism rate. Meanwhile, the host larvae significantly affected the number of offspring (df = 4, 10, F = 125.32, P < 0.001). When 2nd or 3rd instar *Anoplophora glabripennis* larvae were used as hosts for breeding, the number of offspring harvested per cage exceeded 2500, which was the optimal number among all treatments. These results indicate that using 2nd or 3rd instar *Anoplophora glabripennis* larvae as hosts for breeding *Anoplophora glabripennis* parasitic wasps allows the female wasps to exert the strongest parasitic effect, producing more offspring per inoculation. Furthermore, the 3rd instar host produces the largest number of offspring, making it the most suitable host for breeding wasps.

[0044] Example 2

[0045] (1) Cut a 2cm diameter willow wood into 15cm long sections. Make a 1cm x 1.5cm diagonal cut into the willow wood section, removing the xylem but retaining the bark. Place one 3rd instar larva of the Asian longhorn beetle into one of the cuts and cover it with bark. Then fix the bark of the cut with Parafilm. Inoculate 12 Asian longhorn beetle larvae into each sample wood.

[0046] (2) Place 25 sample woods with host plants in a 100-mesh mesh insect rearing cage with dimensions of 20cm×20cm×20cm.

[0047] (3) Parallel experimental setup: Mated female *Bretschneidera sinensis* were introduced into rearing cages at the following ratios (number of females: number of hosts): Treatment 1: 4:1; Treatment 2: 2:1; Treatment 3: 1:1; Treatment 4: 1:2; Treatment 5: 1:4; Treatment 6: 1:6. After introduction, the rearing cages were placed in an artificial climate chamber at 30℃, L:D = 14:10h, and RH = 70%. Each rearing cage was counted as one replicate.

[0048] (4) Two days after receiving the bees, the female bees are removed, and the insect rearing cage and sample wood are kept in the artificial climate chamber until the young bees develop and emerge.

[0049] (5) After the offspring were fully emerged, the sample wood was dissected. The parasitism rate of *Baracus styrensis* on the larvae of *Baracus styrensis* was calculated under different inoculation ratios. Successful parasitism was defined as the presence of a parasitic wasp cocoon inside the incision. The number of offspring and the female ratio that could be produced under different inoculation ratios were also calculated. The female ratio was calculated as the percentage of female offspring out of the total number of offspring.

[0050] Each treatment was repeated 3 times, and the results are shown in Tables 2 and 3:

[0051] Table 2. Parasitism rate of *Begonia sclerotiorum* under different bee species ratios.

[0052]

[0053] Table 3. Number of offspring bees and female ratio per cage under different bee-insect ratios.

[0054]

[0055] The results in Tables 2 and 3 show that when the ratio of *Braconchae stolonifer* to host was 1:1 to 1:4 (treatments 3, 4, and 5), the parasitism rate was above 90%, significantly higher than other treatments (df = 5, 12, F = 105.46, P < 0.001). Treatment 2 had a wasp-to-host ratio of 2:1, but its parasitism rate was actually lower than the treatments with lower wasp density, only about 50%. This result indicates that when the number of female wasps in a confined space is too high, it may cause competition for the host among female wasps, leading to a decrease in their parasitism ability. When the wasp-to-host ratio in treatment 1 further increased to 4:1, its average parasitism rate was less than 40%, indicating that under high female wasp density, female wasps compete more fiercely for host resources, impairing parasitism. Meanwhile, when the wasp-to-insect ratio in treatment 6 decreased to 1:6, its parasitism rate dropped to 66.33%, indicating that at this ratio, the female wasps were insufficient to parasitize the vast majority of the hosts, failing to achieve optimal reproductive efficiency. Regarding the number of offspring obtained, treatments 3, 4, and 5 significantly outnumbered other treatments (df = 5, 12, F = 50.28, P < 0.001), consistent with their high parasitism rates. There was no significant difference in the female ratio of offspring produced under different inoculation ratios (df = 5, 12, F = 0.62, P = 0.690). In conclusion, when artificially breeding *Braconchae lanceolata*, an inoculation ratio of 1:1 to 1:4 results in a high parasitism rate and also yields more offspring, making it a suitable inoculation ratio for artificial wasp breeding. Overall, when the bee-to-insect ratio is 1:4, the parasitism rate and the number of offspring produced are not significantly different from those treated with bee-to-insect ratios of 1:1 and 1:2, but it can save on the input of inoculated mother bees and is the optimal inoculation ratio for artificial bee breeding.

[0056] Example 3

[0057] (1) Cut a 2cm diameter willow wood into 15cm long sections. Make a 1cm x 1.5cm diagonal cut into the willow wood section, removing the xylem but retaining the bark. Place a 3rd instar longhorn beetle larva into one of the cuts and cover it with bark. Then fix the bark of the cut with Parafilm. Inoculate 12 longhorn beetle larvae into each sample wood.

[0058] (2) Place 25 sample woods with host plants in a 100-mesh mesh insect rearing cage with dimensions of 20cm×20cm×20cm.

[0059] (3) Parallel experimental setup: 75 mated female *Bretschneidera sinensis* were introduced into each rearing cage and cultured in an artificial climate chamber under the following conditions: Treatment 1: 22℃, L:D = 14:10h, RH = 70%; Treatment 2: 22℃, L:D = 10:14h, RH = 70%; Treatment 3: 26℃, L:D = 14:10h, RH = 70%; Treatment 4: 26℃, L:D = 10:14h, RH = 70%; Treatment 5: 30℃, L:D = 14:10h, RH = 70%; Treatment 6: 30℃, L:D = 10:14h, RH = 70%; Treatment 7: 34℃, L:D = 14:10h, RH = 70%; Treatment 8: 34℃, L:D = 10:14h, RH = 70%. Each rearing cage was counted as one replicate.

[0060] (4) Two days after receiving the bees, the female bees are removed, and the insect rearing cage and sample wood are kept in the artificial climate chamber until the young bees develop and emerge.

[0061] (5) After the offspring wereps emerged, the sample wood was dissected. The parasitism rate of *Blastodon sclerotiorum* on the larvae of *Blastodon sclerotiorum* was determined under different environmental conditions, with the presence of a parasitic wasp cocoon inside the incision indicating successful parasitism. The number of offspring wasps that could be bred under different environmental conditions was determined. The time required to breed one generation of *Blastodon sclerotiorum* wasps under different environmental conditions was determined, calculated as the interval from the time of receiving the wasps to the emergence of the first offspring wasp.

[0062] Each treatment was repeated 3 times, and the results are shown in Tables 4 and 5:

[0063] Table 4 Parasitism rate under different reproductive environment conditions

[0064]

[0065]

[0066] Table 5. Offspring number and generation cycle under different reproductive environmental conditions.

[0067]

[0068] Table 4 shows that temperature and light conditions significantly affect the parasitism rate of *Begonia radiata* (df = 7, 16, F = 118.73, P < 0.001). Treatments 1 and 2 were conducted at 22℃, under which female wasps exhibited low activity and weak parasitism, resulting in a low overall parasitism rate. Similarly, in treatments 7 and 8, at 34℃, female wasps were prone to heat stress and death under sustained high temperatures, leading to poor parasitism. Temperatures between 26 and 30℃ were optimal for female parasitism, resulting in a higher parasitism rate. Table 4 also indicates that long-day conditions significantly enhance the parasitism of female wasps. At a lower temperature of 22℃, long-day treatment 1 showed better parasitism than short-day treatment 2. At suitable temperatures of 26–30℃, long-day treatments also showed better parasitism than short-day treatments. As shown in Table 5, the parasitism rate was highest under conditions of 26–30℃ and long daylight, and the number of offspring produced by treatments 3 and 5 was significantly higher than that of other treatments (df = 7.16, F = 63.36, P < 0.001). Furthermore, we found that increasing temperature significantly shortened the reproductive cycle of *Begonia glabripennis* (df = 7.16, F = 238.98, P < 0.001). When the ambient temperature was 30℃ or higher, the parasitoid wasp only needed about 12 days to reproduce one generation, which was about 4 days shorter than under 26℃ conditions and about 11 days shorter than under 22℃ conditions. In conclusion, an ambient temperature of 26–30℃, a long daylight period of 14:10h, and a relative humidity of 70% resulted in a high parasitism rate and a large number of offspring for *Begonia glabripennis*, making these suitable environmental conditions for breeding this parasitoid wasp. At a temperature of 30℃, a photoperiod of 14:10h, and a relative humidity of 70%, not only can a high parasitism rate be obtained and more offspring be produced, but the reproductive cycle is also the shortest. This promotes the concentrated breeding of parasitic wasps in a short period of time and is the optimal breeding condition.

[0069] Example 4

[0070] (1) Cut a 2cm diameter willow wood into 15cm long sections. Make a 1cm x 1.5cm diagonal cut into the willow wood section, removing the xylem but retaining the bark. Place a 3rd instar longhorn beetle larva into one of the cuts and cover it with bark. Then fix the bark of the cut with Parafilm. Inoculate 12 longhorn beetle larvae into each sample wood.

[0071] (2) Place 25 sample woods with host plants in a 100-mesh mesh insect rearing cage with dimensions of 20cm×20cm×20cm.

[0072] (3) 75 mated female Brachiosa stylipoda were introduced into each insect rearing cage and placed in an artificial climate chamber at 30℃, L:D=14:10h, RH=70% for cultivation. This was recorded as one replicate.

[0073] (4) Parallel experimental setup: Different parasitism cycles were set up, i.e., the female bees were removed after different time periods of parasitism, including treatment 1: 1 day; treatment 2: 2 days; treatment 3: 4 days; treatment 4: 6 days; and treatment 5: 8 days. The rearing cages and sample wood were kept in the artificial climate chamber until the larvae developed to adulthood. The number of surviving female bees at the time of removal was recorded, and the survival rate was calculated.

[0074] (5) After the offspring werep emerged, dissect the sample wood. Calculate the parasitism rate of *Baracus stylens* larvae on *Baracus stylens* under different parasitism cycles. Successful parasitism is defined as the presence of a parasitic wasp cocoon inside the incision. Calculate the number of offspring wasps that can be produced under different parasitism cycles.

[0075] Each treatment was repeated 3 times, and the results are shown in Tables 6 and 7:

[0076] Table 6. Parasitism rate and offspring number under different inoculation and parasitism cycles.

[0077]

[0078] Table 7. Number and survival rate of female wasps removed from the rearing cage under different inoculation and parasitism cycles.

[0079]

[0080]

[0081] Table 6 shows that the inoculation parasitism period significantly affected the parasitism rate (df = 4, 10, F = 81.19, P < 0.001) and the number of offspring produced (df = 4, 10, F = 37.33, P < 0.001). Treatment 1, where the female wasps were parasitized for only 1 day, had significantly lower parasitism rate and fewer offspring produced compared to other treatments. When the parasitism period was 2 days or longer, a parasitism rate of approximately 91% was achieved, producing approximately 2600 offspring. Therefore, if the parasitism period is only 1 day, the female wasps are insufficient to maximize their parasitism and reproductive capacity. Table 7 shows that when the inoculation time for female wasps was 6 days or longer, the mortality rate of the female wasps was already high. Treatment 4 had a parasitism cycle of 6 days, at which point approximately 73% of the female bees remained surviving; Treatment 5 had a parasitism cycle of 8 days, at which point less than 50% of the female bees remained surviving; both were significantly lower than the survival rates of female bees with parasitism cycles of 1–4 days (df = 4, 10, F = 89.80, P < 0.001). In summary, when the parasitism cycle is 1 day, the parasitism of the female bees is not fully realized, resulting in a low parasitism rate and a small number of offspring. While a parasitism rate and number of offspring can be obtained with a parasitism cycle of 6 days or more, there is no significant difference compared to treatments with parasitism cycles of 2–4 days. This indicates that if the parasitism cycle reaches 2 days after a single inoculation parasitism process, the parasitism rate of the female bee and the number of offspring produced do not increase due to the continued coexistence of the female bee and the host in the same space. More importantly, when the parasitism period is 6 days or longer, a large number of female wasps die, which is not conducive to the reuse of female wasps and increases the breeding cost of *Begonia glabripennis*. Only when the parasitism period is 2-4 days can the best parasitism effect and offspring production be achieved, and more than 90% of the female wasps survive after the first inoculation. These surviving female wasps can be used again for inoculation and breeding, achieving the maximum utilization rate of female wasps. Within the limited lifespan of the female wasp, a parasitism period of 2 days allows for more repeated use of the female wasps, making it the best choice for breeding.

[0082] Example 5

[0083] (1) Cut a 2cm diameter willow wood into 15cm long sections. Make a 1cm x 1.5cm diagonal cut into the willow wood section, removing the xylem but retaining the bark. Place a 3rd instar longhorn beetle larva into one of the cuts and cover it with bark. Then fix the bark of the cut with Parafilm. Inoculate 12 longhorn beetle larvae into each sample wood.

[0084] (2) Place 25 sample woods with host plants in a 100-mesh mesh insect rearing cage with dimensions of 20cm×20cm×20cm.

[0085] (3) Seventy-five mated female *Bretschneidera sinensis* were introduced into each rearing cage and placed in an artificial climate chamber at 30°C, L:D = 14:10h, and RH = 70%. Two days after introduction, the females were removed, and the rearing cages and sample wood were kept in the artificial climate chamber until the larvae developed to adulthood.

[0086] (5) Parallel experimental setup: Newly emerged female wasps were collected in the same rearing cages as described above and placed in a low-temperature incubator under the following conditions: Treatment 1: 4℃; Treatment 2: 6℃; Treatment 3: 8℃; Treatment 4: 10℃; Treatment 5: 12℃; Treatment 6: 14℃; Treatment 7: 16℃. The relative humidity for all treatments was 90%, and there was no light. Each treatment contained 100 female wasps, placed in one rearing cage, and the survival status of the female wasps was recorded daily until all female wasps died.

[0087] Each wasp was counted as a replicate, and the average lifespan of female wasps under different storage conditions was statistically analyzed. The results are shown in Table 8.

[0088] Table 8 Lifespan of female bees under different storage conditions

[0089]

[0090] Table 8 shows that the optimal storage conditions for adult *Braconchaeella spicata* beetles are a temperature of 8–12℃, a relative humidity of 90%, and no light. Under these conditions, the average lifespan of female bees is over 2 months, significantly higher than other treatments (df = 6,693, F = 1283.12, P < 0.001). At temperatures of 6℃ or below, the average lifespan of female bees is less than 20 days, indicating that excessively low temperatures damage the vitality of adult bees. This may be related to frostbite caused by low temperatures, making it unsuitable for adult bee storage. At temperatures of 14℃ or above, female bees are more active and consume a large amount of energy, with an average lifespan of 20–28 days, thus only suitable for short-term storage. Overall, a temperature of 8℃, a relative humidity of 90%, and no light are the most suitable conditions for indoor storage of adult *Braconchaeella spicata* beetles, which is of great significance for the preservation and extended shelf life of this parasitic beetle after it has reproduced into a large population.

[0091] Example 6

[0092] (1) Based on the breeding methods and conditions determined in the previous experiment, the pedicel-shouldered star beetle was obtained and properly preserved.

[0093] (2) During the early larval stage of the Asian longhorn beetle, five independent willow forest plots with relatively consistent damage from the Asian longhorn beetle were selected. A random sampling survey was conducted to investigate the number of grooves made by the Asian longhorn beetle on 15 willow trees in each plot, and the average number of grooves per tree was calculated as the average larval density of the Asian longhorn beetle per tree.

[0094] (3) Parallel experimental setup: Based on the average density of *Anoplophora glabripennis* larvae per tree in each plot, female *Braconchis glabripennis* were placed at the base of willow trees in each plot according to the following wasp-to-larva ratios: Treatment 1: wasp-to-larva ratio = 3:1; Treatment 2: wasp-to-larva ratio = 2:1; Treatment 3: wasp-to-larva ratio = 1:1; Treatment 4: wasp-to-larva ratio = 1:2; Treatment 5: wasp-to-larva ratio = 1:3. Specifically, the female *Braconchis glabripennis* were placed in plastic centrifuge tubes, which were then placed at the base of the affected trees with the tube openings facing upwards and the caps removed.

[0095] (4) Fifteen days after releasing the bees, five willow trees were randomly selected from each plot for dissection. The number of parasitized *Aegilops glabripennis* larvae was recorded, and the parasitism rate was calculated. Parasitism was defined as the presence of *Aegilops glabripennis* stalk-belly braconid wasp larvae on the beetle's body within the borer tunnel, or the presence of *Aegilops glabripennis* stalk-belly braconid wasp cocoons within the borer tunnel. As an example, Figure 3 and Figure 4 The images show young larvae of *Braconchae lanceolata* parasitizing the larvae of the long-horned beetle *Anoplophora lanceolata* in the wild, and the pupae of *Braconchae lanceolata* after parasitizing the larvae. Each dissected willow tree was counted as a replicate to compare the parasitism effects under different release ratios. The results are shown in Table 9.

[0096] Table 9. Control effects of different release ratios on *Begonia styracifolium*.

[0097]

[0098] Table 9 shows that the parasitism effect of *Braconchaeopteryx glabripennis* on the larvae of *A. glabripennis* varies significantly under different release ratios (df = 4.20, F = 33.45, P < 0.001). When the wasp-larva ratio is 3:1 to 1:1, i.e., the number of released female wasps is equal to or greater than the number of *A. glabripennis* larvae, the average parasitism rate can reach over 45%. However, when the wasp-larva ratio decreases to 1:2 to 1:3, the parasitism rate drops significantly; at a ratio of 1:2, the average parasitism rate drops to 32%; and at a ratio of 1:3, the average parasitism rate drops to 17%. In summary, treatment 3, i.e., releasing wasps and larvae in equal proportions, not only achieves a better parasitism effect but also saves on the number of female wasps used. It reduces costs while meeting the control requirements, making it the preferred release ratio for the field application of *Braconchaeopteryx glabripennis*.

Claims

1. A method of rearing Apristota chinensis, characterized by, The method comprises the following steps: (1) using Anoplophora glabripennis 2-3 instar larvae as hosts, inoculating them on willow wood segments to obtain host-bearing sample wood; (2) placing the host-bearing sample wood in a mesh rearing cage, the mesh being 100 mesh, and the rearing cage having a size of 20 cm x 20 cm x 20 cm; (3) introducing mated Anoplophora glabripennis parasitoid female bees into the rearing cage and placing them in an artificial climate chamber for cultivation; wherein the ratio of Anoplophora glabripennis parasitoid female bees to host Anoplophora glabripennis larvae is 1:4, and the artificial climate chamber has a temperature of 26-30 DEG C, a light cycle of L:D = 14-16:10-8 h, and a relative humidity of RH = 50-70%; (4) removing the female bees after they have completed parasitism; leaving the rearing cage and sample wood in the artificial climate chamber until the larvae develop to the pupal stage; (5) collecting newly hatched male and female bees and storing them in a low-temperature incubator for use in forest prevention and control.

2. The breeding method according to claim 1, characterized by, In the first step, the Anoplophora glabripennis larvae are 3 instar larvae.

3. The breeding method according to claim 1, characterized by, In the first step, the host-bearing sample wood is prepared as follows: a willow with a diameter of 1-3 cm is cut into a wood segment with a length of 5-25 cm; a diagonal cut is made into the wood segment with a knife, the cut has a size of 0.5-1.5 x 1-2 cm, the cut wood is removed but the bark is retained; one Anoplophora glabripennis larva is placed in one cut and the bark is covered, and the cut bark is fixed with Parafilm.

4. The breeding method according to claim 3, characterized by, In the first step, the host-bearing sample wood is prepared as follows: a willow with a diameter of 2 cm is cut into a wood segment with a length of 10-20 cm; a diagonal cut is made into the wood segment with a knife, the cut has a size of 1 cm x 1.5 cm, the cut wood is removed but the bark is retained; one Anoplophora glabripennis larva is placed in one cut and the bark is covered, and the cut bark is fixed with Parafilm.

5. The breeding method according to claim 1, characterized by, In the second step, 25 host-bearing sample woods are introduced into each rearing cage.

6. The breeding method according to claim 1, characterized by, In the third step, the artificial climate chamber has a temperature of 30 DEG C, a light cycle of L:D = 14:10 h, and a relative humidity of RH = 70%.

7. The breeding method according to claim 1, characterized by, In the fourth step, the female bees complete parasitism in 2-4 days.

8. The breeding method according to claim 7, characterized by, In the fourth step, the female bees complete parasitism in 2 days.

9. The breeding method according to claim 1, characterized by, In the fifth step, the low-temperature incubator has a temperature of 8-12 DEG C, a relative humidity of RH = 80-90%, and no light.

10. The breeding method according to claim 1, characterized by, In the fifth step, the low-temperature incubator has a temperature of 8 DEG C, a relative humidity of RH = 90%, and no light.

11. Use of Anoplophora glabripennis parasitoid obtained by the breeding method of any one of claims 1-9 in the control of Anoplophora glabripennis.

12. The use of claim 11, comprising the following steps: (1) investigating the Anoplophora glabripennis population density in the forest during the early instar stage of Anoplophora glabripennis; (2) placing Anoplophora glabripennis parasitoid female bees at the base of trees damaged by Anoplophora glabripennis according to the Anoplophora glabripennis population density; Optionally, further comprising the step of evaluating the biological control effect: (3) 15 days after the bees were released, the bark around the notched of the Anoplophora nobilis were dissected to investigate the parasitism of the Anoplophora nobilis by the Cenocoelius nipponica, so as to evaluate the biological control effect.

13. Use according to claim 12, characterized in that, In the first step, the random sampling method is used to investigate the Anoplophora nobilis population density, and the average number of notches per plant represents the average larval density per plant; in the second step, the female Cenocoelius nipponica is placed in a plastic centrifuge tube, the centrifuge tube is placed at the base of the injured tree, the tube opening is upward, and the tube cover is removed.

14. Use according to claim 13, characterized in that, In the second step, the release ratio of the female Cenocoelius nipponica to the Anoplophora nobilis is 3:1 to 1:

1.

15. Use according to claim 14, characterized in that, In the second step, the release ratio of the female Cenocoelius nipponica to the Anoplophora nobilis is 1:1.

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