An artificial bee breeding method to improve the mating rate and reproductive efficiency of the pine sawyer beetle *Cyprinus spp.*

Abstract

This invention discloses a method for artificially breeding *Mesochrysoma pineatus* to increase the mating rate and obtain more female offspring. The method significantly improves the mating and fertilization rate of female *Mesochrysoma pineatus* by adjusting the airflow and light intensity of the mating environment, thereby increasing the proportion of female offspring. This invention also determines the optimal host body weight, the ratio of infected wasps to bees, and the suitable temperature for parasitism and development during breeding, which can significantly increase the parasitism rate, obtain more offspring, and shorten the development cycle of the offspring, allowing for the reproduction of more parasitic wasps in a shorter time. This invention, while ensuring successful mating of adult *Mesochrysoma pineatus*, improves the parasitism rate of female wasps during artificial breeding, resulting in more offspring, especially female offspring, and shortening the breeding cycle, making it applicable to the large-scale production of this parasitic wasp.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for forest pests, specifically to an artificial breeding method for improving the mating rate and reproductive efficiency of the parasitic natural enemy of the pine sawyer beetle, the pine sawyer beetle. Background Technology

[0002] The pine sawyer beetle (Monochamus alternatus) is a major pest of many pine trees. Its larvae are hidden and bore into the host plants. A more serious impact of the pine sawyer beetle lies in its adult stage, which transmits the pine wilt nematode (Bursaphelenchus xylophilus), the primary vector for pine wilt disease. Controlling vector insects is a core part of integrated pest management for pine wilt disease, as the pine sawyer beetle, once it emerges, is ready to transmit the nematode. Therefore, eliminating the pine sawyer beetle in its larval stage is the main control strategy for pine wilt disease, primarily through controlling the pine sawyer beetle. During its larval stage, the pine sawyer beetle feeds hidden under the bark and inside the xylem, making it difficult for conventional control measures to reach and be effective. Through long-term co-evolution with host pests, natural enemy insects have developed unique host-finding mechanisms, enabling them to accurately locate and control hosts. Therefore, biological control techniques, primarily using natural enemy insects, are currently the most effective way to control these hidden borers. Currently, the flower-haired parasitic beetle *Dastarcus helophoroides* and various *Sclerodermus* spp. are used for the biological control of the pine sawyer beetle, achieving some success, but also encountering some problems. For example, the flower-haired parasitic beetle's main parasitic target is the pine sawyer beetle pupa, and the ability to accurately determine the developmental stage of the pine sawyer beetle in the forest has a significant impact on the control effect. *Sclerodermus* has a broad host spectrum but lacks specialization; the adults are basically wingless, resulting in insufficient dispersal ability, and it mainly parasitizes second-instar and younger pine sawyer beetle larvae, resulting in generally poor control effectiveness in the field. Therefore, there is an urgent need to develop new natural enemy products with strong specialization and dispersal ability to effectively control pine sawyer beetle larvae.

[0003] *Cyanopterus ninghais*, a parasitic wasp on the larvae of the pine sawyer beetle, is a winged adult with strong dispersal capabilities. It exclusively parasitizes 3rd-5th instar larvae of the pine sawyer beetle. Forest surveys have revealed a natural parasitism rate of up to 30% on pine sawyer beetle larvae, with a single parasitic beetle producing up to 15 offspring. This makes it a valuable new natural enemy for pine sawyer beetle larval control. Enhanced biological control requires the release of natural enemies through flooding, and artificial breeding that overcomes these natural enemies, particularly obtaining more female individuals, is fundamental to ensuring the effectiveness of biological control. The parasitic wasp exhibits a haploid-diploid sex determination mechanism, meaning fertilized eggs develop into females, while unfertilized eggs develop into males. Field surveys have shown that the natural female-to-male ratio of *Cyanopterus ninghais* in the pine sawyer beetle population is close to 1:1. However, in previous indoor breeding experiments, it was found that when wild-collected individuals were used for inoculation using conventional methods, the proportion of female offspring was often less than 10%. Furthermore, we discovered that this wasp has strict environmental requirements when mating indoors. Unsuitable environments prevent female wasps from mating and fertilizing, resulting in a low or nonexistent female offspring, thus impairing breeding efficiency. During indoor breeding, we also found that female wasps exhibit a strong preference for the size of the host larvae of the pine sawyer beetle; only when the host size is suitable can offspring parasitic wasps be successfully raised. Currently, there are no reported methods for the artificial breeding of *Mesona chinensis*, and our previous research also revealed the specific environmental and host requirements of this parasitic wasp during mating and parasitism. Based on these findings, through extensive research, we have identified conditions that can improve the mating rate and female offspring ratio of *Mesona chinensis*, provide a suitable host-to-wasp ratio, and regulate environmental conditions during parasitism and development. We have developed a technology that enables the rapid and stable breeding of this parasitic wasp. The breakthrough in artificial breeding methods for the pine sawyer beetle, the braconid wasp, is of great significance for the biological control of the pine sawyer beetle and the integrated prevention and control of pine wilt disease. Summary of the Invention

[0004] Currently, there are no techniques or methods for the artificial breeding of *Condor braconidia* from the pine sawyer beetle. In the early stages of breeding this beetle, it was found that the offspring population was predominantly male, with females accounting for less than 10%. This was later identified as parthenogenesis due to unsuccessful mating of the female bees. Further investigation revealed that the mating success rate is closely related to environmental conditions. Simultaneously, the bee's preference for host size was not previously understood. During breeding, unsuitable host sizes often resulted in low parasitism rates, fewer offspring, and failure to fully utilize the reproductive potential of the female bee. Furthermore, an unsuitable host-to-be ratio also caused intraspecific competition or insufficient parasitism. In addition, exploring breeding conditions that ensure high parasitism rates for the female bee and high offspring survival rates while minimizing the generation cycle is crucial for improving breeding efficiency. These issues are all key to the large-scale breeding of *Condor braconidia* from the pine sawyer beetle. Therefore, the purpose of this invention is to provide a method for improving the mating rate of *Contortus pineaemonae*, thereby increasing the proportion of female offspring, and achieving rapid and large-scale production of *Contortus pineaemonae* by providing the most suitable host size, the optimal inoculation ratio, and the appropriate development temperature.

[0005] To achieve the above objectives, the present invention provides the following technical solution.

[0006] Technical Solution: An artificial breeding method for improving the mating rate and reproductive efficiency of the pine sawyer beetle, characterized in that the method includes the following steps: (1) Collect newly emerged male and female adult bees and transfer them to the mating room at a male-to-female ratio of 1:1 to complete mating, with a mating period of 3 days; (2) Use pine sawyer beetle larvae as the breeding host and pine wood segments as sample wood, inoculate pine sawyer beetle larvae into the sample wood to obtain wood segments with host; (3) Transfer the wood segments with host and the mated female pine sawyer beetle to the parasitism room to complete parasitism, with a parasitism period of 3 days; (4) After completing parasitism, transfer the wood segments to the emergence room for continued cultivation until the offspring parasitism emerges; (5) After the offspring emerge, transfer the male and female adult bees to the mating room, use 20% honey water to supplement their nutrition, and obtain a population that can be used in the wild after mating.

[0007] In a specific embodiment, the temperature in the mating chamber is 26–28°C, the relative humidity is 50–70%, and the air velocity is 6000–8000 cm⁻¹. 3 The photoperiod is L:D = 24:0h, and the illuminance is 8000–12000 Lux. The preferred conditions are a temperature of 27°C, a relative humidity of 60%, and an air velocity of 8000 cm⁻¹. 3 / min, photoperiod L:D=24:0h, illuminance 12000Lux.

[0008] In a specific embodiment, the weight of the host pine sawyer beetle larvae is between 400 and 500 mg, preferably 450 mg.

[0009] In a specific embodiment, the method for preparing the insect-infested wood segment is as follows: Make a U-shaped incision along the longitudinal direction of the wood segment with a knife; pry open the bark from the U-shaped opening without cutting it off; carve a groove measuring 3.5cm × 1cm × 1cm (length × width × depth) in the xylem along the edge of the U-shaped incision. Place the pine sawyer beetle larva into the groove and secure the opening end with PARAFILM sealing film.

[0010] In a specific embodiment, the ratio of the number of female braconid wasps to the number of host pine sawyer beetle larvae is 1:1 to 1:2.5, preferably 1:2.

[0011] In a specific embodiment, the conditions in the parasitic environment are: temperature 22–24°C, relative humidity 50–70%, and natural light. Preferably, the temperature is 22°C, relative humidity is 60%, and natural light is provided.

[0012] In a specific embodiment, the conditions in the feathering chamber are: temperature 28–34°C, relative humidity 50–70%, and natural light; preferably, temperature 30–32°C, relative humidity 55–65%, and natural light. More preferably, temperature 32°C, relative humidity 60%, and natural light.

[0013] This invention is based on the biology of the *Mesona chinensis*, a parasitic wasp, and reveals that after emergence, male and female wasps will only mate in a bright environment with a certain airflow velocity, thus producing female offspring. Unlike most parasitic wasps that are directly used for inoculation and reproduction after emergence, this invention creates suitable environmental conditions for mating, significantly improving the mating success rate, increasing the proportion of female offspring, and ensuring the efficiency of artificial breeding of this parasitic wasp. The research found that the *Mesona chinensis* thrives at 27°C and an airflow velocity of 8000 cm⁻¹. 3 In an environment with a light intensity of 12000 Lux and full brightness, both male and female adult wasps are most active, have a higher mating success rate, and produce the largest female-to-female ratio in offspring. Studies have shown that female wasps assess whether to engage in parasitism and the number of eggs to be laid by weighing the size of the host. This invention also clarifies the optimal host body weight, wasp-to-host ratio, and parasitism environmental conditions for wasp breeding. It was found that the optimal host size for *Mesona chinensis* parasitoides is 450 mg, and the optimal ratio of female wasp to host was 1:2. Under natural light and an ambient temperature of 22°C, the best parasitism rate and the largest number of offspring can be obtained. The ambient temperature during juvenile development significantly affects the development rate of the parasitoid wasp and the survival rate of offspring. This invention found that under natural light and an ambient temperature of 32°C, *Mesona chinensis* parasitoides completes one generation in the shortest time while still producing the most offspring, thus shortening the breeding cycle and achieving the best breeding efficiency while ensuring the survival rate of offspring. Attached Figure Description

[0014] Figure 1 Mating of the pine-brown longhorn beetle and the median-ridged braconid wasp; left: mating begins; right: mating ends.

[0015] Figure 2 The cocoon of the pine-brown longhorn beetle, the constrictor wasp. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below:

[0017] Example 1:

[0018] (1) Parallel experimental setup: Newly emerged male and female adult bees were placed in the following environmental conditions at a 1:1 ratio, including treatment 1: airflow velocity of 8000 cm⁻¹. 3 Treatment 1: Full brightness with an illuminance of 12000 Lux; Treatment 2: Air velocity of 8000 cm / min. 3 / min, fully lit with an illuminance of 8000 Lux; Treatment 3: airflow velocity of 8000 cm 3 / min, fully lit with an illuminance of 4000 Lux; Treatment 4: airflow velocity of 8000 cm 3 / min, no light; Treatment 5: airflow velocity of 6000cm 3 / min, fully lit with an illuminance of 12000 Lux; Treatment 6: airflow velocity of 3000 cm 3 / min, fully lit with an illuminance of 12000 Lux; Treatment 7: Air velocity in a closed environment is 0 cm / min. 3 / min, fully lit with an illuminance of 12000 Lux; Treatment 8: airflow velocity of 3000 cm 3 / min, full brightness and light intensity of 4000 Lux. The ambient temperature for all the above treatments was 27°C and the relative humidity was 60%. As an example, Figure 1 The mating status of the *Mesocarpus spp.* longhorn beetle wasp is shown. The male and female adults were removed 3 days after mating, and the female wasp was used for inoculation and reproduction.

[0019] (2) Cut fresh Masson pine into 20cm logs. Make a U-shaped cut in the bark along the length of the log. Pry open the bark from the U-shaped opening without cutting it off. Carve a groove (3.5cm x 1cm x 1cm, length x width x depth) in the xylem along the edge of the U-shaped cut. Place pine sawyer beetle larvae weighing approximately 450mg into the groove and secure the opening with PARAFILM sealing film. Inoculate 10 pine sawyer beetle larvae into each log.

[0020] (3) For each treatment, 15 mated female wasps were selected and placed in a parasitic room with 3 logs containing pine sawyer beetle larvae at 22°C, 60% relative humidity, and natural light. This was recorded as one replicate. Each treatment was set up with 3 replicates.

[0021] (4) After parasitizing for 3 days, the wood segment was transferred to a molting room with a temperature of 32°C, a relative humidity of 60%, and natural light.

[0022] After the offspring bees had emerged from their molts, the proportion of female offspring bees produced in each treatment and replicate was calculated. The results are shown in Table 1.

[0023] Table 1. Female ratio of offspring of *Spodoptera litura* and *Coprinus spp.* under different mating conditions.

[0024]

[0025]

[0026] Table 1 shows that during mating of male and female *Bretschneidera sinensis*, environmental air velocity and light intensity significantly affect the mating success rate, thus influencing the female ratio of offspring (df = 7, 16, F = 143.14, P < 0.001). Treatment 1 had the highest female ratio of 56.56% in its offspring, approaching the 1:1 female-to-male ratio of the natural wild population. Compared to treatment 1, treatments 2 and 5, with slightly lower light intensity and air velocity, resulted in a decrease in the female ratio of offspring produced by their respective mothers, but still maintained a female ratio of over 40%, slightly lower than the 1:1 female-to-male ratio in the wild population. Compared to treatment 1, treatments 3 and 6 further reduced the light intensity or airflow velocity. It was observed that the female offspring ratio of the queen bees mating in these environments was significantly lower; the female offspring ratio was less than 30% in treatment 3 and less than 20% in treatment 6. When queen bees mated in dark or poorly ventilated environments (treatments 4 and 7), or in environments with low airflow velocity and low light (treatment 8), the female offspring ratio was around 5%, indicating that the vast majority of queen bees underwent parthenogenesis, i.e., mating was unsuccessful. These results indicate that mating of the pine sawyer bee *Begonia pulcherrima* requires a certain level of airflow and light in the environment, and that high airflow velocity and high light intensity can increase the mating success rate of queen bees, thereby obtaining a higher proportion of female offspring.

[0027] Example 2:

[0028] (1) Newly emerged male and female adult bees were placed in a 1:1 ratio at a temperature of 27°C, a relative humidity of 60%, and an air velocity of 8000 cm⁻¹. 3 / min, photoperiod L:D=24:0h, light intensity 12000Lux, mating for 3 days in the mating chamber.

[0029] (2) Parallel experimental setup: Pine bark longhorn beetle larvae of different weight ranges were selected as host test subjects, namely: Treatment 1: <100mg; Treatment 2: 100-200mg; Treatment 3: 200-300mg; Treatment 4: 300-400mg; Treatment 5: 400-500mg; Treatment 6: 500-600mg; Treatment 7: 600-700mg.

[0030] (3) Cut fresh Masson pine into 20cm logs. Make a U-shaped cut in the bark along the length of the log. Pry open the bark from the U-shaped opening without cutting it off. Carve a groove (3.5cm x 1cm x 1cm, length x width x depth) in the xylem along the edge of the U-shaped cut for the host. Inoculate each log with 10 pine sawyer beetle larvae of the same weight range.

[0031] (4) For each treatment, 15 mated female wasps were selected and placed in a parasitic room at 22°C, 60% relative humidity, and natural light along with 3 logs containing pine sawyer beetle larvae of the same weight range. This was recorded as one replicate. Each treatment was repeated 3 times.

[0032] (5) After parasitizing for 3 days, the wood segment was transferred to a molting room with a temperature of 32°C, a relative humidity of 60%, and natural light.

[0033] After the offspring were fully emerged, the parasitism rate of the female wasps on pine sawyer beetle larvae of different sizes was determined by dissecting the wood segments. The presence of a pine sawyer beetle cocoon shell in the groove was considered a sign of parasitism. As an example, Figure 2 The cocoons spun by the parasitic wasp *Begonia pulcherrima* after completing its larval stage are shown. The number of offspring wasps and the female-to-female ratio were counted for each treatment. The results are shown in Tables 2 and 3.

[0034] Table 2. Parasitism rate of *Mesocarpus spp.* larvae of *Betula spp.* at different weights.

[0035]

[0036] Table 3. Number of offspring and female ratio of *Spodoptera litura* larvae parasitized by *Conexis braconidia* at different weights.

[0037]

[0038] Table 2 shows that host body weight significantly affected the parasitism rate of *Mesocarpus spp.* (df = 6, 14, F = 50.23, P < 0.001). With increasing host body weight, the parasitism rate of *Mesocarpus spp.* first increased and then decreased. This result indicates that when the host body weight is too small, the female wasp assesses that the host cannot provide sufficient nutrition for the development of its offspring, and therefore is unsuitable for oviposition, resulting in a low parasitism rate. Conversely, when the host body weight is too large, the host is more active and has a stronger ability to resist parasitizing wasp attacks, making it difficult for the female wasp to parasitize all hosts, thus resulting in a low parasitism rate. Treatment 5, with a host body weight of 400–500 mg, showed the highest parasitism rate of *Mesocarpus spp.* larvae in this group, at 86.67%.

[0039] Tables 2 and 3 show that the host body weight was significantly correlated with the parasitism rate of female wasps, thus significantly affecting the number of offspring of female *Bretschneidera sinensis* wasps (df = 6, 14, F = 124.11, P < 0.001). Furthermore, when the host was too small, the nutrition it could provide to the larvae was limited, resulting in fewer eggs laid by the female wasps and fewer offspring per host. When the host was too large, the female wasps consumed too much energy in subduing the host, thus reducing the energy available for reproduction and consequently decreasing the number of eggs laid, leading to a lower number of offspring. Treatment 5 had a host body weight of 400–500 mg. When *Bretschneidera sinensis* wasps parasitizing the larvae of this group of *Bretschneidera sinensis* wasps, an average of 109 offspring were produced per replicate, significantly higher than other groups. Simultaneously, this group also had a higher female-to-female ratio in the offspring, achieving a sex ratio structure of 1:1 in the wild population. In summary, the experimental results show that when the pine sawyer beetle larvae weigh 400–500 mg, the female wasp achieves the highest parasitism rate and the largest number of offspring, thus maximizing its reproductive potential. From the perspective of parasitic wasp breeding, selecting pine sawyer beetle larvae weighing 400–500 mg for breeding allows for a higher yield of offspring per inoculation, ensuring efficient breeding.

[0040] Example 3:

[0041] (1) Newly emerged male and female adult bees were placed in a 1:1 ratio at a temperature of 27°C, a relative humidity of 60%, and an air velocity of 8000 cm⁻¹. 3 Mating lasts for 3 days in a mating chamber with a photoperiod of L:D = 24:0h and an illumination intensity of 12000 Lux.

[0042] (2) Cut fresh Masson pine into 20cm logs. Make a U-shaped cut in the bark along the length of the log. Pry open the bark from the U-shaped opening without cutting it off. Carve a groove (3.5cm x 1cm x 1cm, length x width x depth) in the xylem along the edge of the U-shaped cut. Place pine sawyer beetle larvae weighing approximately 450mg into the groove and secure the opening with PARAFILM sealing film. Inoculate 10 pine sawyer beetle larvae into each log.

[0043] (3) Parallel experimental setup: After mating, female wasps were inoculated into host-bearing wood segments at the following ratios (number of female wasps: number of hosts): Treatment 1:3:1; Treatment 2:2:1; Treatment 3:1:1; Treatment 4:1:2; Treatment 5:1:3. After inoculation, the wood segments were placed in a parasitism room at 22℃, 60% relative humidity, and natural light. Three wood segments were selected as one replicate, and each treatment was replicated three times.

[0044] (4) After parasitizing for 3 days, the wood segment was transferred to a molting room with a temperature of 32°C, a relative humidity of 60%, and natural light.

[0045] After the offspring bees emerged, the parasitism rate was calculated by dissecting the wood segments and determining the parasitism rate under different bee inoculation ratios. The presence of a *Mesona chinensis* cocoon shell in the groove was considered parasitism. The number of offspring bees and the female ratio were also calculated for each replicate of each treatment. The results are shown in Tables 4 and 5.

[0046] Table 4 Parasitism rate under different bee inoculation ratios

[0047]

[0048] Table 5. Number of offspring bees and proportion of female offspring under different inoculation ratios.

[0049]

[0050] Table 4 shows that when the parasitism ratio was 1:1 to 1:2 (treatments 3 and 4), the parasitism rate of *Begonia pineinae* larvae on *Begonia pineinae* was significantly higher than in other groups, at 88.89% and 86.67%, respectively (df = 4, 10, F = 107.71, P < 0.001). In treatments 1 and 2, the number of female parasitists exceeded the number of hosts, yet the parasitism rate decreased, with the parasitism rate decreasing as the parasitism ratio increased. This indicates that under high-density female parasitism conditions, individual female parasitists compete for hosts, disrupting the parasitism process and impairing breeding efficiency. Meanwhile, in treatment 5, the number of female parasitists was far less than the number of hosts, and its average parasitism rate was also less than 50%. This indicates that when the number of female parasitists is too small, they lack the ability to parasitize the vast majority of hosts, thus impairing the efficiency of a single breeding cycle. Table 5 shows that treatments 3 and 4 achieved the highest parasitism rates, and their average number of offspring wasps was significantly higher than other treatments (df = 4, 10, F = 82.25, P < 0.001), indicating the highest breeding efficiency. The female-to-female ratio of offspring did not differ significantly among the treatments (df = 4, 10, F = 0.07, P = 0.990). In summary, a bee-to-larvae ratio of 1:1 to 1:2 achieves optimal parasitism rates and the number of offspring wasps, making it a suitable ratio for large-scale bee breeding. However, a bee-to-larvae ratio of 1:2 can save on the number of queen bees required, and this ratio should be prioritized for inoculation when the number of queen bees is limited.

[0051] Example 4:

[0052] (1) Newly emerged male and female adult bees were placed in a 1:1 ratio at a temperature of 27°C, a relative humidity of 60%, and an air velocity of 8000 cm⁻¹. 3 Mating lasts for 3 days in a mating chamber with a photoperiod of L:D = 24:0h and an illumination intensity of 12000 Lux.

[0053] (2) Cut fresh Masson pine into 20cm logs. Make a U-shaped cut in the bark along the length of the log. Pry open the bark from the U-shaped opening without cutting it off. Carve a groove (3.5cm x 1cm x 1cm, length x width x depth) in the xylem along the edge of the U-shaped cut. Place pine sawyer beetle larvae weighing approximately 450mg into the groove and secure the opening with PARAFILM sealing film. Inoculate 10 pine sawyer beetle larvae into each log.

[0054] (3) Parallel experimental setup: Fifteen mated female wasps were selected and placed in a parasitism chamber with three logs containing host materials under the following conditions: Treatment 1: 18℃; Treatment 2: 20℃; Treatment 3: 22℃; Treatment 4: 24℃; Treatment 5: 26℃; Treatment 6: 28℃. All treatments were conducted under natural light and with a relative humidity of 60%. Each set of three logs was considered one replicate, and each treatment was replicated three times.

[0055] (4) After parasitizing for 3 days, the wood segment was transferred to a molting room with a temperature of 32°C, a relative humidity of 60%, and natural light.

[0056] After the offspring bees had emerged, the parasitism rate was statistically analyzed under different temperature conditions by dissecting wood segments. The presence of a *Mesona chinensis* cocoon shell in the groove was considered indicative of parasitism. The results are shown in Table 6.

[0057] Table 6. Parasitism rate of *Mesocarpus spp.* under different parasitism environments and temperatures.

[0058]

[0059] Table 6 shows that the parasitism rate of female wasps on the larvae of the pine sawyer beetle varied significantly under different temperature conditions (df = 5, 12, F = 81.20, P < 0.001); the parasitism rate first increased and then decreased with increasing temperature. When the temperature was too low (18–20℃), the activity of female wasps was very low, resulting in a low parasitism rate. When the temperature was too high, the larvae of the pine sawyer beetle quickly produced a large amount of frass in the grooves of the wood and quickly burrowed into the xylem, making it difficult for female wasps to parasitize them. Overall, treatments 3 and 4, i.e., the artificial breeding and parasitism process, occurred at an environment with a temperature of 22–24℃, and the parasitism rate reached the highest.

[0060] Example 5:

[0061] (1) Newly emerged male and female adult bees were placed in a 1:1 ratio at a temperature of 27°C, a relative humidity of 60%, and an air velocity of 8000 cm⁻¹. 3 Mating lasts for 3 days in a mating chamber with a photoperiod of L:D = 24:0h and an illumination intensity of 12000 Lux.

[0062] (2) Cut fresh Masson pine into 20cm logs. Make a U-shaped cut in the bark along the length of the log. Pry open the bark from the U-shaped opening without cutting it off. Carve a groove (3.5cm x 1cm x 1cm, length x width x depth) in the xylem along the edge of the U-shaped cut. Place pine sawyer beetle larvae weighing approximately 450mg into the groove and secure the opening with PARAFILM sealing film. Inoculate 10 pine sawyer beetle larvae into each log.

[0063] (3) Select 15 mated female bees and place them with 3 pieces of wood with host in a parasitic room at 22℃, 60% relative humidity and natural light for 3 days.

[0064] (4) Parallel experimental setup: After parasitism was completed, the log segments were moved to a molting chamber under the following conditions: Treatment 1: 26℃; Treatment 2: 28℃; Treatment 3: 30℃; Treatment 4: 32℃; Treatment 5: 34℃; Treatment 6: 36℃. All treatments were conducted under natural light and with a relative humidity of 60%. Each set of three log segments was counted as one replicate, and each treatment was replicated three times.

[0065] The time interval from the time the bees were moved into the emergence chamber to the start of emergence for each treatment was recorded as the juvenile period. After the progeny bees completed emergence, the number of progeny was counted. The results are shown in Table 7:

[0066] Table 7. Larval stage duration and offspring number of *Bretschneidera sinensis* at different temperatures.

[0067]

[0068] Table 7 shows that the ambient temperature significantly affects the larval stage of the *Pterocarya stenoptera* (a type of beetle) (df = 5, 12, F = 51.08, P < 0.001). When the ambient temperature is 26–28℃, the larval stage lasts approximately 20 days; when the ambient temperature rises to 30℃ or higher, the larval stage is shortened by about one week. However, while the larval stage is significantly shortened with increasing temperature, the number of offspring bees decreases significantly when the temperature reaches 34℃ or higher (df = 5, 12, F = 46.93, P < 0.001). This result indicates that high temperatures cause some larvae to die, preventing them from completing their life cycle. In conclusion, treatments 3 and 4, where larvae develop in an environment of 30–32℃, can maintain a high survival rate of larvae while shortening the production and breeding cycle, thus achieving optimal bee breeding efficiency.

Claims

1. A method for artificially breeding *Apis cerana* to improve the mating rate and reproductive efficiency of *Apis cerana*, characterized in that, The method includes the following steps: (1) Collect newly emerged male and female adult bees and transfer them to the mating area at a female-to-male ratio of 1:1 to complete mating. The mating period is 3 days. During this period, the temperature is 26-28℃, the relative humidity is 50-70%, and the air velocity is 6000-8000 cm. 3 / min, photoperiod L:D=24:0h, illuminance 8000~12000Lux; (2) Using the pine sawyer beetle larvae as the breeding host, and using pine logs as sample wood, the pine sawyer beetle larvae were inoculated into the sample wood to obtain the logs with the host; among them, the weight of the pine sawyer beetle larvae was 400-500 mg. (3) Transfer the wood segment with the host and the mated female pine sawyer beetle to the parasitic space to complete the parasitism. The parasitism period is 3 days. The temperature is 22-24℃ and the relative humidity is 50-70%. (4) After parasitism is completed, the wood segment is transferred to the emergence room for further cultivation until the offspring parasitic wasps emerge; (5) After the offspring bees emerge, the male and female adult bees are transferred to the mating area and given honey water to supplement their nutrition. After mating, a population that can be used in the wild is obtained. In step (3), the ratio of the number of female *Begonia spp.* corydalis to the number of larvae of the host *Begonia spp.* is 1:1 to 1:2.

5. The conditions for the feathering process in step (4) are: temperature 30-32℃, relative humidity 55-65%, and natural light.

2. The bee breeding method according to claim 1, characterized in that, The mating conditions in step (1) are: 27°C, 60% relative humidity, and 8000 cm⁻¹ air velocity. 3 / min, photoperiod L:D=24:0h, illuminance 12000Lux.

3. The bee breeding method according to claim 1, characterized in that, In step (2), the weight of the pine beetle larvae is 420–480 mg.

4. The bee breeding method according to claim 1, characterized in that, In step (2), the preparation of the host wood segment is as follows: use a knife to make a "U" shaped cut in the bark along the longitudinal direction of the wood segment, pry open the bark from the "U" shaped opening end but do not cut it off; dig a groove in the wood along the edge of the "U" shaped cut; put the pine sawyer beetle larva into the groove, and fix the opening end with a breathable sealing film.

5. The bee breeding method according to claim 4, characterized in that, The groove measures 3.5cm in length, 1cm in width, and 1cm in depth; the breathable sealing film is PARAFILM sealing film.

6. The bee breeding method according to claim 1, characterized in that, In step (3), the ratio of the number of female parasitic wasps of the pine sawyer beetle to the number of pine sawyer beetle larvae is 1:

2.

7. The bee breeding method according to claim 1, characterized in that, The conditions for the parasite in step (3) are: temperature 22℃, relative humidity 60%, and natural light.

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