A method for propagating Japanese ladybugs and its application

Abstract

This invention relates to a method for propagating the Japanese ladybug and its application, belonging to the field of insect population propagation technology. The method for propagating the Japanese ladybug includes introducing Japanese ladybugs to lay eggs on eggplant leaf discs, which hatch into larvae; feeding Mediterranean mealybug eggs in a culture dish to obtain adult Japanese ladybugs; mating and egg-laying in a concentrated manner to obtain Japanese ladybug eggs; and repeating this cycle to complete the propagation of the Japanese ladybug. The Japanese ladybugs propagated by this invention show significantly improved growth, development, and reproduction, with a significantly increased survival rate, and their predation ability against whiteflies is comparable to that of natural populations.

Description

Technical Field

[0001] This invention relates to the field of insect population propagation technology, and in particular to a method for propagating the Japanese ladybug and its application. Background Technology

[0002] Whiteflies ( Bemisia tabaci The whitefly is a major global pest, often referred to as a "super pest," causing billions of dollars in economic losses to agricultural production annually. It exhibits high levels of resistance to a wide range of conventional and novel insecticides, making it extremely difficult to control.

[0003] Japanese ladybug ( Serangium japonicu The Japanese ladybug (Hypophthalmia japonica) is an important predatory natural enemy of the whitefly, widely distributed in my country. It possesses strong predatory ability, consumes large quantities of food, has a short growth cycle, and a long adult lifespan, making it highly valuable for biological control. However, the traditional propagation model relying on planting plants, raising whiteflies, and then raising Japanese ladybugs suffers from drawbacks such as being time-consuming, labor-intensive, seasonally limited, and requiring large areas of land, hindering large-scale production. Using alternative feeds for Japanese ladybug rearing encounters bottlenecks such as a sharp decline in adult reproductive capacity, low egg production, and severe constraints on insect production and utilization. Furthermore, the difficulty in distinguishing the sex of adult Japanese ladybugs and the inability to directly observe the eggs with the naked eye further complicates reproductive research. Therefore, there is an urgent need to develop propagation methods for Japanese ladybugs based on alternative feeds. Summary of the Invention

[0004] The purpose of this invention is to provide a method for propagating Japanese ladybugs and its application, in order to solve the problems of high difficulty in propagating adult Japanese ladybugs, low reproductive capacity, low egg production, and serious restriction on insect production and utilization in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for propagating the Japanese ladybug, comprising the following steps: (1) Pour 0.5-1.0 cm of agar hydrogel into the bottom of the culture dish, place eggplant leaf discs with whiteflies on it, introduce Japanese ladybugs to lay eggs on the eggplant leaf discs, and hatch Japanese ladybug larvae. (2) The larvae of Japanese ladybug were placed in a culture dish and fed with Mediterranean mealy borer eggs to obtain adult Japanese ladybugs; (3) The adult Japanese ladybugs are transferred to a culture dish and mated together for 7-9 days. They then lay eggs to obtain Japanese ladybug eggs. This process is repeated to complete the propagation of Japanese ladybugs.

[0006] Preferably, the culture vessel in steps (1) to (3) is made of polypropylene.

[0007] Preferably, the concentration of the agar hydrogel in step (1) is 1% to 2%.

[0008] Preferably, the whiteflies in step (1) include whitefly eggs and / or whitefly nymphs.

[0009] Preferably, the diameter of the eggplant leaf disc in step (1) is 2 to 4 cm.

[0010] Preferably, the temperature for rearing in step (2) is 25-27°C, the relative humidity is 55%-65%, and the light exposure time is 10-16 h / d.

[0011] Preferably, the number of adult Japanese ladybugs in step (3) is 10 to 12 per culture dish.

[0012] This invention also provides the application of the aforementioned propagation method in the propagation of Japanese ladybugs.

[0013] The present invention has the following technical effects and advantages: This invention's propagation method involves directly placing Mediterranean whitefly eggs in polypropylene culture dishes, using agar hydrogel for humidification, and employing small-scale group mating and regular egg-laying checks to promote the long-term survival of the Japanese ladybug, overcome reproductive barriers, and successfully lay eggs for subsequent generations. Based on previous research, this invention finds that the leaf hair structure of plant leaves enhances the adhesion of the Japanese ladybug, causing it to prefer laying eggs on leaves with leaf hair structures, and that egg production is positively correlated with adhesion. Therefore, this invention creatively uses polypropylene culture dishes with a raised bottom; the raised area and surrounding surface are relatively rough, facilitating attachment of the Japanese ladybug and significantly increasing egg production. The propagated Japanese ladybugs show significantly improved growth, development, and reproduction, with a significantly increased survival rate, and their predation ability against whiteflies is comparable to that of natural populations. Attached Figure Description

[0014] Figure 1 A polypropylene sauce cup (with raised bottom) for propagating Japanese ladybugs; Figure 2 The effect of the propagation method of the present invention on the weight of adult Japanese ladybugs; Figure 3 The effect of the propagation method of the present invention on the predatory function of Japanese ladybug is shown in the figure, where A is the Bt-F1 generation and B is the Ek-F8 generation. Detailed Implementation

[0015] This invention provides a method for propagating the Japanese ladybug, comprising the following steps: (1) Pour 0.5-1.0 cm of agar hydrogel, preferably 0.5 cm, into the bottom of the culture dish; place an eggplant leaf dish with whiteflies on it, introduce Japanese ladybugs to lay eggs on the eggplant leaf dish, and hatch Japanese ladybug larvae. (2) The larvae of Japanese ladybug were placed in a culture dish and fed with Mediterranean mealy borer eggs to obtain adult Japanese ladybugs; (3) Transfer adult Japanese ladybugs to a culture dish and mate them for 7-9 days, preferably 7 days, to lay eggs and obtain Japanese ladybug eggs. Repeat this process to complete the propagation of Japanese ladybugs.

[0016] In this invention, the culture vessels described in steps (1) to (3) are made of polypropylene.

[0017] In this invention, the bottom of the culture vessel described in steps (1) to (3) preferably has at least one protrusion, and more preferably has one protrusion.

[0018] In this invention, the culture vessel mentioned in steps (1) to (3) includes one or more of the following: culture dish, sauce cup, and plastic bowl.

[0019] In this invention, the diameter of the culture dish is 3-4 cm, preferably 3.5 cm; the height of the culture dish is 2-4 cm, preferably 2 cm. The sauce cup has a volume of 20-30 mL, preferably 25 mL; The volume of the plastic bowl is 400-500mL, preferably 450mL.

[0020] In this invention, the concentration of the agar hydrogel in step (1) is 1% to 2%, preferably 1.2%.

[0021] In this invention, the whiteflies mentioned in step (1) include whitefly eggs and / or whitefly nymphs.

[0022] In this invention, the diameter of the eggplant leaf disc in step (1) is 2 to 4 cm, preferably 3.3 cm.

[0023] In this invention, the temperature for rearing in step (2) is 25-27°C, preferably 26°C; the relative humidity for rearing is 55%-65%, preferably 60%; and the light exposure time for rearing is 10-16 h / d, preferably 14 h / d.

[0024] In this invention, the number of adult Japanese ladybugs in step (3) is 10 to 12 per culture dish, preferably 10 per culture dish.

[0025] This invention also provides the application of the aforementioned propagation method in the propagation of Japanese ladybugs.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] In the test materials of this invention, the Torubam variety of eggplant ( Solanumtorvum Germplasm resources were purchased from Henan Chunqiu Seed Industry Technology Co., Ltd., and cultivated in the glass greenhouse of the Fujian Academy of Agricultural Sciences Strait Modern Agriculture Research Institute Demonstration Park (26°13′N, 119°34′E). The Japanese ladybug was collected in 2009 from a sweet potato field in the suburbs of Fuzhou City, Fujian Province (25°15′N, 117°30′E). From 2009 to 2017, it was reared indoors on cotton seedlings inoculated with whiteflies to 120 generations, and from 2018 to 2023, it was reared indoors on Torubam eggplant plants to 53 generations. The Mediterranean mealybug was used as a prey substitute. Ephestia kuehniella ) eggs.

[0028] Example 1: Determination of body weight and reproductive characteristics of Japanese ladybug In polypropylene petri dishes ( Φ Pour a 1.2% agar solution into the bottom of a plate (h=3.5cm, h=2cm) and let it solidify into a 0.5cm agar hydrogel; then place an eggplant leaf dish containing whitefly eggs and nymphs on top of it. Φ =3.3cm), and inoculated with Japanese ladybug eggs (Bt-F1 generation) laid within 24 hours for hatching to obtain Japanese ladybug larvae; the Japanese ladybug larvae were then transferred to a 25mL polypropylene sauce cup with a raised bottom (e.g., Figure 1 As shown, the larvae of *S. japonicus* were reared in an artificial climate chamber at 26℃, 60% relative humidity, and 14h / d light. They were fed *Mediterranean mealybug* eggs, and the growth and development of the larvae were observed daily until adult *S. japonicus* emerged. Ten adult *S. japonicus* were placed in a 450mL polypropylene plastic bowl containing a sufficient amount of *Mediterranean mealybug* eggs and mated together for 7 days. Afterward, they were reared separately and their oviposition was observed for 2 days. For individuals that could not lay eggs, they were mated together again for 7 days, and then reared separately and their oviposition was observed for 2 days to obtain Ek-F1 generation eggs. Individuals that could not lay eggs were dissected after death to determine their sex. The above steps were repeated to obtain Ek-F2 to Ek-F8 generation eggs. Ninety eggs from Ek-F1 to Ek-F8 generation were randomly selected for hatching and rearing, and their growth and development were recorded daily until adult *S. japonicus* emerged from each generation. The body weight and reproductive status of adult Japanese ladybugs at each generation were measured and recorded. Japanese ladybugs fed entirely with whitefly eggs and nymphs (Bt-F1 generation) served as a control. Results are as follows: Figure 2 As shown in Table 1.

[0029] Table 1. Effects of successive rearing on the reproduction of Japanese ladybugs. The results of body weight measurement showed that the body weight of adult Japanese ladybugs varied under different treatments ( F =4.43、 df =8, 620、 P <0.001), different sex ratios ( F =13.74、 df =1, 620、 P <0.001) and the interaction between processing and sex ratio ( F =12.64、 df =8, 620、 P Significant differences were observed in all data (<0.001). Multiple comparisons showed that, except for the significantly larger female adult weights in the Ek-F3 and Ek-F4 generations compared to the Ek-F8 generation, there were no significant differences in female adult weights among generations. Compared to the Bt-F1 generation, the female adult weights in the Ek-F5, Ek-F7, and Ek-F8 generations were significantly reduced, while other generations showed no significant changes. The male adult weights in the Ek-F7 generation were significantly larger than those in other generations, while the male adult weights in the Ek-F1 generation were significantly smaller. The weight of male adults in the Ek-F4 to Ek-F8 generations was significantly smaller than that in the Bt-F1 generation. Compared with the Bt-F1 generation, the weight of male adults in the Ek-F4 to Ek-F8 generations was significantly increased. Except for the Ek-F4 to Ek-F6 generations, there were significant differences in the weight of male and female adults in the other generations and the Bt-F1 generation. Except for the Ek-F7 generation, where the weight of male adults was significantly greater than that of female adults, the weight of female adults in the Ek-F1 to Ek-F3, Ek-F8, and Bt-F1 generations was significantly greater than that of male adults. Reproduction results showed that after rearing with Mediterranean mealybug eggs, reproductive parameters of *Ladybug japonicus*, including single female oviposition, pre-oviposition period, total pre-oviposition period, oviposition days, oviposition duration, and adult lifespan, were significantly improved. However, the sex ratio of females to males did not change significantly. The single female oviposition of generations Ek-F1 to Ek-F8 generally showed an upward trend. The single female oviposition of generation Ek-F1 was not significantly different from that of generation Bt-F1, while the single female oviposition of generations Ek-F2 to Ek-F8 was significantly greater than that of generation Bt-F1. 79.17% of adult females in generation Ek-F1 laid eggs, while 89.29%, 82.05%, and 87.80% of adult females in generations Ek-F2, Ek-F4, and Ek-F8, respectively, laid eggs. All adult females in generations Ek-F5 to Ek-F8 and generation Bt-F1 laid eggs. The pre-oviposition period and total pre-oviposition period of adults in generations Ek-F1 to Ek-F5 showed a significant shortening with each generation, decreasing from 96.63 days and 113.53 days in the Ek-F1 generation to 8.44 days and 22.87 days in the Ek-F5 generation, respectively. The periods in generations Ek-F5 to Ek-F8 were relatively stable, indicating that after five generations of rearing and domestication, the pre-oviposition period and total pre-oviposition period of *Ladybug japonicus* tended to stabilize. Compared with the Bt-F1 generation, there was no significant change in the pre-oviposition period of adults in the Ek-F8 generation. Although the total pre-oviposition period in the Ek-F8 generation was still significantly prolonged, the average difference between the two was only 1.30 days. The number of days and the duration of oviposition in generations Ek-F1 to Ek-F8 were significantly longer than those in the Bt-F1 generation. Except for the Ek-F4 generation, which had significantly longer oviposition days and duration than other generations, there were mostly no significant differences between generations. The lifespan of adults in generations Ek-F1 to Ek-F8 was significantly longer than that of the Bt-F1 generation. The lifespan of female adults showed a decreasing trend, while the lifespan of male adults showed a trend of first increasing and then decreasing. Female adults in generations Ek-F1, Ek-F2, and Ek-F4 had significantly longer lifespans than other generations, while female adults in generations Ek-F7 and Ek-F8 had significantly shorter lifespans. Male adults in generation Ek-F5 had significantly longer lifespans than other generations, while male adults in generations Ek-F7 and Ek-F8 had significantly shorter lifespans. There was no significant difference in lifespan between male and female adults in the Bt-F1 generation, but female adults in generations Ek-F1 to Ek-F8 had significantly longer lifespans than male adults. Furthermore, using a polypropylene sauce cup with a raised bottom was more conducive to the attachment of the Japanese ladybug, thus significantly increasing the number of eggs laid by a single female.

[0030] Example 2: Determination of growth, development, and survival rate of Japanese ladybugs The developmental parameters and survival rates of each generation of Japanese ladybugs in Example 1 were measured, and the results are shown in Table 2.

[0031] Table 2. Effects of successive rearing on the developmental period and survival rate of *Ladybug japonicus*. The results of the developmental period parameters showed that after the Japanese ladybug fed on the eggs of the Mediterranean mealybug, the developmental period of its life cycle, including the pre-adult stage (egg to adult emergence), egg stage, larval stage, and pupal stage, varied significantly among generations after rearing. Overall, the developmental period showed a trend of shortening from Ek-F1 to Ek-F6 and lengthening from Ek-F6 to Ek-F8. During the pre-adult developmental period, the Ek-F1 to Ek-F3 generation had the longest developmental period (16.78–17.23 days), while the Ek-F6 generation had the shortest (13.50 days). The Ek-F1 to Ek-F3 generation was significantly longer than the Ek-F4 to Ek-F8 generation; the Ek-F6 generation was significantly shorter than the other generations. Compared with the Bt-F1 generation, except for the significantly shortened Ek-F6 generation and the no significant changes in the Ek-F5 and Ek-F7 generations, all other generations were significantly longer. During egg development, the Ek-F2 generation had the longest development period (4.34 days), while the Ek-F5 and Ek-F6 generations had the shortest (3.20–3.28 days). Compared to the Bt-F1 generation, the Ek-F2–Ek-F3 and Ek-F8 generations were significantly longer, while the Ek-F4–Ek-F7 generations were significantly shorter. During larval development, the Ek-F1 generation (9.09 days) and Ek-F3 generation (9.06 days) were significantly longer than other generations, while the Ek-F6 generation (6.95 days) was significantly shorter. Compared to the Bt-F1 generation, the Ek-F6 generation was significantly shorter, the Ek-F7 generation showed no significant change, and all other generations were significantly longer. Among the four larval instars, the developmental periods of the 1st, 2nd, and 3rd instars showed a decreasing trend across generations, while the 4th instar, with the longest developmental period, first decreased and then increased. Compared with the Bt-F1 generation, except for the Ek-F3 generation of 2nd instar larvae, the 1st and 2nd instar larvae showed significantly prolonged development in the Ek-F1 to Ek-F4 generations, while there was no significant difference or significant shortening in the Ek-F5 to Ek-F8 generations. The 3rd instar larvae showed no significant change in the Ek-F1 to Ek-F7 generations, but significantly shortened in the Ek-F8 generation. The 4th instar larvae showed no significant change in the Ek-F4 and Ek-F7 generations, but significantly shortened in the Ek-F6 generation, while significantly prolonged development in other generations. During pupal development, the Ek-F2 generation (4.56 days) and Ek-F8 generation (4.44 days) were significantly longer than other generations, while the Ek-F6 generation (3.23 days) was significantly shorter. Compared with the Bt-F1 generation, the Ek-F6 generation was significantly shorter, while the Ek-F1 and Ek-F7 generations showed no significant change, and all other generations were significantly prolonged.

[0032] Survival rate measurements showed that the survival rate of *Ladybug japonicus* after feeding on *Mediterranean mealybug* eggs varied significantly across generations during the pre-adult, egg, larval, and pupal stages. The survival rates during the pre-adult and larval stages exhibited relatively large variations across generations, while the survival rates during the egg and pupal stages showed relatively more gradual changes. The pre-adult survival rate in the Ek-F1 generation was only 51.1%, significantly increasing to 87.0% in the Ek-F8 generation. The larval survival rate in the Ek-F1 generation was 56.1%, significantly increasing to 95.8% in the Ek-F8 generation. The pre-adult survival rate generally showed an upward trend from Ek-F1 to Ek-F5 generations, remaining above 76% from Ek-F5 to Ek-F8 generations. Compared to the Bt-F1 generation, the pre-adult survival rate showed no significant change from Ek-F1 to Ek-F3 generations, while it significantly increased from Ek-F4 to Ek-F8 generations. The larval survival rate varied across generations in a similar pattern to that of the early adult stage. For egg hatching rates, except for the Ek-F6 generation, there were no significant differences between other generations or between each generation and the Bt-F1 generation. For pupal survival rates, except for the Ek-F7 and Ek-F8 generations which were significantly lower than the Bt-F1 and Ek-F4 generations, there were no significant differences between other generations or between each generation and the Bt-F1 generation.

[0033] Example 3: Determination of population parameters of Japanese ladybug Population parameters of each generation of Japanese ladybugs in Example 1 were determined, and the results are shown in Table 3.

[0034] Table 3 Effects of successive rearing on population parameters of Japanese ladybug. The results showed that after feeding on Mediterranean mealybug eggs, the net growth rate (R0) of *Syngonium japonicum* significantly increased from generation Ek-F1 to Ek-F3, and remained stable from generation Ek-F3 to Ek-F8. The intrinsic growth rate (r) and cyclic growth rate (λ) showed the same trend, significantly increasing from generation Ek-F1 to Ek-F8. The average generation time (T) significantly shortened from generation Ek-F1 to Ek-F5, and remained relatively stable from generation Ek-F5 to Ek-F8, although there were fluctuations. This indicates that compared to *Syngonium japonicum* feeding on its natural prey, the whitefly, after successive generations of rearing, the net growth rate of *Syngonium japonicum* in generation Ek-F8 was significantly higher, while the average generation time remained significantly longer, although there were no significant differences in the intrinsic growth rate and cyclic growth rate.

[0035] Example 4: Determination of predation rate of Japanese ladybug Take cotton leaves containing 1-2 day old whitefly eggs and punch them into leaf discs using a hole puncher. Φ =3.3cm), placed face down in a polypropylene petri dish covered with 0.5cm of 1.2% agar hydrogel. Φ =3.5cm, h=2cm). Under a stereomicroscope, based on the number of whitefly eggs on each leaf disc, they were roughly divided into 6 ranges: approximately 100, 200, 400, 600, 800, and 1000 and above. 50-60 day old female adult Japanese ladybugs (Ek-F8 generation) that had laid eggs after being starved for 24 hours were placed in polypropylene culture dishes. A 1cm diameter hole was made in the center of the lid and sealed with gauze. They were then cultured for 24 hours in an artificial climate chamber at 26℃, 60% relative humidity, and 14h / d light. After removing the Ek-F8 generation female adults, the number of remaining whitefly eggs was observed under a stereomicroscope. Japanese ladybugs reared on cotton leaves (Bt-F1 generation) were used as a control to determine the attack rate. a ) and prey processing time ( T h The result is as follows Figure 3 As shown in Table 4; in Table 4, the same letter after each treatment coefficient indicates that they do not overlap within the 95% confidence interval.

[0036] Table 4. Effects of successive rearing on the predation function response parameters of Japanese ladybugs to whitefly eggs. The results showed that the predatory functional responses of female adult Japanese ladybugs (Bt-F1 and Ek-F8 generations) to whitefly eggs both belonged to the Holling III type; there were no significant differences in attack rate and prey treatment time in the predatory functional responses. This indicates that the predatory ability of Japanese ladybugs obtained using the propagation method of this invention against whiteflies is comparable to that of the natural population.

[0037] As can be seen from the above embodiments, the present invention provides a method for propagating Japanese ladybugs and its application. The Japanese ladybugs propagated according to the method of the present invention exhibit significantly improved growth, development, and reproduction, a significantly increased survival rate, and predation ability against whiteflies comparable to that of the natural population.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for propagating the Japanese ladybug, characterized in that, Includes the following steps: (1) Pour 0.5-1.0 cm of agar hydrogel into the bottom of the culture dish, place eggplant leaf discs with whiteflies on it, introduce Japanese ladybugs to lay eggs on the eggplant leaf discs, and hatch Japanese ladybug larvae. (2) The larvae of Japanese ladybug were placed in a culture dish and fed with Mediterranean mealy borer eggs to obtain adult Japanese ladybugs; (3) The adult Japanese ladybugs are transferred to a culture dish and mated together for 7-9 days. They then lay eggs to obtain Japanese ladybug eggs. This process is repeated to complete the propagation of Japanese ladybugs.

2. The propagation method according to claim 1, characterized in that, The culture vessels mentioned in steps (1) to (3) are made of polypropylene.

3. The propagation method according to claim 2, characterized in that, The concentration of the agar hydrogel in step (1) is 1% to 2%.

4. The propagation method according to claim 3, characterized in that, The whiteflies mentioned in step (1) include whitefly eggs and / or whitefly nymphs.

5. The propagation method according to claim 4, characterized in that, The diameter of the eggplant leaf disc in step (1) is 2-4 cm.

6. The propagation method according to claim 1, characterized in that, The temperature for rearing in step (2) is 25-27℃, the relative humidity is 55%-65%, and the light exposure time is 10-16h / d.

7. The propagation method according to claim 1, characterized in that, The number of adult Japanese ladybugs in step (3) is 10-12 per culture dish.

8. The application of the propagation method according to any one of claims 1 to 7 in the propagation of Japanese ladybugs.

Images