Method for rapidly releasing pre-pupal diapause of hoplia coeruleipennis ula

Abstract

The present application belongs to the technical field of insect diapause release and development regulation, and relates to a method for quickly releasing pre-pupa diapause of aegoliptus tristis. The present application provides a method for quickly releasing pre-pupa diapause of aegoliptus tristis, which comprises the following steps: S1. nest preparation and screening; S2. nest dissection; S3. ecdysone microinjection; S4. pre-pupa incubation and culture after injection; S5. diapause release efficiency and development synchrony evaluation. Through the precise and controllable ecdysone microinjection system and the standardized incubation and culture technology, about 50% of the surviving pre-pupae can successfully release diapause without 4-5 DEG C long-term low-temperature treatment, and the relatively synchronous adult emergence can be obtained within one month after injection.

Description

Technical Field

[0001] This invention belongs to the field of insect diapause release and development regulation technology, and relates to a method for rapidly releasing alfalfa leafcutter bee prepupal diapause. Background Technology

[0002] Most leguminous crops have a typical butterfly-shaped flower structure, with the stamens and pistils tightly enclosed by the keel petals. Their pollination process relies on a specific flower-flipping mechanism to achieve effective pollination. This is especially true for alfalfa (…). Medicago sativa Leguminous crops that require strict cross-pollination, such as soybeans (… Glycine max Leguminous crops that rely on hybrid vigor for seed production have a rigid demand for pollinating insects with high pollination efficiency and strong adaptability during the seed production process.

[0003] Alfalfa leafcutter bees ( Megachile rotundata This is a solitary pollinating bee and a core pollinating bee species for the seed production of leguminous crops such as alfalfa and hybrid soybeans. This bee species can trigger pollen release through precise flower-visiting behavior, using its abdominal pollen brush to carry pollen, and achieving highly efficient cross-pollination through continuous flower-visiting activities, resulting in outstanding pollination efficiency. It also possesses the commercial characteristics of gregarious nesting and controllable density, making it highly suitable for large-scale field planting scenarios. Its application plays a crucial supporting role in improving the seed production capacity of leguminous crops, ensuring the independent supply of germplasm resources, and supporting the high-quality development of the grassland and livestock industry.

[0004] Alfalfa leafcutter bees are typical facultative diapause insects. Their offspring develop from eggs to prepupae and then enter diapause to overwinter. Currently, the commercial application, colony recovery, and storage of these bees all revolve around standardized cultivation and developmental regulation of diapause prepupae. In existing diapause-breaking techniques for alfalfa leafcutter bees, diapause prepupae recovered from the field need to be treated at 4-5℃ for more than 3 months to break diapause. However, prepupae treated with short-term low-temperature treatment suffer from problems such as long emergence periods and asynchronous emergence. To break prepupa diapause, improve emergence synchronization, and maintain long-term prepupa survival, continuous low-temperature treatment at 4-5℃ for 7-10 months is typically required, followed by incubation at 29-30℃ for 18-25 days the following year. This allows for synchronized pupation and emergence of the bee colony, resulting in a sufficient number of adults for scientific research or release into the field for crop pollination.

[0005] However, the aforementioned traditional diapause release techniques have obvious limitations: First, the overall treatment cycle is too long and the timeliness is poor. It usually takes more than 8 months from low-temperature storage to adult emergence, which cannot meet the year-round uninterrupted scientific research needs of using adults as experimental materials, nor can it be adapted to flexible pollination experiments, thus limiting the application scenarios and flexibility; Second, long-term low-temperature storage requires strict temperature and humidity control precision, and is mainly suitable for large-scale centralized storage scenarios, which increases the equipment investment and operating costs of scientific research experiments.

[0006] In summary, there is an urgent need to develop an efficient, convenient, and timely method for rapidly relieving prepupal diapause in alfalfa leafcutter bees, significantly shortening the diapause relieving cycle and meeting the flexible bee usage needs for scientific research and pollination. Summary of the Invention

[0007] This invention aims to provide a method for rapidly inducing diapause in alfalfa leafcutter bee prepupae. The method uses screened and confirmed diapause prepupae as the target, bypassing the traditional method's long-term low-temperature incubation at 4-5°C to induce diapause through microinjection of ecdysone. After ecdysone injection, the prepupae are rapidly induced to break diapause, completing pupation and emergence within one month, thus effectively shortening the cycle from diapause induction to adult acquisition. This method is simple to operate and suitable for small-batch, multi-batch treatments, and can be used for the rapid replenishment of pollinated alfalfa leafcutter bee adults, as well as for obtaining synchronized test insects in developmental regulation research.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] On the one hand, the present invention provides a method for rapidly relieving the prepupal diapause of alfalfa leafcutter bees, comprising the following steps: S1. Nest cell preparation and selection: Place the alfalfa leaf cut honeycomb boards or paper straws collected from the field that year in an artificial climate chamber (temperature 29~30℃, relative humidity 60%±5%, photoperiod L:D=0:24) for more than 2 months to allow non-diapause individuals to develop into adults, obtaining nest cells containing diapause prepupae to be selected; then use a nest cell punching tool to vertically insert into the nest hole of the pretreated nest board to punch out the nest cells to be selected, or cut open the outer wall of the paper straw with scissors to remove the nest cells to be selected; select the diapause prepupae nest cells by gently pinching the side wall of the nest cells with your fingers; S2. Nest cell dissection: Clean the leaves at the top of the selected diapause prepupae cells, select diapause prepupae cells with a diameter ≥5mm and a length ≥7mm, and use micro forceps and micro scissors to radially circumferentially remove about 1mm of the top of the cell to form a flat opening at the top of the cell. Place the cell with the opening facing upward in a 96-well plate for later use. S3. Ecdysone Microinjection: Using micro-forceps, the thorax and abdomen of the diapause prepupa are held and slowly removed from the pyloric chamber in the 96-well plate. Under a stereomicroscope, the ecdysone working solution is injected into the intersegmental membrane of the second to third segment from the bottom on the dorsal side of the prepupa's abdomen using a microinjector. During injection, the microneedle is inserted at a 30° to 50° angle to the horizontal, with an insertion depth (depth into the larva) of 1.5 to 2 mm. The dosage of ecdysone for each diapause prepupa is 0.05 μg, and the injection volume is 0.138 μL. After injection, the needle is slowly withdrawn to avoid loss of body fluids. S4. Post-injection prepupa incubation and culture: Using micro-tweezers, diapause prepupae injected with molting hormone were picked up and placed back into their original nest cells with their heads facing up and abdomens facing down. This provided a stable microenvironment for the prepupae's development, avoiding physical damage, environmental fluctuations, and microbial infection. The nest cells were then transferred to a 96-well plate to obtain diapause prepupae in their original nest cells after injection. The plates were sealed and ventilation holes were made (after sealing with a single layer of plastic wrap, a small ventilation hole was made in the plastic wrap corresponding to each nest cell using a No. 1 insect needle). The 96-well plate was placed in an artificial climate chamber (temperature 29~30℃, relative humidity 60%±5%, photoperiod L:D=0:24) for continuous culture for 30 days. S5. Evaluation of diapause resolution efficiency and developmental synchronicity: Starting from the day of injection, the number of healthy prepupae, dead individuals, pupated individuals, and emerging individuals were counted daily. The survival rate, pupation rate, emergence rate, average pupation time, average emergence time, coefficient of variation of pupation time, and coefficient of variation of emergence time of diapause prepupa after injection of ecdysone were calculated to evaluate the diapause resolution efficiency, pupation synchronicity, and emergence synchronicity.

[0010] Furthermore, in step S1, the nest board is made of polystyrene, the nest hole diameter is 6 mm, and the depth is 7 cm or 7.5 cm; the paper straw has an outer diameter of 6 mm, an inner diameter of 5.7 mm, and a length of 7.5~7.8 cm; the nest chamber punching tool has a diameter of 5.5 mm and a length of 12 cm~20 cm.

[0011] Furthermore, in step S2, the single-well specifications of the 96-well plate are a depth of 1 cm and a diameter of 7 mm; the blade length of the micro-scissors is <1 cm and the blade thickness is <0.15 mm; the tip diameter of the micro-tweezers is <0.15 mm.

[0012] The above-mentioned nest chamber size limit is intended to ensure that the prepupa has the body size basis to develop into a healthy adult, while also being compatible with the 96-well plate specification and microdissection operation; the single hole specification of the 96-well plate is 1 cm deep and 7 mm in diameter; the blade length of the micro-scissors is <1 cm and the blade thickness is <0.15 mm, and the tip diameter of the micro-tweezers is <0.15 mm, which can achieve precise circumcision of the nest chamber without damaging the diapause prepupa inside.

[0013] Furthermore, in step S3, the ecdysone is β-ecdysone (20-hydroxyecdysone).

[0014] Furthermore, in step S3, the preparation method of the ecdysone working solution is as follows: a stock solution is prepared using dimethyl sulfoxide as a solvent, and then the stock solution is diluted with sterile ultrapure water to form the working solution.

[0015] Furthermore, in step S3, the ecdysone working solution is prepared as follows: a stock solution with a concentration of 20 μg / μL is prepared using dimethyl sulfoxide (DMSO) as a solvent, and then the stock solution is diluted with sterile ultrapure water (ddH2O) to a working solution of 0.3623 μg / μL. DMSO can fully dissolve ecdysone, which has poor water solubility. After dilution with sterile ultrapure water, the concentration of DMSO can be significantly reduced, thereby mitigating its toxicity to diapause prepupae.

[0016] Furthermore, in step S5, The method for determining a healthy prepupa is as follows: the insect body is curved in a C-shape, the body surface is smooth, and the body segments are clearly defined; The method for determining the death of an individual is as follows: the insect body is yellowed, shriveled and softened, sunken and deformed, and the body segments are blurred; The method for identifying pupated individuals is as follows: the insect body changes from a C-shaped curve to a long spindle shape, and a transparent head shell appears on the head; The method for identifying emerging individuals is as follows: the mouthparts, antennae, legs, and wings of the adult insect are fully developed and begin to move. Among them, the male adult has yellowish-white head hairs, no pollen brush on the abdomen, and a flat abdomen end, while the female adult has darker head hairs, a pollen brush on the abdomen, and a sharp abdomen end.

[0017] Furthermore, in step S5, the calculation method for relevant indicators after injection of ecdysone into the diapause prepupa is as follows: Survival rate = (Total number of diapause prepupae tested - Number of dead individuals) / Total number of diapause prepupae tested × 100%.

[0018] The pupation rate = number of pupated individuals / (total number of diapause prepupae tested - number of dead individuals) × 100%.

[0019] Emergence rate = Number of emerged individuals / (Total number of diapause pupae tested - Number of dead individuals) × 100%.

[0020] Average pupation time = total time taken by each individual pupating / number of individuals pupating.

[0021] Average molting time = Total time taken for each molting individual / Number of molting individuals.

[0022] Coefficient of variation of pupation time = standard deviation of pupation time / average pupation time × 100%.

[0023] Coefficient of variation of feathering duration = standard deviation of feathering duration / average feathering duration × 100%.

[0024] On the other hand, the present invention provides the application of the method described in the pollination, seed production, breeding or scientific research of leguminous crops.

[0025] The invention also provides the application of the method described herein in shortening the diapause release period of alfalfa leafcutter bees.

[0026] The invention also provides the application of the method described herein in improving the synchronicity of pupation and emergence.

[0027] The invention also provides the application of the method described herein in the preparation of adult alfalfa leafcutter bees for pollinating leguminous crops.

[0028] The invention also provides the application of the method described herein in providing synchronized test insects for the study of the diapause development regulation mechanism of alfalfa leafcutter bees.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) It can quickly relieve the prepupal diapause of alfalfa leafcutter bees, significantly shorten the time cycle for obtaining adults, and eliminate the need for long-term low-temperature treatment of more than 7 months required by traditional methods. It can relieve diapause and obtain adults in just 1 month, breaking through the bottleneck of poor timeliness and limited application scenarios of traditional technology. It can meet the needs of bees for scientific research and pollination throughout the year.

[0030] (2) Through a precise and controllable molting hormone microinjection system and standardized incubation and cultivation technology, about 50% of the surviving prepupae can be successfully released from diapause, significantly improving the synchronicity of pupation and emergence. At the same time, the method of this invention is simple to operate and highly adaptable. A single operation can process more than 300 diapause prepupae. Through multiple batches of treatment, sufficient bee quantity can be provided for scientific research experiments and pollination services.

[0031] (3) By using optimized ecdysone working solution concentration, refined microinjection operation, and combined with the plan of returning to the original nest chamber for incubation, mechanical damage and physiological toxicity to the prepupa can be minimized as much as possible, while providing a natural nest chamber environment for its development, thus ensuring the survival and healthy development of the prepupa after injection. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of steps S1 to S4 in the method for rapidly relieving prepupal diapause of alfalfa leafcutter bees provided in an embodiment of the present invention.

[0033] 1.1: An artificial climate chamber providing an environment with a temperature of 29-30℃, relative humidity of 60%±5%, and photoperiod L:D=0:24; 1.2: Alfalfa cutter honeycomb boards recovered from the field; 1.3: Alfalfa cutter honeycomb paper pipettes recovered from the field; 1.4: Cell punching tool; 1.5: Scissors; 1.6: Screened diapause prepupae; 2.1: A 1 mm girdled cut at the top of the cell; 2.2: Microscopic scissors; 2.3: Microscopic tweezers; 2.4: Diapause prepupae with an open top; 2.5: 96-well plate; 3.1: Diapause prepupa after injection; 3.2: Microinjector; 4.1: Diapause prepupa in the original cell of the 96-well plate after injection.

[0034] Figure 2The original nest chamber contained healthy prepupae, pupated individuals, dead individuals, male adults, and female adults.

[0035] Figure 3 The survival and development of diapause prepupae of alfalfa leafcutter bees under different doses of ecdysone treatment. a represents the cumulative survival rate; b represents the cumulative pupation rate; c represents the cumulative emergence rate; d represents the pupation duration; and e represents the emergence duration.

[0036] Figure 4 The survival and development of alfalfa leafcutter bee diapause prepupae under different low-temperature treatment durations are shown. a represents the survival rate; b represents the cumulative pupation rate; c represents the cumulative emergence rate; d represents the pupation duration; and e represents the emergence duration.

[0037] Different lowercase letters in the figure indicate that the differences between treatments were significant after one-way ANOVA and Tukey HSD multiple comparison tests. P <0.05). Detailed Implementation

[0038] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention clearer. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0039] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials are all commercially available.

[0040] Example 1 like Figure 1 As shown, this embodiment provides a method for rapidly relieving the prepupal diapause of alfalfa leafcutter bees, including the following steps: S1. Nest Cell Preparation and Selection: Select alfalfa leaf-cutting honeycomb boards 1.2 or paper straws 1.3 recovered from alfalfa fields in Ordos City, Inner Mongolia Autonomous Region in June of the current year. First, place them in an artificial climate chamber 1.1 (temperature 29~30℃, relative humidity 60%±5%, photoperiod L:D=0:24) for continuous treatment for more than 2 months to allow non-diapause individuals to develop into adults, obtaining nest cells containing diapause prepupae for selection. Then, use a nest cell punching tool 1.4 to punch out the nest cells to be selected from the pretreated nest board, or use ordinary scissors 1.5 to cut open the outer wall of the paper straw to remove the nest cells to be selected. By gently pinching the side wall of the nest cell with your fingers, the cells can be selected. Under pressure, empty nests, nests containing pollen balls, and nests containing dead larvae were removed, as were nests with obviously collapsed sidewalls (obvious collapse means the sidewall collapse exceeds 1 / 4 of the nest diameter), nests that could not recover their original shape after being pressed, or nests with a soft texture and no obvious support. The following nests were selected: diapause pre-pupae nests containing firm bee cocoons, which provided obvious support when lightly squeezed (obvious support means the bee cocoons inside the nest block the pressure of the fingers, preventing the nest from being easily crushed, and which basically recovered their original shape after being released), nests with only slight indentation on the sidewalls (slight indentation means the sidewall collapse does not exceed 1 / 4 of the nest diameter), and which basically recovered their original shape after being released. S2. Nest cell dissection: Clean the leaves at the top of the selected diapause prepupae cells. Select diapause prepupae cells with a diameter ≥5 mm and a length ≥7 mm. Use micro forceps and micro scissors to radially circumferentially remove about 1 mm of the top of the cell 2.1, so that the top of the cell forms a flat opening. Place the diapause prepupae cells with the top opening 2.4 (cells with the opening facing upwards or open cells with the top facing upwards) in a 96-well plate 2.5 for later use. S3. Ecdysone Microinjection: Using micro-forceps 2.3, the thorax and abdomen of the diapause prepupa are held and slowly removed from the apical opening of the nest chamber in the 96-well plate. Under a stereomicroscope, a microinjector 3.2 is used to inject the ecdysone working solution into the intersegmental membrane of the second to third segment from the bottom on the dorsal side of the prepupa's abdomen. During injection, the microneedle is inserted at an angle of 30° to 50° to the horizontal, with an insertion depth (depth into the larva) of 1.5 to 2 mm. The dosage of ecdysone for each diapause prepupa is 0.05 μg, and the injection volume is 0.138 μL. After injection, the needle is slowly withdrawn to avoid loss of body fluids. S4. Post-injection prepupa incubation culture: Using micro-tweezers, diapause prepupae 3.1 injected with molting hormone were picked up and placed back into their original nest cells with their heads facing up and abdomens facing down. This provided a stable microenvironment for the prepupae's development, avoiding physical damage, environmental fluctuations, and microbial infection. The nest cells were then transferred to a 96-well plate to obtain diapause prepupae 4.1 in their original nest cells after injection. The plates were sealed and ventilation holes were made (after sealing with a single layer of plastic wrap, a small ventilation hole was made in the plastic wrap corresponding to each nest cell using a No. 1 insect needle). The 96-well plate was placed in an artificial climate chamber (temperature 29~30℃, relative humidity 60%±5%, photoperiod L:D=0:24) and cultured for 30 days. S5. Evaluation of diapause resolution efficiency and developmental synchronicity: Starting from the day of injection, the number of healthy prepupae, dead individuals, pupated individuals, and emerging individuals were counted daily. The survival rate, pupation rate, emergence rate, average pupation time, average emergence time, coefficient of variation of pupation time, and coefficient of variation of emergence time of diapause prepupa after injection of ecdysone were calculated to evaluate the diapause resolution efficiency, pupation synchronicity, and emergence synchronicity.

[0041] In step S1, the nest board is made of polystyrene, with a nest hole diameter of 6 mm and a depth of 7 cm or 7.5 cm; the paper straw has an outer diameter of 6 mm, an inner diameter of 5.7 mm, and a length of 7.5~7.8 cm; the nest chamber punching tool is made of stainless steel, with a diameter of 5.5 mm and a length of 12 cm~20 cm.

[0042] In step S2, the single-well specifications of the 96-well plate are 1 cm in depth and 7 mm in diameter; the blade length of the micro scissors is <1 cm and the blade thickness is <0.15 mm; the tip diameter of the micro tweezers is <0.15 mm.

[0043] In step S1, the size of the nest chamber is limited to ensure that the prepupa has the body size to develop into a healthy adult, while also being compatible with the 96-well plate specification and the nest chamber dissection operation in step S2; the single hole specification of the 96-well plate is 1 cm deep and 7 mm in diameter; the blade length of the micro-scissors is <1 cm and the blade thickness is <0.15 mm, and the tip diameter of the micro-tweezers is <0.15 mm, which can achieve precise circumcision of the nest chamber without damaging the diapause prepupa inside.

[0044] In step S3, the ecdysone is β-ecdysterone, also known as 20-hydroxyecdysone, with CAS number 5289-74-7.

[0045] In step S3, the ecdysone working solution is prepared as follows: a stock solution with a concentration of 20 μg / μL is prepared using dimethyl sulfoxide (DMSO) as a solvent, and then the stock solution is diluted with sterile ultrapure water (ddH2O) to a working solution of 0.3623 μg / μL. DMSO can fully dissolve ecdysone, which has poor water solubility. Diluting with sterile ultrapure water can significantly reduce the concentration of DMSO and mitigate its toxicity to diapause prepupae.

[0046] In step S5 The method for determining a healthy prepupa is as follows: the insect body is curved in a C-shape, the body surface is smooth, and the body segments are clearly defined; The method for determining the death of an individual is as follows: the insect body is yellowed, shriveled and softened, sunken and deformed, and the body segments are blurred; The method for identifying pupated individuals is as follows: the insect body changes from a C-shaped curve to a long spindle shape, and a transparent head shell appears on the head; The method for identifying emerging individuals is as follows: the mouthparts, antennae, legs, and wings of the adult insect are fully developed and begin to move. Among them, the male adult has yellowish-white head hairs, no pollen brush on the abdomen, and a flat abdomen end, while the female adult has darker head hairs, a pollen brush on the abdomen, and a sharp abdomen end.

[0047] In step S5, the calculation method for relevant indicators after injecting ecdysone into diapause prepupa is as follows: Survival rate = (Total number of diapause prepupae tested - Number of dead individuals) / Total number of diapause prepupae tested × 100%.

[0048] The pupation rate = number of pupated individuals / (total number of diapause prepupae tested - number of dead individuals) × 100%.

[0049] Emergence rate = Number of emerged individuals / (Total number of diapause pupae tested - Number of dead individuals) × 100%.

[0050] Average pupation time = total time taken by each individual pupating / number of individuals pupating.

[0051] Average molting time = Total time taken for each molting individual / Number of molting individuals.

[0052] Coefficient of variation of pupation time = standard deviation of pupation time / average pupation time × 100%.

[0053] Coefficient of variation of feathering duration = standard deviation of feathering duration / average feathering duration × 100%.

[0054] In step S4, images of healthy prepupae, pupated individuals, dead individuals, male adults, and female adults within the original nest chamber of the 96-well plate are shown below. Figure 2 As shown, the method of the present invention for rapidly relieving the prepupal diapause of alfalfa leafcutter bees can successfully induce the prepupal diapause of alfalfa leafcutter bees to release from diapause, complete pupation, and emerge as healthy adults with sex differentiation within 30 days.

[0055] Example 2 Based on the method for rapidly relieving prepupal diapause of alfalfa leafcutter bees described in Example 1, this embodiment compares and tests the effects of different doses of ecdysone treatment on the survival rate, pupation rate, emergence rate, average pupation time, average emergence time, coefficient of variation of pupation time, and coefficient of variation of emergence time of diapause prepupae.

[0056] Except for the dosage of ecdysone injection in step S3, all other operating steps were completely consistent with those in Example 1. This example set up 11 experimental groups, including 4 treatment groups and 7 control groups. Each group had 100 diapause prepupae, and the injection volume of each diapause prepupa was 0.138 μL. Four treatment groups were set up according to a 2-fold ecdysone dosage gradient, with each diapause prepupa injected with a dose of 0.0125 μg, 0.025 μg, 0.05 μg (consistent with Example 1), and 0.1 μg, respectively. The concentrations of the ecdysone working solution used were 0.0906 μg / μl, 0.1812 μg / μl, 0.3623 μg / μl (consistent with Example 1), and 0.7246 μg / μl, respectively. Seven control groups included a control group without injection, a control group with sham injection (no reagent was injected after needle insertion), a control group injected with sterile ultrapure water, and four control groups injected with an equal volume of solvent (the ratio of DMSO and sterile ultrapure water was exactly the same as the working solution in the above four groups, but without ecdysone) into the ecdysone working solution.

[0057] 1. Survival of alfalfa leafcutter bees under different doses of ecdysone treatment (see...) Figure 3 (a) like Figure 3As shown in Figure a, the survival rate of diapause pupae in the uninjected control group reached 97%, significantly higher than all other groups, indicating that the injection operation itself causes certain physical damage to diapause pupae of alfalfa leafcutter bees. Specifically, the survival rates of the sham-injected group and the sterile ultrapure water injection group were 72% and 66%, respectively; the survival rates of the four ecdysone treatment groups (0.0125 μg, 0.025 μg, 0.05 μg, and 0.1 μg) were 73%, 68%, 61%, and 43%, respectively, while the survival rates of the corresponding solvent control groups were 72%, 58%, 59%, and 56%, respectively. This suggests that, under the same injection volume, the injection system containing DMSO solvent may further affect the survival of diapause pupae, and this effect is related to the DMSO ratio in the working solution and the ecdysone treatment dose gradient. For example, the effects of low-dose DMSO (0.0125 μg group) were solely due to physical damage from injection. Medium-dose DMSO (0.025 μg and 0.05 μg groups) exhibited some chemical toxicity to prepupae, further reducing survival rates. High-dose (0.1 μg group) showed the lowest survival rate, indicating that excessive DMSO injection produces strong chemical toxicity, which, along with physical damage, significantly reduces prepupa survival rates. Therefore, the dosage selection of ecdysone needs to consider both diapause relief efficiency and post-injection survival rate.

[0058] 2. Pupation and emergence of alfalfa leafcutter bees under different doses of ecdysone treatment (see...) Figure 3 (in Chinese) like Figure 3 As shown in Figure b, none of the surviving individuals in the control group exhibited pupation or eclosion after 30 days of incubation, indicating that the injection procedure, injection solvent, and sterile ultrapure water did not induce the prepupa to break diapause. However, the pupation rates among the surviving individuals in the four ecdysone treatment groups (0.0125 μg, 0.025 μg, 0.05 μg, and 0.1 μg) were 12.3%, 20.6%, 49.2%, and 46.5%, respectively. Figure 3 (b) The average pupation times were 8.4 days, 8.1 days, 8.5 days, and 8.8 days, respectively, and there was no significant difference in pupation time among the four treatment groups. F 3,69 =0.401, P =0.752)( Figure 3 (d). Furthermore, all pupae in the above four treatment groups were able to emerge normally ( Figure 3 (c) The average molting time was 24.0 days, 24.6 days, 23.3 days and 24.3 days, respectively, and there was no significant difference in molting time. F 3,69 =1.614, P =0.194)( Figure 3(e). The above results indicate that injection of 0.0125 μg to 0.1 μg of ecdysone can induce the alfalfa leafcutter bee prepupa to break diapause to varying degrees, but injection of 0.05 μg has the best efficiency in inducing the alfalfa leafcutter bee to break diapause.

[0059] 3. Synchronization of pupation and emergence of alfalfa leafcutter bees under different doses of ecdysone treatment (see Table 1) As shown in Table 1, the coefficients of variation for pupation time in the four ecdysone treatment groups (0.0125 μg, 0.025 μg, 0.05 μg, and 0.1 μg) were 16.9%, 21.4%, 23.0%, and 24.3%, respectively, and the coefficients of variation for eclosion time were 5.9%, 8.8%, 9.1%, and 9.0%, respectively. This indicates that the dosage of ecdysone injection has little effect on the synchronicity of pupation and eclosion, and once the diapause state is successfully relieved, their development rates are relatively consistent.

[0060] Table 1. Coefficients of variation for pupation and eclosion under different doses of ecdysone treatment.

[0061] In summary, the injection dose of 0.05 μg ecdysone achieves the optimal balance between post-injection survival rate, diapause relief efficiency, and developmental synchronicity. Its overall performance is significantly better than other tested doses, fully verifying the scientific validity and rationality of the dose used in Example 1. This dose is the preferred treatment dose for rapidly relieving prepupal diapause of alfalfa leafcutter bees according to the present invention.

[0062] Example 3 Based on Example 1, this example uses the traditional low-temperature diapause release method as a control and sets different low-temperature treatment duration gradients to explore its effects on the survival rate, pupation rate, emergence rate, average pupation time, average emergence time, pupation time coefficient of variation, and emergence time coefficient of variation of alfalfa leafcutter bee diapause prepupae.

[0063] Six 96-well plates with diapause pre-pupa chambers with top openings were obtained from steps S1 and S2 in Example 1. One of these 96-well plates was directly placed in an artificial climate chamber with a temperature of 29-30°C, relative humidity of 60%±5%, and photoperiod L:D=0:24 as described in step S4 of Example 1 for incubation. This group was recorded as 0 days of low-temperature treatment (no low-temperature treatment). The remaining five 96-well plates were placed in a low-temperature incubator for treatment under the following conditions: temperature 4-5°C, photoperiod L:D=0. :24. After 15, 30, 90, 150 and 210 days of low temperature treatment, one 96-well plate was randomly selected from each plate. More than 50 pre-pupae were randomly selected from the plate and transferred to a new 96-well plate. The plates were then placed in the same incubation environment as described above and continuously cultured until all adults emerged. The above five groups were recorded as 15 days, 30 days, 90 days, 150 days and 210 days of low temperature treatment, respectively. The 210-day low temperature treatment was the traditional diapause release method, and the other durations were gradient control treatments.

[0064] 1. Survival of alfalfa leafcutter bees under different low-temperature treatment durations (see...) Figure 4 (a) like Figure 4 As shown in Figure a, the survival rates of the low-temperature treatments for 0 days, 15 days, 30 days, 90 days, 150 days, and 210 days were 33.7%, 38.0%, 52.2%, 84.5%, 63.8%, and 71.2%, respectively. This indicates that the duration of low-temperature treatment has a significant impact on the survival rate of diapause prepupae of alfalfa leafcutter bees. Short-term low-temperature treatment of 0 to 30 days cannot effectively break their diapause state, causing the prepupae to fail to survive during the incubation process. However, low-temperature treatment of 90 days or more can relieve the diapause of alfalfa leafcutter bees and better maintain their survival during the incubation stage.

[0065] 2. Pupation and emergence of alfalfa leafcutter bees under different low-temperature treatment durations (see...) Figure 4 (be).

[0066] like Figure 4 As shown in Figure b, although some individuals in all treatment groups were able to develop into pupae and adults ( Figure 4 (bc), but there were significant differences in pupation and emergence rates among the treatments. The average pupation time for the low-temperature treatments at 0, 15, 30, 90, 150, and 210 days was 68.7 days, 35.7 days, 35.5 days, 19.4 days, 10.8 days, and 8.1 days, respectively. Among them, the pupation time at 150 and 210 days was significantly lower than that of the other treatments, and the pupation time at 90 days was significantly lower than that at 0 days. F 5,201 =21.112, P <0.001) Figure 4(d). Furthermore, the average eclosion durations for each treatment were 84.9 days, 51.2 days, 50.9 days, 33.2 days, 26.8 days, and 23.0 days, respectively. The eclosion durations for the low-temperature treatments (150 days and 210 days) were significantly shorter than those for the other treatment groups, and the eclosion duration for the 90-day low-temperature treatment was significantly shorter than that for the 0-day low-temperature treatment. F 5,201 =21.935, P <0.001) Figure 4 (e). The above results indicate that the pupation and emergence rates of diapause prepupae of alfalfa leafcutter bees are significantly positively correlated with the duration of low-temperature treatment. Low-temperature treatment for 90 days or more can effectively induce the prepupae to break diapause and significantly accelerate their development during incubation.

[0067] 3. Synchronization of pupation and emergence of alfalfa leafcutter bees under different low-temperature treatment durations (see Table 2).

[0068] As shown in Table 2, the coefficients of variation for pupation duration in the low-temperature treatment groups of 0, 15, 30, 90, 150, and 210 days were 57.8%, 106.7%, 139.2%, 11.0%, 20.7%, and 24.1%, respectively, while the coefficients of variation for eclosion duration were 46.5%, 74.9%, 97.7%, 8.3%, 6.6%, and 6.4%, respectively. This indicates that the duration of low-temperature treatment has a significant regulatory effect on the synchronicity of pupation and eclosion in alfalfa leafcutter bees. The synchronicity of pupation and eclosion is extremely poor under short-term low-temperature treatment of 0–30 days, while the development rate of individuals is highly consistent under low-temperature treatment of 90 days or more, with the 210-day treatment group showing the best eclosion synchronicity.

[0069] Table 2. Coefficients of variation for pupation and eclosion under different low-temperature treatment durations.

[0070] In summary, low-temperature treatment for 210 days is a traditional method for relieving diapause in alfalfa leafcutter bees. It can stably relieve prepupal diapause and accelerate the synchronization of emergence. It is one of the most mature technical solutions for large-scale regulation of alfalfa leafcutter bee development. However, this method requires 7 months of continuous low-temperature treatment, and the overall diapause relief cycle is extremely long, with a significant shortcoming in timeliness.

[0071] Example 4 Based on Examples 2 and 3, this example uses five indicators—survival rate, eclosion rate, average eclosion time, coefficient of variation of eclosion time, and total treatment time—to further compare and evaluate the differences between the microinjection of ecdysone hormone method of this invention and the traditional low-temperature method for relieving diapause, in terms of efficiency of diapause release, synchronicity of eclosion, and timeliness. The total treatment time was calculated starting from the entry of the diapause prepupa into the treatment stage.

[0072] As shown in Table 3, the survival rate, emergence rate, average emergence time, coefficient of variation of emergence time, and total treatment time of the traditional low-temperature diapause release method were 71.2%, 100.0%, 23.0 days, 6.4%, and 8 months, respectively. In contrast, the corresponding indicators of the microinjection of ecdysone hormone method of this invention were 61.0%, 49.2%, 23.3 days, 9.1%, and 1 month, respectively. It is evident that the average emergence time of the two methods is similar, with the main differences lying in the emergence rate, diapause release efficiency, and treatment cycle. The traditional method, using long-term low-temperature treatment, allows all surviving individuals to develop into adults under incubation conditions, exhibiting significant advantages in emergence rate and synchronicity, making it suitable for planned large-scale bee colony storage and release. However, this method has a long treatment cycle and poor timeliness. In contrast, the method of this invention does not require 7 months of low-temperature treatment, significantly shortening the total treatment time and highlighting its timeliness advantage. Provided there are sufficient diapause pre-pupa chambers, a large number of adults can be obtained rapidly, synchronously, and in multiple batches throughout the year, meeting the efficient and immediate bee needs of scientific research experiments and pollination applications.

[0073] Table 3. Comparison of the efficiency of diapause relief and developmental synchronicity between the method of the present invention and the traditional method.

[0074] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Claims

1. A method for rapidly relieving prepupal diapause in alfalfa leafcutter bees, characterized in that, Includes the following steps: S1. Nest preparation and selection: Place the alfalfa leaf cut honeycomb boards or paper straws collected from the field in the current year in an artificial climate chamber for continuous treatment for more than 2 months to allow non-diapause individuals to develop into adults, and obtain nest cells containing diapause prepupae to be selected; then use a nest cell punching tool to vertically insert into the nest hole of the pretreated nest board to punch out the nest cells to be selected, or use scissors to cut open the outer wall of the paper straw to remove the nest cells to be selected; select the diapause prepupae nest cells by gently pinching the side wall of the nest cell with your fingers; S2. Nest cell dissection: Clean the leaves at the top of the selected diapause prepupae cells, select diapause prepupae cells with a diameter ≥5 mm and a length ≥7 mm, and use micro forceps and micro scissors to radially circumferentially remove the top of the cell to form a flat opening at the top of the cell. Place the cell with the opening facing upward in a 96-well plate. S3. Ecdysone microinjection: Using micro-forceps, the thorax and abdomen of the diapause prepupa are held and slowly removed from the apical opening of the nest chamber in the 96-well plate. Under a stereomicroscope, the working solution of ecdysone is injected into the intersegmental membrane of the second to third segment from the bottom on the dorsal side of the prepupa's abdomen using a microinjector. During injection, the microneedle is inserted at an angle of 30° to 50° to the horizontal, and the depth of insertion is 1.5 to 2 mm. After injection, the needle is slowly withdrawn. S4. Post-injection prepupa incubation culture: Use micro forceps to pick up the diapause prepupa after injection of molting hormone, and place it back into the original nest cell with its head facing up and abdomen facing down; then transfer the nest cell into a 96-well plate to obtain the diapause prepupa in the original nest cell of the 96-well plate after injection. After sealing and setting ventilation holes, place the 96-well plate in an artificial climate chamber for continuous culture for 30 days. S5. Evaluation of diapause relief efficiency and developmental synchronicity: Starting from the day of injection, the number of healthy prepupae, dead individuals, pupated individuals, and emerging individuals were counted daily. The survival rate, pupation rate, emergence rate, average pupation time, average emergence time, coefficient of variation of pupation time, and coefficient of variation of emergence time of diapause prepupa after injection of ecdysone were calculated.

2. The method according to claim 1, characterized in that, In step S1, the nesting board is made of polystyrene.

3. The method according to claim 1, characterized in that, In step S2, the blade length of the micro-scissors is <1 cm and the blade thickness is <0.15 mm; the tip diameter of the micro-tweezers is <0.15 mm.

4. The method according to claim 1, characterized in that, In step S3, the ecdysone is β-ecdysone.

5. The method according to claim 1, characterized in that, In step S3, the preparation method of the ecdysone working solution is as follows: a stock solution is prepared using dimethyl sulfoxide as a solvent, and then the stock solution is diluted with sterile ultrapure water to form the working solution.

6. The application of the method described in any one of claims 1 to 5 in the pollination, seed production, breeding, or scientific research experiments of leguminous crops.

7. The application of the method according to any one of claims 1 to 5 in shortening the diapause release period of alfalfa leafcutter bees.

8. The application of the method according to any one of claims 1 to 5 in improving the synchronicity of pupation and emergence.

9. The use of the method according to any one of claims 1 to 5 in the preparation of adult alfalfa leafcutter bees for pollinating leguminous crops.

10. The application of the method according to any one of claims 1 to 5 in providing synchronized test insects for the study of the diapause development regulation mechanism of alfalfa leafcutter bees.

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