A method for recovering alfalfa leaf-cutting beehive chambers and monitoring offspring development

Abstract

The present invention discloses a method for recovering honeycomb cells and monitoring offspring development of alfalfa leafcutter bees, comprising the following steps: 1. placing honeycombs and hanging honeycomb panels; 2. inserting paper straws into the honeycomb panels; 3. releasing adult bees; 4. recovering paper straws; 5. cultivating larvae; 6. cultivating prepupae; and 7. performing data statistics and analysis. The method for recovering honeycomb cells and monitoring offspring development provided by the present invention can promptly and quickly recover newly constructed honeycombs by female bees, monitor the development of offspring in real time, and obtain information such as the diapause rate, offspring mortality rate, natural enemy damage rate, empty nest rate, and pollen ball percentage of offspring recovered within a selected time period. This provides a simple and rapid monitoring technology for timely and accurate understanding of the causes of bee losses during the propagation and application of alfalfa leafcutter bees. The method provided by the present invention can guide those skilled in the art to take timely measures to reduce offspring losses, and provide more efficient and high-quality pollination services for alfalfa seed production by protecting and utilizing alfalfa leafcutter bees.

Description

Technical Field

[0001] The invention belongs to the technical field of artificial breeding and monitoring of alfalfa leafcutter bees, and particularly relates to a method for recovering alfalfa leafcutter bee hive chambers and monitoring the development of offspring. Background Art

[0002] Alfalfa (Medicago sativa) is known as the king of forage grasses, boasting high yields and excellent quality, making it a popular food for a variety of livestock and poultry. However, due to the unique structure of its floral organs, with the pistils and stamens enclosed by the keel, and its self-incompatibility and cross-pollination, alfalfa requires specialized pollinators to press open the keel and spread pollen. This results in very low pollination and seed set rates and uneven seed quality in the field. The lack of effective pollinators has become a major bottleneck limiting the improvement of alfalfa seed yield and quality.

[0003] Leafcutter bees ( Megachile ) is recognized by the alfalfa seed industry as the best pollinating insect group for alfalfa pollination. my country began to introduce Canadian commercial populations of alfalfa leafcutter bees for alfalfa pollination in 1988. It has been applied on a large scale many times in Beijing, Jilin, Inner Mongolia and Heilongjiang to establish bee breeding systems. The application technology of alfalfa leafcutter bees has been greatly improved, but it is still limited by the fact that the second generation of bees do not diapause, the lack of efficient natural enemy trapping technology and artificial bee breeding technology, which makes it impossible to achieve large-scale breeding.

[0004] The pollination period of alfalfa leafcutter bees in alfalfa fields is one to two months. Female bees build nests and give birth to offspring. After the offspring develop to the pre-pupae stage, some enter a state of diapause and development stagnates, while others continue to develop into adults. During this period, parasitic wasps and other natural enemies pose a serious threat. Existing technologies generally use nesting plates to attract female bees to build nests. However, these plates are not capable of spot-checking newly built nesting chambers. During the spot-checking process, the integrity of the nest chambers is often damaged, making it impossible to accurately detect the internal conditions of the nest chambers. The only option is to wait until the field pollination service is completed and then collect the nest plates. Computed tomography or manual dissection of the nest chambers is then used for testing. This method can only capture the development status of the offspring at a specific time point or a few points in time, and the developmental progress of the offspring cannot be recorded in real time, which also increases the cost of the experiment. In addition, existing technologies cannot evaluate the developmental status of offspring produced by female bees at different time periods, making it very easy to miss the optimal time for larval sampling and determining the developmental period of the offspring. This also increases experimental error for experiments that require controlling the offspring development temperature, leading to inaccurate results.

[0005] Timely and rapid recovery of nest chambers built by female bees, real-time monitoring of offspring development, and accurate detection of parasitic wasp and predator damage are crucial for determining the offspring developmental period and predator damage rates. Therefore, a low-cost, simple, and highly accurate method for nest chamber recovery and offspring development monitoring is urgently needed to address these issues. Summary of the Invention

[0006] In order to solve the problems existing in the above-mentioned background technology, the present invention provides a method for recovering alfalfa leafcutter bee cells and monitoring the development of offspring, which is used to monitor the development of offspring in real time and accurately detect the damage caused by parasitic wasps and predators, while providing data reference for the artificial breeding of alfalfa leafcutter bees.

[0007] The "A×B specifications" mentioned in this specification all mean A vertical holes × B horizontal holes, where A and B are both positive integers, A=B or A≠B.

[0008] Specifically, the present invention adopts the following technical solutions:

[0009] The present invention provides a method for recovering an alfalfa leaf-cutter honeycomb chamber and monitoring offspring development, comprising the following steps:

[0010] S1. Place the hive and hang the honeycomb:

[0011] A test plot was selected at the early flowering stage of alfalfa. A beehive was placed in the center of the alfalfa field, facing southeast. Eight to ten honeycomb boards were hung on the inner wall of the beehive at a height of 1.5 to 1.6 meters. Each honeycomb board had a 120×30 hole, each with the same diameter of 6 mm and a depth of 7 cm. The honeycomb boards were patterned and coated with blue or black colors.

[0012] S2. Nesting paper straws:

[0013] On the nest board facing east or southeast, each nest board is divided into three rectangular areas: upper, middle and lower.

[0014] Specifically, the rectangular area division method is as follows:

[0015] The upper part is a rectangular area of ​​39×30, the middle part is a rectangular area of ​​42×30, and the lower part is a rectangular area of ​​39×30;

[0016] Select one small rectangular area each in the upper, middle, and lower rectangular areas. Specifically, the method for selecting the small rectangular areas is as follows:

[0017] The small rectangular area on the top is located as follows: 10 holes on the top edge of the vertical release plate, and 5 holes on each of the left and right edges of the horizontal release plate.

[0018] The positions of the small rectangular areas in the middle are: 27 holes in the small rectangular area on the top of the nest-leaving plate for inserting paper straws in the vertical direction, and 5 holes on each of the left and right edges of the nest-leaving plate in the horizontal direction;

[0019] The position of the small rectangular area at the bottom is: 28 holes in the small rectangular area for inserting paper straws in the middle of the vertical departure plate, 10 holes at the bottom edge of the departure plate, and 5 holes on each of the left and right edges of the horizontal departure plate.

[0020] Take the 5×5 holes at the four corners of each small rectangular area as the experimental holes, and insert paper straws into the holes respectively; the specifications of the paper straws inserted into the nest chamber are: outer diameter 6mm, inner diameter 5.7mm, and length 7.5-7.8cm.

[0021] S3. Release of adult bees:

[0022] The brooding tray containing 30,000 to 40,000 cells is brought into the hive, the tray lid is opened, and the adult bees are released, allowing the female bees to build nests in the paper straws;

[0023] The above-mentioned bee hatching tray needs to be incubated indoors before opening. The incubation conditions are: temperature 29-30°C, relative humidity 60%±5%, darkness, and incubation time 18-25 days.

[0024] S4. Recycle paper straws:

[0025] Every 7 days, the sealed paper straws that have produced offspring on the nest board are collected, the corresponding position information of each paper straw is recorded, and new paper straws are replaced to continue attracting female bees to build nests. The paper straws removed from the nest board are tied with rubber bands and placed flat in a cardboard box and taken back. Several holes are pierced on the 6 sides of the cardboard box.

[0026] S5. Larval culture:

[0027] In a cool, dark area of ​​the laboratory, cut open a paper straw at the smooth, sealed end. Remove all nest chambers from the paper straws brought back in Step 4. Use tweezers to remove the lids from each nest. Arrange the individual nests in the order they were built (the sealed end is the last nest built, the innermost nest is the first nest built) on a 96-well plate. Cover the plate with the 96-well plate cover or wrap the plate with a single layer of transparent plastic wrap. Use a No. 1 insect needle to poke a small hole in the plastic wrap directly opposite each nest. Place the 96-well plate horizontally in an incubator set at 29-30°C, 60% ± 5% relative humidity, and incubate in the dark. Observe the development of the progeny until they have completed cocooning (the progeny in the cocoon are now called prepupae). Record the developmental duration of the egg and 1st to 5th instar larvae to obtain the developmental history of each instar and the entire larval stage. 96-well plate specifications: flat bottom, 1 cm depth, 7 mm diameter.

[0028] S6. Prepupal Culture:

[0029] The offspring prepupae of alfalfa leafcutter bees in the cocoon are transferred into transparent capsules made of corn starch or glutinous rice. A small hole is pierced at each end of the capsules with an insect needle. The capsules are then affixed to cardboard in the order of the prepupae in the nest. The cardboards are then placed horizontally and transferred into a dark incubator at a temperature of 29-30°C and a relative humidity of 60%±5% for further incubation. The development of the prepupae is observed through the capsules. Prepupae that do not continue to develop within 30 days are recorded as diapause individuals, while offspring that continue to develop to the pupal or adult stage are recorded as non-diapause individuals.

[0030] The method provided by the present invention can count the number of dead young larvae, dead old larvae, dead prepupae, as well as the number of empty nests, pollen balls, parasitized and preyed nest chambers in the process of recovering offspring and culturing offspring from eggs to prepupae.

[0031] S7. Data Statistics and Analysis:

[0032] Statistics were collected on the offspring diapause rate, offspring mortality rate, natural enemy damage rate, empty nest rate and pollen ball ratio, the causes of bee losses during pollination and nesting were clarified, and changes in the alfalfa leafcutter bee population were monitored.

[0033] The offspring diapause rate = number of diapause individuals / (number of diapause individuals + number of non-diapause individuals) × 100%.

[0034] Offspring mortality rate = (younger larvae + dead older larvae + dead prepupae) / (younger larvae + dead older larvae + dead prepupae + number of diapause individuals + number of non-diapause individuals) × 100%;

[0035] Natural enemy damage rate = (number of parasitized species + number of predators) / total number of nests × 100%;

[0036] Empty nest rate = number of empty nests / total number of nests × 100%;

[0037] Pollen ball ratio = number of pollen balls / total number of nests × 100%.

[0038] On the other hand, the present invention also seeks to protect the application of the above method in artificial breeding and population protection of alfalfa leafcutter bees.

[0039] Compared with the prior art, the present invention "a method for recovering alfalfa leaf-cutting honeycomb cells and monitoring offspring development" has the following beneficial effects:

[0040] 1. The damage caused by natural enemies and the death of offspring in the nest chambers recovered during the selected time period and test plots can be accurately obtained, so as to timely assess the losses and take corresponding measures.

[0041] 2. The accumulation of monitoring data on the development of offspring of alfalfa leafcutter bees can obtain the developmental history of eggs, various instars and the entire larval stage, providing effective data reference for research such as sampling of offspring at different developmental stages and obtaining test larval materials.

[0042] 3. By using paper straws, the separation of the nest chamber containing offspring from the nest board can be faster. The peak period of female bees building nests and laying eggs can be determined according to the number of recycled paper straws. In addition, the offspring of alfalfa leafcutter bees in the field can be brought back in a timely and convenient manner and their development can be monitored indoors, which simplifies the steps of offspring development monitoring and reduces the cost of the experiment.

[0043] 4. By using transparent capsules made of corn starch or glutinous rice, the activities of the prepupae of the alfalfa leafcutter bee are not restricted, and there is no toxicity or physical damage. The morphological changes of the offspring prepupae can be visually inspected, and the development of the offspring can be observed in real time. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a diagram showing how the honeycomb panels are hung in the hive and the upper, middle, and lower areas of the panels. 1.1 is the honeycomb panel, 1.1.1 is the upper area, 1.1.2 is the middle area, and 1.1.3 is the lower area.

[0045] Figure 2 Schematic diagram of the rectangular areas and cells for inserting paper straws. 2.1.1, 2.1.2, 2.1.3, and 2.1.4 are the four 5×5 cells for inserting paper straws. 2.2 is the area without paper straws, 2.3 is the area with paper straws, and 2.3.1 is the paper straw.

[0046] Figure 3 Culture eggs or larvae in 96-well plates.

[0047] Figure 4 For prepupae, stick them on the cardboard in the order in which they are in the paper straws.

[0048] Figure 5 This is the time dynamic curve of the cumulative proportion of larvae of each instar during the development process from the egg stage to the prepupal stage (50 offspring).

[0049] Figure 6 This is a dynamic diagram of the diapause rate of offspring in paper straws recovered from sample sites 1 and 2.

[0050] Figure 7 The mortality rate dynamics of offspring in paper straws recovered from plots 1 and 2.

[0051] Figure 8 This is a dynamic graph of the natural enemy damage rate in paper straws recovered from sample sites 1 and 2.

[0052] Figure 9Dynamic graph of the empty nest rate in paper straws recovered from sample sites 1 and 2.

[0053] Figure 10 This is a dynamic graph of the proportion of pollen balls in paper straws recovered from sample sites 1 and 2. DETAILED DESCRIPTION

[0054] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0055] In the description of the present invention, it should be understood that the terms "upper", "middle" and "lower" etc. indicating orientations or positional relationships are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0056] Example 1

[0057] This embodiment provides the specific steps of the method for recovering alfalfa leafcutter honeycomb cells and monitoring offspring development.

[0058] 1. Place the hive and hang the honeycomb:

[0059] During the early flowering period of alfalfa, alfalfa planting areas in Pingluo County, Shizuishan City, Ningxia Hui Autonomous Region (sample site 1, survey time was June 3-July 14, 2022) and Ordos City, Inner Mongolia Autonomous Region (sample site 2, survey time was June 1-July 12, 2022) were selected as experimental plots. In each plot, three beehives were placed in the center of the alfalfa field, with the beehive facing southeast, and 8-10 honeycomb boards (such as 1000 honeycomb boards) were hung vertically on the inner wall of the beehive. Figure 1 ), the hanging height is 1.5 to 1.6 m, the nest board has textures and blue and black coatings, each nest board has 120×30 holes, the hole diameter is 6 mm, and the hole depth is 7 cm.

[0060] 2. Nesting paper straws:

[0061] In each honeycomb, select 3 honeycomb boards facing east or southeast, and divide each honeycomb board into 3 rectangular areas: upper, middle and lower (e.g. Figure 1 (as marked in 1.1.1, 1.1.2, and 1.1.3), where:

[0062] The upper part is a rectangular area with 39 vertical holes and 30 horizontal holes;

[0063] The middle part is a rectangular area with 42 vertical holes and 30 horizontal holes;

[0064] The lower part is a rectangular area with 39 vertical holes and 30 horizontal holes.

[0065] Select a small rectangular area with 20 holes × 15 holes in each of the upper, middle and lower areas (such as Figure 2 ),in,

[0066] Select the upper rectangular area as follows: 10 holes on the top edge of the vertical separation plate, and 5 holes on each of the left and right edges of the horizontal separation plate;

[0067] The positions of the selected central rectangular area are: 27 holes in the rectangular area where the paper straws are inserted on the upper part of the nesting plate in the vertical direction, and 5 holes on each of the left and right edges of the nesting plate in the horizontal direction;

[0068] The position of the lower rectangular area is selected as follows: 28 holes in the rectangular area where paper straws are inserted in the middle of the vertical nesting plate, 10 holes at the bottom edge of the nesting plate, and 5 holes on each of the left and right edges of the horizontal nesting plate.

[0069] The 5×5 holes at the four corners of each small rectangular area are used as test holes (such as Figure 2 Insert 100 paper straws into the small holes (marked in 2.1.1, 2.1.2, 2.1.3, and 2.1.4). The specifications of the paper straws inserted into the cells are: 6 mm outer diameter, 5.7 mm inner diameter, and 7.5-7.8 cm long.

[0070] 3. Release adult bees

[0071] After indoor incubation, the incubation tray containing 30,000 to 40,000 honeycomb cells is transported to the beehive, the tray cover is opened, the adult bees are released, and the female bees are allowed to build nests in the paper straws; the indoor incubation conditions are: temperature 29-30°C, relative humidity 60%±5%, darkness, and incubation time 18-25 days.

[0072] 4. Recycle paper straws

[0073] Collect the paper straws that have produced offspring and are smoothly sealed with leaves on the brood board once every 7 days, remove the paper straws from the brood board with tweezers, record the nesting location information of each paper straw, and replace them with new paper straws to continue attracting female bees to build nests. Tie the paper straws removed from the brood board with rubber bands and place them flat in a cardboard box to take back.

[0074] The cardboard box is 30cm long, 17cm wide, 19cm high, and about 3.5mm thick. Several holes are pierced on the six sides with scissors or large tweezers. It has good pressure resistance, heat insulation and air permeability, so that the paper straws can withstand extreme external temperatures or physical extrusion during the recycling process, ensuring the normal development of the offspring.

[0075] The purpose of the rubber band to fix the paper straws is to reduce the vibration of the paper straws in the cardboard box and ensure that the offspring reach the room safely and smoothly.

[0076] 5. Larval Culture

[0077] In a cool place indoors, use scissors to cut open the smooth sealed end of the paper straw, take out all the nest chambers in the paper straw brought back in step 4, use tweezers to remove the leaf (nest cover) sealing each nest, and arrange the individual nests in the 96-well plate in the order of nesting (the sealed end is the last one to be built, and the innermost side is the first one to be built), then cover the 96-well plate with a 96-well plate cover or wrap the 96-well plate with a single layer of transparent plastic wrap, and use a No. 1 insect needle to poke a small hole in the plastic wrap opposite each nest; place the 96-well plate horizontally and transfer it to a dark incubator with a temperature of 29-30°C and a relative humidity of 60%±5% for incubation (such as Figure 3 ), during which the development of the offspring is observed until the cocoon is completed (the offspring in the cocoon are called prepupae at this time), and the development time of the eggs and the 1st to 5th instar larvae is recorded to obtain the developmental history of each instar and the entire larval period.

[0078] 96-well plate specifications: flat bottom, 1 cm depth, 7 mm diameter, consistent with the length and diameter of the offspring's nest chamber, to ensure the normal development of larvae in the 96-well plate.

[0079] 6. Prepupal Culture

[0080] Use pointed tweezers or small scissors to insert 1 to 2 mm into the upper edge of the cocoon, cut or shear horizontally in a circle, and completely cut off the upper part of the cocoon. Transfer the pre-pupae of the offspring of the alfalfa leafcutter bee in each cocoon into a capsule. Use an insect needle to poke a small hole at both ends of the capsule. Stick the capsules on the cardboard in the order of the pre-pupae in the nest. Place the cardboard horizontally and transfer it to a dark incubator with a temperature of 29 to 30 ° C and a relative humidity of 60% ± 5% for further cultivation. Observe the development of the pre-pupae through the capsule (such as Figure 4 ), pre-pupae that did not continue to develop within 30 days were recorded as diapause individuals, and progeny that continued to develop to the pupal or adult stage were recorded as non-diapause individuals.

[0081] The capsule is made of corn starch or glutinous rice, is 17 to 21 mm long and 6 to 7 mm in diameter, does not restrict the activities of the prepupae of the alfalfa leafcutter bee, is non-toxic and does no physical harm, is transparent, and can visually inspect the morphological changes of the offspring prepupae.

[0082] The cardboard is 1 to 1.3 mm thick, 29.7 cm long, 21 cm wide, and yellowish-brown. Each piece of cardboard can accommodate all the offspring in 25 to 26 paper straws. When used with capsules, the contrast is higher, making the observation results more accurate.

[0083] The method for recovering and monitoring the development of offspring of alfalfa leafcutter bees of the present invention can obtain the diapause rate of offspring recovered within a selected time period:

[0084] Diapause rate = number of diapause individuals / (number of diapause individuals + number of non-diapause individuals) × 100%.

[0085] Example 2

[0086] Based on Example 1, this example further describes the method of recovering alfalfa leafcutter honeycomb cells and monitoring offspring development in combination with experimental data, specifically:

[0087] 1. The number of sealing paper straws recovered every 7 days during nesting in plots 1 and 2 (see Table 1).

[0088] Table 1 shows that the number of sealed paper straws collected in Sites 1 and 2 first increased and then decreased during the survey period. Site 1 collected a relatively large number of sealed paper straws from June 10th to 30th (over 200, cumulatively accounting for 68.94%), with the highest number of sealed paper straws collected from June 24th to 30th, at 284 (accounting for 25.13%). Site 2 collected a relatively large number of sealed paper straws from June 1st to 28th (over 200, cumulatively accounting for 91.27%), with the highest number of sealed paper straws collected from June 8th to 14th, at 1,667 (accounting for 48.05%). Therefore, the peak nesting period of alfalfa leafcutter bees in sample sites 1 and 2 is in June. During this period, the number of paper straws inserted into the nest boards can be increased or the frequency of recycling can be accelerated to obtain more offspring. The number of sealing paper straws recycled in July in both sites was significantly reduced. The nest boards can be recycled in time according to the recycled quantity, ending the field breeding of alfalfa leafcutter bees and turning to indoor research.

[0089] Table 1. Number of paper straws recovered every 7 days during the survey period in Sites 1 and 2

[0090]

[0091]

[0092] 2. Under dark conditions at a temperature of 29-30°C and a relative humidity of 60% ± 5%, the developmental duration of eggs, 1st instar larvae, 2nd instar larvae, 3rd instar larvae, 4th instar larvae, and 5th instar larvae of A. lucerne in 96-well plates, as well as the total duration of egg development to prepupae (taking 50 offspring cultured from 0-day-old eggs as an example, see Table 2), the proportion of larvae of each instar corresponding to each period (taking 50 offspring cultured from 0-day-old eggs as an example, see Table 2). Figure 5 ).

[0093] Table 2. Development duration of each stage of the alfalfa leafcutter bee

[0094]

[0095] As shown in Table 2, the average developmental time of eggs, first-instar larvae, second-instar larvae, third-instar larvae, fourth-instar larvae and fifth-instar larvae of the alfalfa leafcutter bee was 2.52 days, 1.17 days, 1.26 days, 1.55 days, 1.68 days and 2.74 days, respectively. The total development time from egg to prepupa was 10.91 days. In general, the development time of each instar was less than 3 days, and the transition from younger larvae to older larvae was very rapid.

[0096] Depend on Figure 5 It can be seen that for experiments such as sampling of offspring at different developmental stages and obtaining test larval materials, eggs or embryos can be sampled within 3 days after the start of culture, 1st instar larvae can be sampled within 2 to 3 days of culture, 2nd instar larvae can be sampled within 3 to 4 days, 3rd instar larvae can be sampled within 4 to 6 days, 4th instar larvae can be sampled within 5 to 8 days, 5th instar larvae can be sampled within 7 to 10 days, and prepupae begin to appear from 9 to 12 days, and prepupae can be sampled during this time period or later.

[0097] 3. Diapause rate of offspring recovered every 7 days during nesting period in plots 1 and 2 (taking offspring from 100 randomly selected sealed paper straws recovered every 7 days as an example, see Figure 6 ).

[0098] Depend on Figure 6 It can be seen that the diapause rate of offspring in sample site 1 during nesting period did not change much, with an average diapause rate of 50.69%. The diapause rate of offspring in sample site 2 during nesting period gradually increased with time, with an average diapause rate of 92.03%. Overall, the diapause rate of offspring in sample site 2 in each time period was higher than that in sample site 1, indicating that the female bees released in sample site 2 were more likely to produce diapause offspring, and it was more suitable to expand the alfalfa leafcutter bee in this area.

[0099] Example 3

[0100] Based on Examples 1 and 2, this example further describes the results of the present invention's investigation of offspring mortality and natural enemy damage, using experimental data. Specifically, the experimental results include offspring mortality, natural enemy damage rate, empty nest rate, and pollen ball percentage in paper straws collected every seven days during nesting at Plots 1 and 2. This study clarified the causes of bee losses during pollination and nesting, and monitored population changes in the alfalfa leafcutter bee.

[0101] Offspring mortality rate = (number of young larvae + number of dead older larvae + number of dead prepupae) / (number of young larvae + number of dead older larvae + number of dead prepupae + number of diapause individuals + number of non-diapause individuals) × 100%;

[0102] Natural enemy damage rate = (number of parasitized + number of predators) / total number of nests × 100%;

[0103] Empty nest rate = number of empty nests / total number of nests × 100%;

[0104] Pollen ball ratio = number of pollen balls / total number of nests × 100%.

[0105] The statistical results are as follows Figure 7 、 Figure 8 、 Figure 9 and Figure 10 As shown, progeny mortality, natural enemy damage rate, empty nest rate, and pollen ball percentage were generally higher in Plot 1 than in Plot 2, indicating that the progeny or nests recovered from Plot 1 were of relatively poor quality, resulting in greater losses. Therefore, propagating alfalfa leafcutter bees in Plot 2 is more advantageous. Losses were particularly severe from June 1st to 9th and from July 6th to 14th. During these periods, measures such as trapping and killing natural enemies of alfalfa leafcutter bees in the field can be used to reduce losses.

[0106] In summary, the method for recovering alfalfa leafcutter bee hive chambers and monitoring offspring development provided by the present invention can effectively attract female bees to build nests, and the peak period of female bee nesting can be predicted by the number of recovered paper straws. The offspring of alfalfa leafcutter bees can be brought back from the field in a timely and convenient manner and their development can be monitored in real time. The monitoring data can provide a reference for the implementation of experiments such as sampling offspring at different developmental stages and obtaining test larval materials.

[0107] In addition, the method provided by the present invention can detect and obtain data such as the offspring diapause rate, larval mortality rate, natural enemy damage rate, empty nest rate, and pollen ball ratio in a selected time period, so as to more quickly analyze the causes of offspring loss and take corresponding measures in a timely manner to minimize the loss, thereby increasing the number of alfalfa leaf-cutting peaks that expand in the field and provide pollination services, and further improving the yield and quality of alfalfa.

[0108] The embodiments described above are only some of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A method for recovering alfalfa leaf-cutting honeycomb cells and monitoring offspring development, characterized in that: The steps include: S1. Placement of hives and hanging nest boards: Selected test plots at the early flowering stage of alfalfa, place hives in the center of the alfalfa field, the hives face southeast, and hang 8 to 10 nest boards on the inner wall of the hive, each of which has a 120 × 30 hole, each of which has the same aperture and the same depth. S2. Nesting paper straws in nesting boards: Insert paper straws into four 5×5 holes in a nesting board facing east or southeast to attract female bees to build nests. S3 release of bees: after the indoor incubation of 3 to 40,000 cells of the brooding bee tray transported to the hive, open the tray cover, release the bees, so that the female bees nest in the paper straw; S4. Recycling paper straws: Every seven days, collect sealed paper straws that have produced offspring from the nesting plates. Record the location of each straw and replace them with new ones to continue attracting female bees to build nests. Place the removed straws flat in a cardboard box and take them home. S5. Larval culture: Remove all nest chambers from the paper straws brought back and arrange the offspring in a 96-well plate in the order of nesting. Culture in a dark incubator. Observe the offspring's development during this time and record the developmental duration of the egg and 1st to 5th instar larvae. Calculate the developmental duration of each instar and the entire larval stage. S6. Prepupal Culture: Transfer the offspring prepupae from each cocoon into transparent capsules. Place the capsules on cardboard in the order in which the prepupae were placed in the nest. Continue culture under suitable conditions, observing the development of the prepupae through the capsules. Record the data of both diapause and non-diapause individuals, and compile statistical data. S7. Data statistics and analysis: Statistical analysis of offspring diapause rate, offspring mortality rate, natural enemy damage rate, empty nest rate, and pollen ball ratio to clarify the causes of bee losses during pollination and nesting.

2. The method for recovering alfalfa leaf cutting honeycomb chambers and monitoring progeny development according to claim 1, wherein: In the step S1, 8 to 10 honeycomb boards are vertically hung on the inner wall of the honeycomb. The honeycomb boards are hung at a height of 1.5 to 1.6 meters, with a hole diameter of 6 mm and a hole depth of 7 cm.

3. The method for recovering alfalfa leaf cutting honeycomb chambers and monitoring progeny development according to claim 1, wherein: In step S2, the method for selecting the four 5×5 small holes is as follows: divide the nest plate into three rectangular areas: upper, middle, and lower, select a small rectangular area in each rectangular area, and use the 5×5 small holes at the four corners of each small rectangular area as test holes; the specifications of the paper straws are: outer diameter 6mm, inner diameter 5.7mm, and length 7.5-7.8cm.

4. The method for recovering alfalfa leaf cutting honeycomb chambers and monitoring progeny development according to claim 3, wherein: The rectangular area division method is as follows: The upper part is a rectangular area of ​​39×30, the middle part is a rectangular area of ​​42×30, and the lower part is a rectangular area of ​​39×30; The small rectangular area division method is as follows: The small rectangular area on the top is located as follows: 10 holes on the top edge of the vertical release plate, and 5 holes on each of the left and right edges of the horizontal release plate. The positions of the small rectangular area in the middle are: 27 holes in the rectangular area where paper straws are inserted on the upper part of the nest-leaving plate in the vertical direction, and 5 holes on each of the left and right edges of the nest-leaving plate in the horizontal direction; The position of the small rectangular area at the bottom is: 28 holes in the rectangular area where paper straws are inserted in the middle of the vertical departure plate, 10 holes at the bottom edge of the departure plate, and 5 holes on each of the left and right edges of the horizontal departure plate.

5. The method for recovering alfalfa leaf-cutting honeycomb chambers and monitoring progeny development according to claim 1, wherein: In step S3, the environmental conditions for indoor incubation are: temperature 29-30° C., relative humidity 60%±5%, darkness, and incubation time 18-25 days.

6. The method for recovering alfalfa leaf-cutting honeycomb cells and monitoring progeny development according to claim 1, wherein: In step S4, a plurality of holes are punched on the six sides of the cardboard box.

7. The method for recovering alfalfa leaf-cutting honeycomb cells and monitoring progeny development according to claim 1, wherein: In step S6, the transparent capsule is made of corn starch or glutinous rice, with a length of 17 to 21 mm and a diameter of 6 to 7 mm; the cardboard is 1 to 1.3 mm thick, 29.7 cm long and 21 cm wide, and can accommodate all offspring in 25 to 26 paper straws; the suitable conditions are: temperature 29 to 30°C, relative humidity 60% ± 5%, and darkness.

8. The method for recovering alfalfa leaf-cutting honeycomb cells and monitoring progeny development according to claim 1, wherein: In step S7, the offspring diapause rate, offspring mortality rate, natural enemy damage rate, empty nest rate and pollen ball ratio are calculated as follows: Offspring diapause rate = number of diapause individuals / (number of diapause individuals + number of non-diapause individuals) × 100%; Offspring mortality rate = (younger larvae + dead older larvae + dead prepupae) / (younger larvae + dead older larvae + dead prepupae + number of diapause individuals + number of non-diapause individuals) × 100%; Natural enemy damage rate = (number of parasitized species + number of predators) / total number of nests × 100%; Empty nest rate = number of empty nests / total number of nests × 100%; Pollen ball ratio = number of pollen balls / total number of nests × 100%.

9. Use of the method for recovering cells and monitoring offspring development of alfalfa leafcutter bees according to any one of claims 1 to 8 in artificial breeding and population protection of alfalfa leafcutter bees.

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