A method for individual marking and fecundity determination of alfalfa leafcutter bees

Abstract

The invention discloses a method for individual marking and fertility determination of alfalfa leafcutter bees. The method comprises the following steps: selecting a marking object, fixing the insect body, marking the chest, and mixing male and female adults to mark the individual alfalfa leafcutter bees. The method comprises the following steps: placing beehives and nest boards, transporting male and female adults, releasing them in the field, monitoring nesting activities, and calculating fertility indicators to evaluate the fertility of the alfalfa leafcutter bees. According to tests, the method for individual marking and fertility determination of alfalfa leafcutter bees provided by the invention has little effect on the flight of adult alfalfa leafcutter bees, and 98.13% of female insects can fly freely. In addition, the method for individual marking and fertility determination provided by the invention has a large sample size that can be marked, a high degree of visualization, and can determine the number of nests built throughout the life history of the alfalfa leafcutter bees, which is of great significance for the artificial breeding of alfalfa leafcutter bees.

Description

Technical Field

[0001] The invention belongs to the field of insect breeding and relates to a method for individual marking and fecundity determination of alfalfa leafcutter bees. Background Art

[0002] The alfalfa leafcutter bee is a solitary bee that lives in groups and builds nests. It can effectively trigger the flower ejection mechanism of alfalfa, increasing alfalfa seed yield by more than 3 times. It is the most widely used and most intensively managed pollinating bee in the global alfalfa seed industry.

[0003] The life cycle of the alfalfa leafcutter bee includes four stages: egg, larva, pupa and adult. It is a typical facultative dimorphic insect that hibernates as a pre-pupa. In the summer of the following year, the adult builds a nest in artificial nest boards and various holes in nature. After mating, the female insects will visit flowers and build nests repeatedly for 1 to 2 months.

[0004] At present, the traditional marking method for studying bee colonies is the tagging method, which distinguishes different individuals by sticking unique tags on the dorsal thorax of adults. However, this method is not suitable for smaller bee species such as alfalfa leafcutter bees. Tags and glue of a certain size and weight not only affect the extension and flight of their wings, but also cause the tags to loosen due to the failure of the glue and the vigorous activity of the insect body, thus affecting the observation results. For small insects, the commonly used marking method in existing research is the dye marking method, but because alfalfa leafcutter bees live in groups and build nests, there is currently no example of using the dye marking method to mark and accurately distinguish thousands of individuals on a large scale.

[0005] In addition, regarding the nesting activities of bee colonies at the individual level, the existing technology preliminarily estimates the reproductive capacity and nesting volume of the entire population by establishing the correlation between the total release, emergence rate and total recovery. The work is cumbersome and has large errors. It is not clear what the location, number and time of the nest holes of a single individual in its life history are. Accurately understanding the nesting activities of bee colonies at the individual level is of great practical significance for the breeding of alfalfa leafcutter bees. Therefore, how to monitor and evaluate the individual behavior, fertility and adaptability of alfalfa leafcutter bees has become a practical problem that urgently needs to be solved in the breeding of alfalfa leafcutter bees. Summary of the invention

[0006] The present invention aims to provide a method for individual marking and fecundity determination of alfalfa leafcutter bees, which is relatively safe for alfalfa leafcutter bees, has little effect on the flight and nesting of adults, and can distinguish and observe the nesting conditions of a large sample group of more than 100 bees during the entire nesting period.

[0007] Based on the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The method for individual marking and fertility determination of alfalfa leafcutter bees provided by the present invention comprises individual marking and fertility determination, wherein the individual marking comprises step 1, selecting a marking object; step 2, fixing the insect body; step 3, marking the chest; and step 4, mixing male and female adults.

[0009] Furthermore, the selection of marking objects includes: when the flowering amount of alfalfa field reaches 25%, collecting alfalfa leafcutter bee adults that occur indoors on the same day from 8 am to 12 pm for 3-5 consecutive days, selecting active female adults and male adults with good flying ability, and placing them separately in breathable small mesh cages for standby use, and the small mesh cages for placing the adults and female adults are named male insect mesh cages and female insect mesh cages respectively.

[0010] Furthermore, the insect body fixation includes: opening the opening of the female insect net cage, closing the opening after letting one female adult crawl out each time, carefully clamping the front, middle and hind legs of one side of the three pairs of legs of the female adult with tweezers at the same time and / or clamping the chest with tweezers to keep the insect body stable in the horizontal direction.

[0011] Furthermore, the chest marking includes: using a dye pen to paint unique marks on the back plates of the chests of different female adults.

[0012] Furthermore, the mixing of male and female adults includes: placing the marked female insects into another empty small mesh cage, adding an equal number of empty shells of bee cocoons after the emergence of adults, and then taking out an equal number of male adults from the male mesh cage and placing them into the small mesh cage. This mesh cage is named the male and female mesh cage.

[0013] Furthermore, the specifications of the small net cage used in the above-mentioned selection of marked objects and mixing of male and female adults are: 20 cm long × 10 cm wide × 10 cm high, made of gauze with a mesh size of 40-60, and a closable opening on the top with an opening size of 2 cm × 2 cm.

[0014] Furthermore, the colors of the dye pens used in the above-mentioned chest markings include red, pink, dark blue, light blue, yellow, green and white, the ingredients are acrylic paint, and the pen tip is 0.7mm-1mm; there are 791 types of marks, including 7 single points, 49 upper and lower points, 49 left and right points, 343 regular triangles with three points, and 343 inverted triangles with three points.

[0015] The method for individual marking and fertility determination of alfalfa leafcutter bees provided by the present invention comprises the following steps: step 1, placing beehives and nest boards; step 2, transporting male and female adults; step 3, releasing them in the field; step 4, monitoring nesting activities; and step 5, calculating fertility indicators.

[0016] Furthermore, the placement of the beehive and nest boards includes: placing the beehive in the center of the alfalfa field, with the beehive as the center, and no other beehive is placed within 100m, the beehive is provided with a beehive door, the beehive door faces southeast, a nest board is hung on the inner wall of the beehive opposite the beehive door at a position about 1.5m away, the long end of the nest board is level with the horizontal direction, paper straws are inserted into all nest holes, and the position of each hole is numbered in the vertical and horizontal directions of the nest board with a starting number of 1 and increasing, and each nest hole has a unique nest hole coordinate combined with the vertical and horizontal numbering.

[0017] Furthermore, the transportation of male and female adults includes: covering the male and female net cages with breathable black cloth to avoid direct sunlight, placing the male and female net cages in a cool place in the transport vehicle and fixing them, transporting them to the alfalfa field, and avoiding bumps as much as possible during transportation.

[0018] Furthermore, the field release includes: placing male and female net cages on the ground directly below the nest plate, removing the black cloth, opening the upper opening, and allowing the male and female adults to fly out freely.

[0019] Furthermore, the nesting activity monitoring includes: setting up a camera directly in front of the nest board, recording the marks of female insects carrying leaves or pollen in and out of the nest after the adult insects are released, and recording the corresponding nest hole coordinates, and verifying the recorded female insect marks and the corresponding nest hole coordinates in the subsequent recording process, and finally determining the nesting positions of different female insects and the time period of nesting in the corresponding nest holes.

[0020] Furthermore, the calculation of the reproductive index includes: taking out the paper straws whose openings have been sealed by leaves from the nest plate, marking the female insect number and the coordinates of the nest holes on the paper straws, and taking them back to the indoor incubator for cultivation. After 10-15 days, cutting the paper straws, taking out all the nests, dissecting each nest, detecting and recording the internal conditions of the nests, including surviving larvae, dead larvae, pollen balls, empty nests, predation and parasitism, etc., continuing to cultivate the surviving larvae for 30 days, screening out diapause individuals and non-diapause individuals, and then summarizing the information of all nests built by each female insect, calculating the total number of nests and the number of nest holes of each female insect during the nesting period, as well as the female's reproductive rate and effective reproductive rate, and calculating the population expansion rate and effective expansion rate, and comprehensively evaluating the reproductive capacity of single female insects and the entire population. Female reproductive rate = (total number of nests / number of nesting females) × 100%; female effective reproductive rate = (number of dormant individuals / number of nesting females) × 100%; population expansion rate = [total number of nests / (number of released females + number of released males)] × 100%; population effective expansion rate = [number of dormant individuals / (number of released females + number of released males)] × 100%.

[0021] Furthermore, the nest plate in the method for measuring fertility provided by the present invention is made of polystyrene, with the number of nest holes in the vertical direction not exceeding 30 holes, the number of nest holes in the horizontal direction not exceeding 120 holes, the hole diameter is 6 mm, and the depth is 7 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.

[0022] Furthermore, the parameters of the incubator used in the above-mentioned calculation of the fertility index are: temperature 29°C-30°C, relative humidity 60%, and L:D of 0:24.

[0023] Through the above technical scheme, combined with the embodiments, the method for individual marking and fecundity determination of alfalfa leafcutter bees provided by the present invention has at least the following beneficial effects or advantages:

[0024] 1. The method for individual marking and fertility determination of alfalfa leafcutter bees provided by the present invention is to apply a dot mark on the back of the female insect's thorax without harming the insect body, having little effect on the flight of the female insect, and the flight success rate reaches 98.13%.

[0025] 2. The method for individual marking and fertility determination of alfalfa leafcutter bees provided by the present invention has a large number of marked samples and a high degree of visualization. There are 791 kinds of marks, which are very easy to identify and distinguish through single-point, double-point, triple-point and different color combinations.

[0026] 3. The dye component used in the method for individual marking and fertility determination of alfalfa leafcutter bees provided by the present invention is acrylic paint, which can exist for a long time during the nesting period of the female insects, ensuring the accuracy of observation or monitoring.

[0027] 4. The small mesh cage used in the method for individual marking and fertility determination of alfalfa leafcutter bees provided by the present invention can accommodate more than 200 adults. The cage is made of gauze, which provides a breathable environment for the adults and ensures the vitality of the adults before release. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the types of markings used in the method for individual marking and fecundity determination of alfalfa leafcutter bees described in the present invention.

[0029] Figure 2 The invention discloses the nesting situation of a single female insect in the method for individual marking and fecundity determination of alfalfa leafcutter bee of the present invention.

[0030] Figure 3 The invention discloses the internal conditions of a nest built by a single female insect in the method for individual marking and fecundity determination of alfalfa leafcutter bee of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is described below in conjunction with embodiments; however, the present invention is not limited to the following embodiments.

[0032] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products available on the market.

[0033] Example 1

[0034] This example provides the specific steps of the method for individual marking and fecundity determination of alfalfa leafcutter bees.

[0035] The specifications of the small net cage used in this embodiment are: 20cm long × 10cm wide × 10cm high, made of gauze, with a mesh size of 40-60 mesh, a closable opening on the top, the opening size is 2cm×2cm, and the specifications of the tweezers used are 12cm-15cm, which saves effort and can ensure that the adult insect body is not mechanically damaged.

[0036] (1) Individual marking

[0037] S1. Selection of marking objects: When the flowering amount of the selected alfalfa field (located in Pingluo County, Shizuishan City, Ningxia Hui Autonomous Region) reaches 25%, collect the alfalfa leafcutter bee adults that occur indoors from 8:00 a.m. to 12:00 p.m. for 3-5 consecutive days, select active female and male adults with good flying ability, and place them separately in small breathable mesh cages for use to ensure that the adults are in normal vitality before release. The small mesh cages for placing the adults and female adults are named male mesh cages and female mesh cages, respectively.

[0038] S2. Fixing the insect body: open the opening of the female insect net cage, let one female adult crawl out at a time and then close the opening. Use tweezers to carefully clamp the front, middle and hind legs of one side of the three pairs of legs of the female adult at the same time and / or use tweezers to clamp the chest to keep the insect body stable in the horizontal direction.

[0039] S3. Thoracic marking: Use a dye pen to paint unique marks on the dorsal thorax of different female adults.

[0040] Furthermore, the colors of the dye pen include red, pink, dark blue, light blue, yellow, green and white, the ingredients are acrylic paint, the pen tip is 0.7mm-1mm, and the colors are preferred color combinations, which are conducive to perception and distinction.

[0041] Furthermore, there are 791 types of marks, specifically 7 single points, 49 upper and lower points, 49 left and right points, 343 regular triangles with three points, and 343 inverted triangles with three points, as shown in the attached figure. Figure 1 The mark has a high degree of visualization, and through the arrangement and combination of colors, it can satisfy the marking of individuals with a small sample quantity and also achieve large-scale marking.

[0042] S4. Mixing male and female adults: Put the marked female insects into another empty small mesh cage, add an equal number of empty shells of cocoons after the emergence of adults, and then take out an equal number of male adults from the male mesh cage and put them into the small mesh cage. This mesh cage is named as a male and female mesh cage. The empty shells of cocoons can attract alfalfa leafcutter bees, simulate the environment when the adults just emerge, attract the adults to the empty shells, reduce their energy consumption, and ensure normal vitality. Female insects that have not successfully mated do not build nests, and the males are used to mate with female insects to ensure that the female insects can smoothly start their nesting activities.

[0043] (2) Fertility determination

[0044] S1. Placement of beehives and nest boards: Place a beehive in the center of the selected alfalfa field. With the beehive as the center, no other beehives are placed within a range of 100m. The beehive is provided with a beehive door, which faces the southeast direction. A nest board is hung about 1.5m from the inner wall of the beehive opposite the beehive door. The long end of the nest board is level with the horizontal direction. Paper straws are inserted into all nest holes. The position of each hole is numbered in the vertical and horizontal directions of the nest board with numbers starting from 1 and increasing. Combined with the vertical and horizontal numbers, each nest hole has a unique nest hole coordinate. The correspondence between the female insect numbering and the nesting position is achieved.

[0045] Furthermore, the nest plate is made of polystyrene, with no more than 30 holes in the vertical direction and no more than 120 holes in the horizontal direction. The hole size is 6 mm and the depth is 7 cm. The paper straw has an outer diameter of 6 mm, an inner diameter of 5.7 mm, and a length of 7.5 cm-7.8 cm, providing a place for adult insects to build nests and live, and can attract a large number of female adults to build nests.

[0046] S2. Transportation of male and female adults: Cover the male and female cages with breathable black cloth to avoid direct sunlight, place the male and female cages in the shade of the transport vehicle and secure them, transport the cages to the alfalfa field, and try to avoid bumps during transportation. Adults are phototactic, and the black cloth can reduce their movement frequency, ensuring that the adults arrive safely at the alfalfa field.

[0047] S3. Release in the field: Place the male and female net cages on the ground directly below the nest board, remove the black cloth, open the upper opening, and let the male and female adults fly out freely. Ensure the natural and safe flight of the adults.

[0048] S4. Monitoring of nesting activities: A combination of manual observation and video monitoring was used. A high-definition camera was set up 50 cm away from the nest board. After the adult insects were released, manual observation and video recording were carried out from 8:00 to 19:00 every day. At the same time, the marks of female insects carrying leaves or pollen in and out of the nest, as well as the corresponding nest hole coordinates were recorded. In the subsequent recording process, the recorded marks of female insects and the corresponding nest hole coordinates were verified to finally determine the nesting locations of different female insects and the time periods for nesting in the corresponding nest holes.

[0049] S5. Calculation of reproductive index: Take out the paper straws with the openings sealed by leaves from the nest plate, write the female insect number and nest hole coordinates on the paper straws, and bring them back to the indoor incubator for cultivation. After 10-15 days, cut the paper straws, take out all the nests, dissect each nest, inspect and record the internal conditions of the nests, including surviving larvae, dead larvae, pollen balls, empty nests, predation and parasitism, etc. Continue to cultivate the surviving larvae for 30 days, screen out dormant individuals and non-dormant individuals, and then summarize the information of all nests built by each female insect, calculate the total number of nests and the number of nest holes of each female insect during the nesting period, as well as the female's reproductive rate and effective reproductive rate, and calculate the population expansion rate and effective expansion rate, and make a comprehensive evaluation of the reproductive capacity of single female insects and the entire population.

[0050] Furthermore, the female reproductive rate = (total number of nests / number of nesting females) × 100%; the female effective reproductive rate = (number of dormant individuals / number of nesting females) × 100%; the population expansion rate = [total number of nests / (number of released females + number of released males)] × 100%; the population effective expansion rate = [number of dormant individuals / (number of released females + number of released males)] × 100%.

[0051] Furthermore, the parameters of the incubator are: temperature 29° C.-30° C., relative humidity 60%, L:D 0:24, and the parameters are suitable for the development of offspring and can determine whether the offspring is dormant or not.

[0052] Example 2

[0053] This example compares and tests the effects of different marking methods on the flight of female alfalfa leafcutter bees.

[0054] Newly emerged female and male alfalfa leafcutter bees were selected, and 200 and 160 female adults of alfalfa leafcutter bees were marked by the traditional labeling method and the individual marking method of the present invention, respectively. An equal number of males were released at the same time, and a 120-hole×30-hole nest plate with paper straws inserted was used to attract the females to build nests. The flight success rate of the female adults was measured, and the test results are shown in Table 1.

[0055] Table 1. Flight success rate of female adults marked by different marking methods

[0056]

[0057] According to Table 1, the flight success rate of female adults after marking by the traditional labeling method is 0%, and the flight success rate of the individual marking method described in the present invention is 98.13%, which is significantly higher than the flight success rate after marking by the traditional labeling method. This is because compared with the traditional labeling method, the method of the present invention uses a dye pen to apply dots on the chest of female adults, and does not use glue and labels that affect the movement of the chest muscles of female adults and increase the difficulty of flying. It is beneficial to the movement of the insect body, so it can maximize the normal flight of female adults and complete nesting activities.

[0058] Example 3

[0059] This example compares and tests the nesting conditions and internal conditions of the hive after female beekeepers are marked using different marking methods.

[0060] This embodiment is based on Example 2. Since the traditional labeling method did not obtain female insect individuals that successfully flew, and therefore could not measure the reproductive capacity of the female insects, the reproductive capacity measurement method described in Example 1 was used to measure the nesting conditions of the female insects, and the results are shown in Table 2. The numbers of the nesting female insects and the number of nesting holes were measured, and the results are shown in Table 3.

[0061] Table 2. Nesting conditions of female adults marked by different marking methods

[0062]

[0063] According to Table 2, among the 157 females that successfully flew, a total of 65 (41.4%) females built nests on a given nest board.

[0064] Table 3, the number of female insects building nests and the number of nesting holes

[0065]

[0066]

[0067] Note: Red, pink, dark blue, light blue, yellow, green and white are represented by the letters R, P, B, b, Y, G and W respectively. The “-” is used to distinguish the upper and lower positions of the color marks. The letter before the “-” indicates that the color is on the top, and the letter after the “-” indicates that the color is on the bottom.

[0068] According to Table 3, 25 females built more than 2 nest holes (38.5%), and 11 females built more than 3 nest holes (16.9%). The total number of nests built by a single female was fitted with a Gauss model, and the equation of the total number of nests built by a single female X and the relative frequency Y was obtained. Among them, Y0 is 2.11±0.81, X C was 6.25±0.33, W was 5.02±0.72, A was 124.78±17.73, and the correlation coefficient R 2The average number of nests built by a single female insect was 9.89, with 6.24 nests being the highest frequency. Figure 2 shown.

[0069] All nests built by single females were inspected and the results are shown in the attached Figure 3 As shown in the figure, the proportions of diapause individuals, non-diapause individuals, dead young larvae, dead old larvae, dead prepupae, pollen balls, empty nests, parasitized and preyed upon in the nest were 62.31%±3.54%, 7.47%±2.47%, 6.12%±2.32%, 0.86%±0.44%, 9.41%±1.56%, 10.57%±2.00%, 2.73%±0.96%, 0.53%±0.34% and 0%, respectively. The proportion of diapause individuals was significantly higher than that in other cases (F 8,576 =109.133, P<0.001).

[0070] Table 4. Reproduction rate and multiplication rate of female adults marked by different marking methods

[0071]

[0072]

[0073] According to Table 4, the fertility determination method described in Example 1 was used to obtain that the female reproductive rate and effective reproductive rate were 989.2% and 656.9%, respectively, and the population expansion rate and effective expansion rate were 200.9% and 133.4%, respectively.

[0074] As described above, the present invention can be well implemented. The above embodiments are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A method for individual marking and fecundity determination of alfalfa leafcutter bees, It is characterized in that Includes individual marking and fecundity determination; The individual markers include: Step 1, selecting marking objects: after alfalfa blooms, continuously collect alfalfa leafcutter bee adults that occur indoors on the same day, and select active female and male adults with good flying ability for use; Step 2, fixing the insect body: clamping the front, middle and rear legs of one side of the three pairs of legs of each reserved female adult insect in the selected marked object at the same time and / or clamping the chest to keep the insect body stable in the horizontal direction; Step 3, chest marking: Use dye to paint unique marks on the chest dorsal plate of different fixed female adults; Step 4, mixing male and female adults: the marked female adults, the empty shells of cocoons after adult emergence and the reserved male adults among the selected marked objects are mixed and placed in a net cage, and the net cage is named as a male and female net cage; The fertility assay includes: Step 1, placing beehives and nesting boards: placing beehives in the alfalfa field, wherein the beehives have a beehive door facing the southeast, and nesting boards are hung in the beehives, wherein the longer end of the nesting boards is level with the horizontal direction, and paper straws are inserted into all nesting holes on the nesting boards, and the position of each nesting hole is numbered in the vertical and horizontal directions of the nesting boards, so that each nesting hole has a unique nesting hole coordinate; Step 2, transporting male and female adults: Cover the male and female net cages with breathable black cloth to avoid direct sunlight, place the male and female net cages in a cool place in the transport vehicle and fix them, and transport them to the alfalfa field, and try to avoid bumps during transportation; Step 3, field release: placing the male and female net cages on the ground directly below the nest board, removing the black cloth cover, opening the upper opening, and allowing the male and female adults to fly out freely; Step 4, monitoring of nesting activities: a camera is set up in front of the nest board, and after the adult insects are released, the marks of female insects carrying leaves or pollen in and out of the nest are recorded, and the corresponding nest hole coordinates are recorded at the same time, and the recorded female insect marks and the corresponding nest hole coordinates are verified in the subsequent recording process, and finally the nesting positions of different female insects and the time periods of nesting in the corresponding nest holes are determined; Step 5, calculation of fertility index: take out the paper straws with openings sealed by leaves in the nest plate, write the female insect number and the corresponding nest hole coordinates on the paper straws, bring them back to the room for cultivation, cut the paper straws after cultivation, take out and dissect each nest, inspect and record the internal conditions of each nest, and evaluate the fertility of the female insects.

2. The method for individual marking and fertility determination of alfalfa leafcutter bees according to claim 1, It is characterized in that In the selected marking object, the alfalfa flowering period is when the flowering amount of the alfalfa field reaches 25%.

3. The method for individual marking and fertility determination of alfalfa leafcutter bees according to claim 1, It is characterized in that In the selected marked objects, the continuous collection of alfalfa leafcutter bee adults occurring indoors on the same day is to collect the alfalfa leafcutter bee adults occurring indoors on the same day from 8 am to 12 pm for 3 to 5 consecutive days.

4. The method for individual marking and fertility determination of alfalfa leafcutter bees according to claim 1, It is characterized in that In the mixture of male and female adults, the mixing ratio of the marked female adults, the empty shells of honey bee cocoons after adulthood and the spare male adults among the selected marked objects is 1:1:

1.

5. The method for individual marking and fertility determination of alfalfa leafcutter bees according to claim 1, It is characterized in that The specifications of the net cage are: 20cm long × 10cm wide × 10cm high, made of gauze with a mesh size of 40-60, and a closable opening on the top with an opening size of 2cm×2cm; the marks include 7 single points, 49 upper and lower points, 49 left and right points, 343 regular triangles with three points and 343 inverted triangles with three points; the dye is acrylic paint, including red, pink, dark blue, light blue, yellow, green and white.

6. The method for individual marking and fertility determination of alfalfa leafcutter bees according to claim 1, It is characterized in that No other beehives shall be set up within 100 meters of the beehive. The nest board is located 1.5 meters away from the inner wall of the beehive facing the beehive door, and one nest board is hung inside each beehive.

7. The method for individual marking and fecundity determination of alfalfa leafcutter bees according to claim 1, It is characterized in that In the nesting activity monitoring, the distance between the camera and the nest plate is 50 cm, the camera records from 8:00 to 19:00 every day, and the observation period is 30-40 days.

8. The method for individual marking and fertility determination of alfalfa leafcutter bees according to claim 1, It is characterized in that The nest plate is made of polystyrene, with the number of nest holes in the vertical direction not exceeding 30 holes and the number of nest holes in the horizontal direction not exceeding 120 holes. The nest hole size is 6mm and the depth is 7cm. The paper straw has an outer diameter of 6mm, an inner diameter of 5.7mm, and a length of 7.5-7.8cm. The culture temperature of the indoor culture is 29℃-30℃, the relative humidity is 60%, and the lighting time L:D is 0:24.

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