A rapid method for identifying successful insemination of bumblebee queens

Abstract

The invention relates to the technical field of bumblebee breeding, and provides a method for quickly identifying the successful insemination of a bumblebee queen bee. The method can directly detect the condition of the spermatocyst of a living bumblebee queen bee by directly observing the spermatocyst of the queen bee in vitro without damaging the queen bee, thereby accurately and quickly judging whether the insemination of the queen bee is successful or not, and eliminating or re-inseminating the queen bee according to the identification result, thereby saving time, improving the experimental progress, improving the breeding efficiency and reducing the breeding cost.

Description

Technical Field

[0001] The invention relates to the technical field of bumblebee breeding, in particular to a method for quickly identifying successful insemination of a bumblebee queen. Background Art

[0002] Bumblebees belong to the superfamily Apidae and the genus Bombous. They are important pollinating insects and play an important role in improving the quality and efficiency of crops and maintaining the balance of the ecosystem. Bumblebee pollination has been proven to be an important supporting technology that is indispensable for the safe and efficient production of greenhouse fruits and vegetables. It can not only replace manual labor and save labor, but also reduce the use of hormones and pesticides, improve product quality and safety, promote user income growth, and improve the ecological environment. Breakthroughs have been made in the successive breeding technology of bumblebees at home and abroad, achieving multi-generation breeding and factory production in one year. In the factory production of bumblebees, the successful insemination of the queen bee is a key technical link. How to quickly and accurately determine whether the insemination of the queen bee by artificial insemination or natural mating is successful is crucial for factory artificial breeding. However, the existing technology for identifying whether the insemination of the bumblebee queen is successful has some limitations.

[0003] Common methods currently used include dissecting the queen bee to observe whether there are sperms in its spermathecae, waiting for the queen bee to lay offspring and observing the gender of the offspring after emergence, and using molecular detection technology to detect whether the eggs are diploid. However, these methods have their own limitations: dissecting the queen bee requires killing the queen bee, which causes certain losses; although it is possible to accurately determine whether the queen bee has been successfully fertilized based on the emergence of the queen bee's female offspring, it is necessary to wait for the queen bee to recover after the diapause stage, lay eggs, hatch eggs into larvae, larvae pupate, and pupae emerge before the results can be obtained; and although molecular detection technology can directly detect the ploidy of eggs, it is limited in practical application due to the complex operation and high cost.

[0004] Therefore, a fast, accurate, efficient and reliable technology is needed to identify whether the insemination of a bumblebee queen is successful or not, without damaging the queen. It should be able to directly observe and evaluate whether there are sperm in the spermatheca of the bumblebee queen, so as to accurately judge whether the insemination of the queen is successful. Queens that have not been successfully inseminated artificially or naturally can be identified in time and eliminated or inseminated again, thereby improving reproductive efficiency and reducing breeding costs. Summary of the invention

[0005] The invention aims to provide a rapid identification method for successful insemination of a bumblebee queen.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for rapid identification of successful insemination of a bumblebee queen, the method comprising: anesthetizing the inseminated bumblebee queen, opening the queen's stinger cavity, finding a spermatocyst between the left side of the stinger base and the reproductive opening, and judging whether there are sperm in the spermatocyst based on the color of the spermatocyst.

[0007] Generally speaking, bumblebee insemination can be identified 90 minutes after.

[0008] Furthermore, the queen bee sting cavity is opened under an insect artificial insemination instrument to expose the queen bee reproductive opening, and the spermatheca is found on the left side of the sting base and the right side of the reproductive opening.

[0009] The bumblebees described in the present invention include but are not limited to ground bumblebees, Lanzhou bumblebees, and dense forest bumblebees.

[0010] In a specific embodiment of the present invention, a method for quickly identifying successful insemination of a bumblebee queen comprises: anesthetizing the bumblebee queen after artificial insemination or natural mating, opening the queen stinger cavity under an insect artificial insemination instrument to expose the queen reproductive opening, finding a spermatocyst between the left side of the stinger base and the reproductive opening, and judging whether there are sperm in the spermatocyst based on the color of the spermatocyst; the judgment criteria are as follows: a spermatocyst of successful insemination usually appears white, otherwise, if the spermatocyst appears transparent, it indicates that the insemination is unsuccessful.

[0011] Preferably, the test can be performed 90 minutes after insemination.

[0012] Preferably, the bumblebees may be anesthetized by carbon dioxide.

[0013] In a second aspect, the present invention provides the application of the above method in bumblebee breeding, cultivation and experimental research.

[0014] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0015] The present invention can quickly determine the fertilization status of the bumblebee queen in vitro without killing the queen, waiting for the offspring to emerge from the hive, or performing complex molecular testing. The technology is fast, accurate, easy to operate, and low cost. It can timely identify the queen bee that failed to be fertilized, and eliminate or re-fertilize it according to the identification results, thereby saving time, improving the progress of the experiment, improving the breeding efficiency, and reducing the cost of feeding. The bumblebee worker bees reported by the inventors earlier also have the function of producing females similar to the queen bee (Zhuang et al, 2023). Accordingly, this method is also suitable for the rapid identification of successful fertilization of bumblebee worker bees. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the spermatheca of the bumblebee queen bee that has been successfully inseminated in a preferred embodiment of the present invention.

[0017] Figure 2 This is the spermatheca of the bumblebee queen bee that has failed in fertilization in a preferred embodiment of the present invention.

[0018] Figure 3 This is the spermatheca of the Lanzhou bumblebee queen bee that has been successfully inseminated in a preferred embodiment of the present invention.

[0019] Figure 4 This is the spermatheca of the Lanzhou bumblebee queen that has failed insemination in a preferred embodiment of the present invention.

[0020] Figure 5 This is the spermatheca of the queen bee of the dense forest bumblebee that has been successfully inseminated in the preferred embodiment of the present invention.

[0021] Figure 6 This is the spermatheca of the queen bee of the dense forest bumblebee that has failed to be fertilized in the preferred embodiment of the present invention.

[0022] Figure 7 This is the spermatheca of a bumblebee worker bee that has been successfully fertilized in a preferred embodiment of the present invention.

[0023] Figure 8 It is the spermatheca of bumblebee worker bees that failed in fertilization in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0024] The present invention aims to provide a technology for quickly identifying whether the insemination of a bumblebee queen is successful or not, by observing the situation of the queen's spermatocyst in vitro, so as to solve the limitations of the existing technology and meet the needs of bumblebee research and breeding. The present invention provides a fast, accurate, efficient and reliable means for factory breeding and scientific research of bumblebees, accurately identifies the queen that has failed to be inseminated without killing the queen, and eliminates or re-inseminates according to the identification results, thereby saving time, improving experimental progress, improving breeding efficiency and reducing feeding costs.

[0025] The present invention adopts the following technical solution:

[0026] 1. Select bumblebee queens or worker bees after artificial insemination or natural mating. Identification can be performed 90 minutes after insemination.

[0027] 2. Use an insect artificial insemination instrument or other suitable observation equipment to anesthetize the bumblebee with carbon dioxide. Open the queen bee's reproductive organs and expose the queen bee's reproductive opening under the insect artificial insemination instrument. The spermatheca can be seen between the left side of the stinger base and the reproductive opening.

[0028] 3. Observe the color of the spermatheca to determine whether there are sperm in the spermatheca. The spermatheca of successful fertilization usually appears white, otherwise, if the spermatheca appears transparent, it means that fertilization is unsuccessful.

[0029] It should be noted that, given the unique physiological characteristics of bumblebees, their reproductive cavity and spermathecae are both transparent. This method is not only applicable to queen bees of the genus Bombus under the superfamily Apoidea, but also to identifying whether bumblebee worker bees are successfully inseminated.

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] Example 1 Rapid Identification Method for Successful Insemination of Bumblebee Queen

[0032] Select a bumblebee queen 90 minutes after artificial insemination or natural mating. Use an insect artificial insemination instrument or other suitable observation equipment to fix the bumblebee queen on the insect artificial insemination instrument under a microscope, and adjust the focus of the microscope so that the stinger cavity of the bumblebee queen can be clearly seen under the microscope and exposed within the operating range. Use the operating lever of the insect insemination instrument to gently open the stinger cavity of the bumblebee queen so that the internal structure of the genitals and the spermatheca on the right side of the reproductive opening are clearly visible. Observe the color of the spermatheca to determine whether there are sperm in the spermatheca. Successful insemination is usually manifested by the presence of sperm in the spermatheca, as shown in the figure attached to the instruction manual. Figure 1 The spermatheca of a successfully inseminated queen bee appears white, otherwise, see the figure in the specification. Figure 2 If the spermatheca of the queen bee of the ground bumblebee appears transparent, it means that the insemination was unsuccessful.

[0033] Example 2 Rapid Identification Method for Successful Insemination of Lanzhou Bumblebee Queen

[0034] Select a Lanzhou bumblebee queen 90 minutes after artificial insemination or natural mating. Use an insect artificial insemination instrument or other suitable observation equipment to fix the Lanzhou bumblebee queen on the insect artificial insemination instrument under a microscope, and adjust the focus of the microscope so that the stinger cavity of the Lanzhou bumblebee queen can be clearly seen under the microscope and exposed within the operating range. Use the operating lever of the insect insemination instrument to gently open the stinger cavity of the Lanzhou bumblebee queen so that the internal structure of the genitals and the spermatheca on the right side of the reproductive opening are clearly visible. Observe the color of the spermatheca to determine whether there are sperm in the spermatheca. Successful insemination is usually manifested by the presence of sperm in the spermatheca, see the figure in the instruction manual. Figure 3 The spermatheca of a successfully inseminated Lanzhou bumblebee queen appears white, whereas, as shown in FIG. 4 of the accompanying drawings of the specification, the spermatheca of a Lanzhou bumblebee queen appears transparent, indicating that the insemination was unsuccessful.

[0035] Example 3 Rapid Identification Method for Successful Insemination of Bumblebee Queen Bee

[0036] Select a queen bee of the forest bumblebee 90 minutes after artificial insemination or natural mating. Use an insect artificial insemination instrument or other suitable observation equipment to fix the queen bee of the forest bumblebee on the insect artificial insemination instrument under the microscope, and adjust the focus of the microscope so that the stinger cavity of the queen bee of the forest bumblebee can be clearly seen under the microscope and exposed within the operating range. Use the operating lever of the insect insemination instrument to gently open the stinger cavity of the queen bee of the forest bumblebee so that the internal structure of the genitals and the spermatheca on the right side of the reproductive opening are clearly visible. Observe the color of the spermatheca to determine whether there are sperm in the spermatheca. Successful insemination is usually manifested by the presence of sperm in the spermatheca, see the figure in the instruction manual. Figure 5 The spermatheca of a successfully inseminated queen bee of the dense forest bumblebee appears white, otherwise, see the figure in the specification Figure 6 If the spermatheca of the queen bee of the jungle bumblebee appears transparent, it means that the insemination was unsuccessful.

[0037] Example 4 Rapid Identification Method for Successful Insemination of Bombus terrestris Worker Bees

[0038] Select bumblebee workers 90 minutes after artificial insemination or natural mating. Use an insect artificial insemination instrument or other suitable observation equipment to fix the bumblebee worker bees on the insect artificial insemination instrument under the microscope, and adjust the focus of the microscope so that the stinger cavity of the bumblebee worker bees can be clearly seen under the microscope and exposed within the operating range. Use the operating lever of the insect insemination instrument to gently open the stinger cavity of the bumblebee worker bees so that the internal structure of the genitals and the spermatheca on the right side of the reproductive opening are clearly visible. Observe the color of the spermatheca to determine whether there are sperm in the spermatheca. Successful insemination is usually manifested by the presence of sperm in the spermatheca, see the figure in the instruction manual. Figure 7 The spermatheca of a successfully inseminated bumblebee worker bee appears white, otherwise, see the figure in the specification. Figure 8 If the spermatheca of a bumblebee worker is transparent, it means that fertilization was unsuccessful.

[0039] Comparative Example 1

[0040] This comparative example is substantially the same as Example 1, except that the 90 minutes in Example 1 is replaced by 30 minutes.

[0041] Comparative Example 2

[0042] This comparative example is substantially the same as Example 1, except that the 90 minutes in Example 1 is replaced by 60 minutes.

[0043] Comparative Example 3

[0044] This comparative example is substantially the same as Example 1, except that the 90 minutes in Example 1 is replaced by 120 minutes.

[0045] Comparative Example 4

[0046] This comparative example is substantially the same as Example 1, except that the 90 minutes in Example 1 is replaced by 150 minutes.

[0047] Comparative Example 5

[0048] This comparative example is substantially the same as Example 1, except that the artificial insemination or natural mating in Example 1 is replaced by the injection of physiological saline, and 90 minutes is replaced by 30 minutes.

[0049] Comparative Example 6

[0050] This comparative example is substantially the same as Example 1, except that the artificial insemination or natural mating in Example 1 is replaced by the injection of physiological saline, and the 90 minutes is replaced by 60 minutes.

[0051] Comparative Example 7

[0052] This comparative example is substantially the same as Example 1, except that the artificial insemination or natural mating in Example 1 is replaced by the injection of physiological saline.

[0053] Comparative Example 8

[0054] This comparative example is substantially the same as Example 1, except that the artificial insemination or natural mating in Example 1 is replaced by the injection of physiological saline, and the 90 minutes is replaced by 120 minutes.

[0055] Comparative Example 9

[0056] This comparative example is substantially the same as Example 1, except that the artificial insemination or natural mating in Example 1 is replaced by the injection of physiological saline, and the 90 minutes is replaced by 150 minutes.

[0057] It should be noted that the age of the bumblebee queens used in the above embodiments and comparative examples is all 7 days old.

[0058] Experimental Example 1 Changes in the state of the spermathecae over time after bumblebee insemination and injection of saline

[0059] According to the requirements of each group, 20 unmated queens were artificially inseminated. Each queen in the insemination group was inseminated with 0.1 μL of semen, and each queen in the saline group was injected with 0.1 μL of saline. The treated queens were raised individually in a light-proof breeding room with a temperature of 28°C ± 1°C and a humidity of 60% ± 5%. The spermatocysts were observed after different time periods (see Table 1).

[0060] Table 1 Changes of spermathecae status over time after bumblebee insemination

[0061]

[0062] As can be seen from Table 1, in Comparative Example 1, the spermathecae was observed 30 minutes after fertilization, and the spermathecae was still translucent, indicating that sperm had been transferred to the spermathecae; in Comparative Example 2, the spermathecae was observed 60 minutes after fertilization, and the spermathecae began to appear opaque; in Example 1, the spermathecae was observed 90 minutes after fertilization, and the spermathecae appeared white; in Comparative Example 3, the spermathecae was observed 120 minutes after fertilization, and the spermathecae appeared white; in Comparative Example 4, the spermathecae was observed 150 minutes after fertilization, and the spermathecae appeared white; in Comparative Examples 5-9, physiological saline was injected, and the spermathecae were all transparent.

[0063] By comparing the above-mentioned Example 1 with Comparative Examples 1-4, it can be seen that by observing the spermatheca starting from the 90th minute, whether the insemination is successful or not can be accurately determined.

[0064] Comparative Examples 5-9 were injected with physiological saline, and the spermathecae were transparent at the above time points, which means that no sperm entered the spermathecae and the color of the spermathecae did not change.

[0065] Experimental Example 2: Observing the color of the spermatheca in the bumblebee reproductive cavity to determine the accuracy of successful insemination

[0066] According to the methods of Example 1 and Comparative Example 7, 30 queen bees were treated respectively. In Example 1, each queen bee was inseminated with 0.1 microliters of semen; while in Comparative Example 7, each queen bee was injected with 0.1 microliters of saline. After treatment and before observing the spermatophore, the queens of the two groups were mixed. Subsequently, under the insect artificial insemination instrument, the queen bee sting cavity was opened, and the spermatophore was found between the left side of the sting base and the reproductive opening. The spermatophore conditions of these queen bees were checked one by one to distinguish the inseminated and uninseminated queen bees, according to the color of the spermatophore: white for Example 1 and transparent for Comparative Example 7. Then, the spermatophore of these queen bees was dissected, and the sperm therein was observed to determine the insemination status. The results showed that the accuracy rate of judging whether the insemination was successful or not by observing the color of the spermatophore in the bumblebee reproductive cavity reached 100%.

[0067] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A rapid identification method for successful insemination of a bumblebee queen, characterized in that: The method comprises: anesthetizing the queen bee after insemination, opening the queen bee stinger cavity, finding the spermatheca between the left side of the stinger base and the reproductive opening, and judging whether there are sperms in the spermatheca according to the color of the spermatheca.

2. The method according to claim 1, characterized in that The queen bee was identified 90 minutes after insemination.

3. The method according to claim 1, characterized in that Open the queen bee stinger cavity under the insect artificial insemination instrument, expose the queen bee reproductive opening, and find the spermatheca on the right side of the reproductive opening.

4. The method according to claim 1, characterized in that Bumblebees were anesthetized with carbon dioxide.

5. The method according to claim 1, characterized in that The bumblebees include ground bumblebees, Lanzhou bumblebees, and dense forest bumblebees.

6. The method according to any one of claims 1 to 5, characterized in that: The specific judgment criteria are as follows: if the sperm sac is successfully fertilized, it will appear white; otherwise, if the sperm sac is transparent, it means that the fertilization is unsuccessful.

7. Application of the method according to any one of claims 1 to 6 in bumblebee breeding, breeding and experimental research.

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