A detection device and detection method for social conflict between bumblebee individuals

Abstract

The present invention relates to a detection device and a test method for social conflicts between individual bumblebees, and belongs to the field of insect social behavior testing. The device includes a large box, in which two small boxes are arranged, and the two small boxes are connected through a cylindrical pipe, and the small boxes are all opened on the top surface, and the top surface is covered by a transparent top cover with a slot; a vertical incision is arranged on the upper part of the middle position of the cylindrical pipe, and a partition that can move up and down is inserted at the vertical incision; the lower end of the partition is an irregular shape, and a gap of 1-2mm is maintained with the inner side of the cylindrical pipe, and a thin rope is attached to the upper end of the partition. The present invention also provides a method for experimentally detecting social conflicts between individual bumblebees using the device. The device can provide a winner and loser bumblebee model that is not harmed and stable, laying a key technical foundation for the subsequent development of the conflict winner and loser effect and mechanism. The method is novel in design, easy to operate, and has good repeatability, which greatly improves the efficiency of experimental detection.

Description

Technical Field

[0001] The invention relates to a device and a method for detecting social conflicts among bumblebee individuals, and belongs to the field of insect social behavior testing. Background Art

[0002] Bumblebees are important pollinators of many wild plants and crops, and play a very important role in maintaining the balance of natural ecosystems and global food security. Therefore, understanding bumblebees, protecting bumblebees, and utilizing bumblebees are major issues facing mankind. However, unlike those insects that live as single individuals, bumblebees are social insects that live in groups and are primitively eusocial. The queen bee is the highest level and is responsible for laying eggs; worker bees are usually responsible for raising larvae, cleaning and storing food in the nest, and going out to collect nectar, pollen and water, and are at the bottom of the society (Dornhaus A & Chittka D L, 2001; Dornhaus A et al., 2003). It has been proven through the European ground bumblebee that lower animal insects have a variety of advanced cognition. European ground bumblebees can independently and spontaneously solve the problem of pulling ropes to feed (Dayman, 2020); bumblebees have individual social abilities (Liu Yimiao, 2021), and social isolation can lead to social disorders in bumblebees (Liu Yimiao, 2021). Leadbeater found that European ground bumblebees have the ability of social learning (Leadbeater and Chittka, 2007); Solvi et al. found that European ground bumblebees have cross-modal cognitive abilities (Solvi et al., 2020); European ground bumblebees are also believed to have self-cognition (Ravi et al., 2020; Brebner and Chittka, 2021). Bumblebees are becoming a model organism for exploring cognition, complex social behavior, and social evolution mechanisms (Sadd et al., 2015). These studies have promoted researchers' understanding of bumblebee society.

[0003] Social competition is one of the important driving forces for the social development of bumblebee populations. Understanding and recognizing social competition and conflict in bumblebee society is important for researchers to understand the laws of cross-species social evolution, the laws of bumblebee social development, and the protection of bumblebees. Among them, the winner and loser effects of social conflict have always been a hot topic that researchers are keen to pursue. Winning and losing in competition will affect the individual's physiology and psychology, and will have a significant impact on individual survival and health. However, there is a lack of observations on the winner and loser effects of social conflicts in lower invertebrates. Previous studies, whether from higher animals or lower animals, mostly stayed at the individual and small group level to explore the influence of winners and losers in conflicts on their next victory and their neural mechanisms. However, whether the victory in social conflict can affect the social status of animals at a higher social level has not been reported so far. Through careful comparative analysis, the inventors found that bumblebees are completely different from honeybee, ant and other societies. The life cycle of bumblebee society is short, only 1 year, but it has a complete development stage, which gives researchers the opportunity to continuously observe the development and changes of society in a short period of time. At the same time, unlike bee and ant societies, bumblebee societies are small, with only a few hundred individuals. More importantly, queen bees and worker bees mostly live together on the surface of the hive, which provides a very rare experimental scenario for researchers to observe the relationship between individuals and society, as well as the behavior of winners / losers of social conflicts after they return to society.

[0004] Previous studies on conflicts between insects (including crickets) generally place paired animals in an open field in a certain space to observe conflicts between them. If the paired insects are highly aggressive, this method often leads to fierce fights between individuals, often resulting in animal injuries. Injuries caused by a fierce fight will make it impossible to continue subsequent experiments. For example, bumblebees will bite each other with their foreheads and stab each other with their tail stingers during fierce fights, which may not only cause physical injuries, but also make it impossible to evaluate the effects of winners and losers in the future; in addition, the fighting behavior between bumblebees in the open field is complex and changeable. Researchers not only have difficulties in accurately classifying fighting behaviors, but also have difficulty in quantifying the number and duration of various types of fighting behaviors. Even if they can be quantified, it is time-consuming and labor-intensive, and the experimental efficiency is low; at the same time, if the paired insects are not highly aggressive against each other, it takes a long time to wait for conflicts between them. For example, bumblebee individuals sometimes do not fight for a long time. It can be seen that this uncertainty also greatly affects the efficiency of the experiment.

[0005] In summary, the lack of suitable equipment has greatly limited researchers' understanding of social conflicts in lower invertebrates and their subsequent effects. So, is it possible to develop a conflict device that can both increase the incidence of conflict and limit the intensity of conflict to protect the animals from physical harm; make fighting behaviors simpler and easier to classify; and also make it easy to quantify the number and duration of these fighting behaviors?

[0006] After long-term observation of the behavior of bumblebees, the inventor discovered that bumblebees love to explore holes, drill holes and build nests in the nest; after carefully dissecting the structure of the bumblebee nest, it was discovered that the inside of the hive is a complex pipeline system, through which bumblebees can move in the intricate tunnels of the nest. This phenomenon inadvertently inspired the inventor, if bumblebees happen to meet head-on in the narrow tunnel of the hive. The inventor speculated that space limitations will cause the two bees to conflict in order to pass. Even if a conflict occurs, due to space limitations, the two bees will not be able to use violent tools normally, so there may not be a fight involving biting, and the bumblebees can only move forward or backward, with no choice. To this end, based on the assumption that social insect bumblebee individuals will meet in the dark pipes of the hive and there may be conflicts, the inventor began to try to simulate and design a pipeline device and its testing method that can create conflicts between bumblebee individuals.

[0007] Following this idea, the inventors have tried for a long time, but all the experiments failed. Whether in dark or bright conditions, bumblebees never enter the pipe designed by the inventors. Instead, they show various behaviors outside the pipe, such as flying, standing still / resting, and even have few chances to meet. The inventors conducted in-depth and repeated analysis of the failure and found that bumblebees have no interest in the pipe outside the hive. Tests under single-tube conditions show that it is almost impossible for bumblebees to crawl into the pipe on their own, let alone two bumblebees can meet in the pipe. Therefore, the design of the device must encourage bumblebees to enter the pipe and meet autonomously, which poses a huge technical challenge; although bumblebees drill wax cups and other behaviors regardless of day or night, they are difficult to observe in the hive. For the visualization and convenience of observation, the conflict process of bumblebees in the pipe should be set to be observed under bright conditions; considering that bumblebees will spontaneously show many behaviors that are not conducive to climbing the pipe, such as flying, the technical design of the device must promote its crawling and suppress its flying motivation and behavior; preliminary experiments have shown that bumblebees are relatively docile under dark conditions. Under bright conditions, external environment and human operation can cause stress to bumblebees, which in turn affects their exploration and interest in the pipeline. Currently, there is no corresponding device that can solve the above technical problems. Summary of the invention

[0008] In view of the above technical problems, the present invention provides a device for detecting social conflicts between bumblebee individuals and a method for detecting social conflicts between bumblebee individuals, and the technical solution is as follows:

[0009] A device for detecting social conflicts between individual bumblebees, characterized in that the device comprises a large box, two small boxes are arranged in the large box, the two small boxes are connected by a cylindrical pipe, the two small boxes are free exploration areas, the middle cylindrical pipe is a conflict area, the small boxes are all open on the top surface, and the top surface is covered by a transparent top cover with a card slot; a vertical incision is provided in the upper middle part of the cylindrical pipe, and a partition that can move up and down is installed at the vertical incision; the lower end of the partition is an irregular polygon, and a gap of 1-2 mm is maintained with the inner side of the cylindrical pipe, and a thin rope is attached to the partition.

[0010] The purpose of setting up a large box is to isolate the external environment from interfering with the attention of lower animals during the experiment as much as possible.

[0011] The side and bottom of the small box are made of opaque, smooth, white materials, and the bottom and sides are not light-transmissive; the purpose of establishing a small, low, opaque, smooth, white inner side free exploration area is: 1) to simulate the color of white flowers through color, thereby reducing stress; 2) opacity is to suppress the distraction of the external scene from the bumblebee's attention; 3) because the transparent box can trigger the illusion of the bumblebee, causing it to focus on trying to escape through the transparent wall, resulting in it being unable to focus on exploring the small box. Therefore, the opaque body can focus its attention on exploring the box. 4) The smoothness of the inner side of the opaque body prevents its climbing behavior; 5) The roughness of the bottom of the opaque body reduces its crawling stress; 6) The low box prevents its flying behavior; 7) On the basis of suppressing the above motivations and behaviors, the box with a smaller space can ensure that the bumblebees with small individuals and slow crawling speed can autonomously explore in the box, and can quickly detect the pipe opening, and use their instincts to make them voluntarily enter the pipe. However, the top cover and cylindrical pipe of the small box are made of transparent materials to facilitate visualization; the bottom surface of the cylindrical pipe must be kept rough to increase friction and facilitate the occurrence and quantification of conflict behavior.

[0012] Since the two insects could not arrive at the middle of the test tube at the same time during the test, it was natural to set a partition in the middle. However, when the bumblebee reaches the partition, it will exit the pipe on its own due to obstruction, and the test will fail. How to solve this technical problem? There has been no clue or solution for a long time. However, in other experimental tasks, the inventor accidentally discovered that bumblebees seemed to be particularly interested in physical barriers with gaps. For this reason, the inventor thought of designing a partition that could form a gap with the pipe. As a result, the experiment was successful, so that the two bumblebees could meet head-on at the designated position, realizing the first autonomous and voluntary meeting and looking at each other of insects. Therefore, the thin partition made of specially installed transparent material plays a key regulatory function, ensuring that any insect that arrives at the position first can stay and wait in the area until the two meet and look at each other. A special partition that can move up and down must be installed in the middle of the cylindrical pipe. It is made of transparent material to facilitate the two bumblebees to see each other. The lower end of the special partition is structured in a polygonal shape and maintains a gap of 1-2mm with the inner circular surface of the tube, which stimulates the bumblebees that arrive in the middle position in advance to squeeze the gap, so that they stay in the middle position and wait for the bumblebees that arrive later. Otherwise, it is difficult for the two animals to meet head-on in the middle position. The gap is also conducive to communication between the animals that meet head-on. Once the animals meet head-on in the middle position, a thin rope attached to the top of the partition will be used to pull up the partition vertically.

[0013] In order to enable the bumblebees to enter the cylindrical pipe as quickly and unimpeded as possible, the bottom surface of the cylindrical pipe opening is maintained at the same level as the bottom surface of the small box.

[0014] Preferably, the device further comprises a camera, which is mounted on the inner side of the large box, and the camera lens is kept at the same level as the cylindrical pipe.

[0015] Preferably, the small box is 5cm×5cm×5cm.

[0016] Preferably, the diameter of the cylindrical pipe matches the size of the bumblebee, allowing only one bumblebee to pass through at a time.

[0017] Design idea of ​​the device of the present invention: The inventor boldly imagined whether a bumblebee might conflict with another bee when it was traveling in a narrow dark pipe in the hive. However, there were huge challenges in the manufacturing and testing experiments. Because bumblebees are very easily disturbed by the external environment and produce stress, the instinct of insects to explore and drill holes cannot be expressed, resulting in the failure of the experimental test. In addition, during the test, it is almost impossible for two bees to enter the pipe autonomously at the same time and start fighting at the middle of the pipe at the same time. For this reason, a partition design was added to the device, but the experiment still failed. However, when the lower end of the partition becomes a polygon, so that a certain gap is formed between the partition and the circular inner bottom surface of the pipe, it is found that the bumblebee stay time is significantly extended, and most of them do not retreat, showing that the ingenious gap design stimulates the instinct of the bumblebee, which buys time for the second bee to arrive at the designated location with a delay, ensuring that the two bees can meet head-on at the designated location and the normal progress of subsequent experiments. After hundreds of manufacturing attempts, conflict test attempts and failure analysis, we found a way to eliminate the flying of insects and a technical method to eliminate the interference of environment and operation on bumblebees and this experiment. Finally, we successfully designed a device to detect social conflicts between bumblebee individuals, and through conflict test experiments, we successfully achieved the expected technical goals.

[0018] The present invention also provides a testing method for detecting social conflicts between bumblebee individuals using the above device, comprising the following steps:

[0019] S1. Sample and device preparation: Randomly select healthy, active, and uniform-sized bumblebees from the bumblebee social hive or the small colony established by the experimenter, mark them with colored stickers, and starve them individually in plastic cages for 30-60 minutes; remove the cylindrical pipes with vertical cuts in the device from the two small boxes, replace them with complete cylindrical pipes without vertical cuts, and after installation, disinfect them for use;

[0020] S2. Adaptation stage

[0021] For the worker bees to be tested, a single bumblebee is gently moved into the free exploration area on one side of the device under dark conditions with the lights off, and allowed to crawl and explore freely for 30 seconds. Usually, the behavior of the bumblebee can be stabilized; then, under lighting conditions, the bumblebee is adapted to the tube and its behavior is encouraged. A single bumblebee autonomously and voluntarily shuttles back and forth in the tube 10 times under lighting conditions. Each time, a drop of sugar water is added to the circular tube mouth of the free exploration area on the other side as a reward for the insect's voluntary crossing of the tube. Tube adaptation generally takes 10 minutes; if 10 tube drillings are not completed within 10 minutes, the bumblebee is abandoned; after the scene adaptation is completed, the bumblebee can be put back into the hive or return to the small group established by the experimenter according to the experimenter's experimental purpose;

[0022] S3, testing phase:

[0023] The next day, the cylindrical tube with vertical cutouts was replaced, and the partition (5) was fitted with the vertical cutouts. Under dark conditions, the two randomly paired bumblebees were moved to the free exploration areas on both sides respectively, and allowed to adapt for another 1 minute. Then, under light conditions, the camera video recording was turned on. The two bumblebees being tested were able to enter the tube on their side autonomously, voluntarily, and relatively quickly. When the two bumblebees met at the partition, the partition was gently lifted with a rope. The video recorded the pushing behavior of the two bumblebees in the tube, the winning and losing results, and the fighting time until the end of the conflict. The bumblebee pushed out of the tube was the loser, and vice versa, the winner. The test phase was set to 5 minutes. After the test was completed each day, the bumblebees could be put back into the hive or returned to the small group established by the experimenter according to the experimenter's experimental purpose. The number of conflict tests and the number of test days within a day could be determined according to the needs of the experimenter.

[0024] S4. Data collection and analysis: Video image data is transmitted to a computer in real time. Adobe Premiere software is used to analyze and define behaviors frame by frame, and to quantify the number of occurrences of various behaviors, their duration, and win / loss results. This behavioral data analysis can be used to analyze the process of win / loss.

[0025] During the adaptation phase, the bumblebees adapted to a complete cylindrical tube without vertical cuts. If a cylindrical tube with vertical cuts was used during the adaptation phase, the bumblebees would squeeze in the gap at the vertical cuts, not explore the other side of the tube, and not conflict with other bumblebees, resulting in implementation failure.

[0026] Compared with the prior art, the present invention has achieved beneficial effects:

[0027] 1) The present invention breaks through the limitations of existing methods and can for the first time conduct non-physical harm conflict testing between individuals in lower animal societies.

[0028] 2) The present invention can provide a paradigm or inspiration for inter-individual conflict testing in other lower animals.

[0029] 3) The present invention not only increases the incidence of conflict, but also makes the intensity of conflict controllable. The death and disability rate of bumblebees after the conflict is low, which is conducive to subsequent experiments. The types of conflict behaviors become simpler and objectively identifiable; conflict behaviors are easier to quantify. Therefore, the present invention provides a device and method that can quantify conflicts and easily determine the winners and losers.

[0030] 4) This method is novel in design, simple in manufacturing, low in cost, easy in operation, and has good repeatability, which greatly improves the experimental detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1It is a schematic diagram of the structure of the device of the present invention, wherein: 1- small box, 2- cylindrical pipe, 3- top cover, 4- vertical cutout, 5- partition, 6- thin rope;

[0032] Figure 2 This is a schematic diagram of the partition structure of the present invention;

[0033] Figure 3 Schematic diagram of various conflict behaviors and their duration between the winner and loser of two bumblebees paired based on this device

[0034] Figure 4 The number of occurrences and their respective durations of several behaviors of winning / losing bees in a small bumblebee colony during a conflict process obtained based on the device;

[0035] Figure 5 This is a ranking result diagram of the social hierarchy within a small group of bumblebees obtained based on the device;

[0036] Figure 6 This is a behavioral diagram of the victorious / losing bees after they return to their original society, where a represents biting the egg sac; b represents laying eggs; c represents drilling the wax cup; and d represents incubation. DETAILED DESCRIPTION

[0037] The technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0038] Embodiment 1: A detection device for detecting social conflicts between bumblebee individuals

[0039] like Figure 1 and Figure 2 As shown, this embodiment provides a device for detecting social conflicts between bumblebee individuals.

[0040] A device for detecting social conflicts between individual bumblebees, the device comprising a large box, two small boxes 1 are arranged in the large box, the two small boxes 1 are connected through a cylindrical pipe 2, the two small boxes 1 are free exploration areas, the middle cylindrical pipe 2 is a conflict area, the small boxes 1 are all open on the top surface, and the top surface is covered by a transparent top cover 3 with a card slot; a vertical cutout 4 is arranged on the middle position of the cylindrical pipe 2, and a partition 5 that can move up and down is installed at the vertical cutout 4; the lower end of the partition 5 is an irregular polygon, and a gap of 1-2mm is maintained with the inner side of the cylindrical pipe 2, and a thin rope 6 is attached to the partition. The large box is set to isolate external interference as much as possible.

[0041] Since the two insects cannot arrive at the middle of the test tube at the same time, in order to solve this technical problem, a partition made of specially installed transparent material plays a key regulatory function, ensuring that any insect that arrives at the position first can stay and wait in this area until the two meet and look at each other. A partition that can move up and down must be installed in the middle of the cylindrical pipe. It is made of transparent material, which is conducive to the two bumblebees to see each other. The lower end of the partition is structured into a polygon and maintains a gap of 1-2mm with the inner circular surface of the tube, which stimulates the bumblebee that arrives in the middle position in advance to squeeze the gap, so that it stays in the middle position and waits for the bumblebee that arrives later. Otherwise, it is difficult for the two animals to meet head-on in the middle position. The gap is also conducive to communication between animals that meet head-on. Once the animals meet head-on in the middle position, a thin rope attached to the top of the partition will be used to pull up the partition vertically.

[0042] In order to enable the bumblebees to adapt to the device and enter the cylindrical pipe as quickly as possible, the bottom surface of the cylindrical pipe opening is on the same level as the bottom surface of the small box.

[0043] The side and bottom of the small box are made of opaque materials, and the side is not light-transmissive; the top cover and cylindrical pipe of the small box are made of transparent materials; the bottom of the cylindrical pipe must be kept rough. The purpose of establishing a small opaque free exploration area is to stimulate and ensure that the small and slow-crawling bumblebees can quickly explore the pipe opening and voluntarily enter the pipe. The device also includes a camera, which is installed on the inner side of the large box, and the lens of the camera is kept at the same level as the cylindrical pipe. The small box is 5cm×5cm×5cm. The diameter of the cylindrical pipe matches the size of the bumblebee, and only one bumblebee is allowed to pass through at a time. The top cover of the small box is made of acrylic material with a thickness of 0.5cm and is made of transparent acrylic plate, so that the experimenter can easily observe the behavior trajectory of the insects in the guidance room.

[0044] The small box has smooth sides to prevent insects from climbing around and distracting attention; the bottom of the small box is rough to facilitate insects to crawl freely in the box; the small box is low to prevent them from flying.

[0045] The cylindrical pipe is made of transparent organic glass, with a length of 12 cm and a thickness of 0.1 cm. The partition is made of organic plastic with a thickness of 0.1 cm.

[0046] Example 2: Study on the influence of conflict victory on the social status of bumblebee workers based on the above-mentioned conflict device and the characteristics of bumblebee colonies

[0047] Bumblebees (B. terrestris) were purchased from Biobest, Belgium. The hives were moved into wooden boxes of 50 cm × 35 cm × 25 cm for breeding (nest boxes). Wooden boxes of 60 cm × 43 cm × 30 cm were used as flight boxes for free movement and foraging. The nest boxes and flight boxes were connected by a 20 cm long and 4 cm × 4 cm cross-section plexiglass channel. The bee colony was reared under the conditions of room temperature of 26 ± 2 °C, 50 ± 5% relative humidity, and a 12 h:12 h light and dark rhythm. Each bee colony was fed 5 g of pollen (Jiulong Bee Industry, Xuchang, China) and 200 ml of 50% sucrose solution per day. The experimental subjects were mainly experimental bumblebees that were normally raised in the nest. In order to avoid the rhythm affecting the state of the bumblebees, the experimental time was from 9:30 am to 20:00 pm every day.

[0048] 2.1 The test method for detecting social conflict between bumblebee individuals using the above device comprises the following steps:

[0049] S1. Sample preparation:

[0050] Worker bees with similar weight and size, good health and good activity were selected from the beehive in the harmonious period and marked, and divided into a conflict group (4) and a control group (4). Before adaptation, the device and the experimental background box were cleaned with detergent and 75% alcohol to remove impurities and odors. They were raised alone in a plastic cage and starved for 30-60 minutes.

[0051] S2, Adaptation stage:

[0052] For the worker bees to be tested, in the dark with the lights off, carefully and gently move a single insect into the free exploration area on one side of the device, and allow it to crawl and explore freely for 30 seconds. Usually, the insect behavior can be stabilized; during the adaptation process, always pay attention to the stress state of the bumblebees to be tested; then, under lighting conditions, carry out tube penetration adaptation (the tube does not have a vertical cut) and the incentive for this behavior. A single bumblebee autonomously and voluntarily shuttles back and forth in the tube 10 times under lighting conditions. Each time, a drop of sugar water is added to the circular tube mouth of the free exploration area on the other side as a reward for the insect's voluntary passage through the tube. Tube penetration adaptation generally takes 10 minutes. If 10 tube drillings are not completed within 10 minutes, the animal will be abandoned. After the scene adaptation is completed, the bumblebees can be returned to the hive or returned to the small group established by the experimenter separately according to the experimenter's experimental purpose.

[0053] S3, testing phase:

[0054] The next day, the pipe was replaced with a vertical cutout, and the partition (5) was fitted with the vertical cutout. Under dark conditions, the two randomly paired bumblebees were moved to the free exploration area on both sides to adapt for another 1 minute. Then, under light conditions, the camera video was turned on to record the video. The two bumblebees tested were able to enter the pipe on their side autonomously, voluntarily, and relatively quickly. When the two worker bees met at the partition, the partition was gently lifted with a rope. The video recorded the pushing behavior of the two bumblebees in the pipe, the winning and losing results, and the fighting time until the end of the conflict. The bumblebee that was pushed out of the pipe was the loser, and vice versa, it was the winner. The test phase was set to 5 minutes;

[0055] After each day's test, the bumblebees were returned to the hive. The test lasted for at least 4 days. Before the next test, they were allowed to rest in the breeding box for one hour to reduce the potential direct impact of recent wins and losses. A circular design was used to randomize the test order to ensure the accuracy and reliability of the experimental results. The conflict group worker bees were tested in pairs in the conflict tube for 4 days, and each conflict test usually took less than 1 minute; the control group worker bees also needed to go through the same conflict device, but the difference was that the control bees went through the tube alone and did not meet their opponents, which usually took less than 30 seconds.

[0056] The order of the two workers in each conflict test should be randomly assigned. Use the following cycle design to randomize the test order (A, B, C, and D are the numbers of the workers being tested): Day 1: AB, CD, BC, AD, BD, AC; Day 2 AD, CB, DC, BA, AC, DB; Day 3 DB, CA, AD, BC, CD, AB; Day 4 DC, AB, BD, AC, DA, CB. For ease of recording, this is indicated by “>”. After each test, both the conflict and control workers need to rest for one hour before starting the next test to reduce the potential direct impact of recent wins or losses. For example, after testing AB and CD on the first day, let all bees rest before testing BC. Repeat the above steps for the next 3 days of testing. The ranking on the first day is not fixed and may change in the next few days, so a 4-day test cycle is needed to fix the consequences of the conflict (the ranking of the winners and losers). In rare cases, neither bumblebee will retreat within 1 minute; in this case, the conflict test should be stopped; adjustments should be made based on the specific situation. If all worker bees maintain the same ranking for 3 consecutive days, it is considered that a stable ranking has been obtained. Conflict victory and conflict failure are evaluated based on the number of victories over the same experimental group. In the 4-day conflict test, the bee with the most wins is the conflict victory bee, and the bee with the least wins is the conflict failure bee. The test results and time of each trial, as well as the conflict victory bees and conflict failure bees, were recorded. After each test, the experimental device was cleaned with 75% ethanol.

[0057] S4. Data collection and analysis

[0058] The video image data was transmitted to the computer in real time, and Adobe Premiere software was used to analyze and define the behaviors in the tube frame by frame (see Table 1 below), as well as to quantify the number of occurrences of various behaviors, their duration, and the results of wins and losses. This behavioral data analysis can be used to analyze the process of wins and losses. After the experiment ended for 4 days, a ranking change chart was drawn based on the number of wins each day. Finally, the single-factor variance method was used to analyze the number and time of occurrence of various behaviors.

[0059] Table 1. Definition of simple behaviors during bumblebee conflicts

[0060]

[0061] This embodiment breaks through the technical bottleneck and develops a research paradigm for conflict behavior testing of low-level social insects, as well as a method for evaluating the social hierarchy among worker bees in a small colony, and for the first time evaluates the impact of conflict victory on the social status of bumblebee workers themselves.

[0062] 2.2 Analysis of research results:

[0063] 2.2.1 Ranking chart after conflict test

[0064] like Figure 3 As shown, through the conflict device, a ranking level diagram for four days is obtained. It can be seen that although the ranking of the tested bumblebees fluctuates, the rankings of three days in the four-day test are the same, and its overall ranking remains unchanged.

[0065] 2.2.2 The device only induces simple, objectively discernible conflict behaviors

[0066] The behaviors in the tube were analyzed frame by frame using Adobe Premiere software to identify the types of conflict behaviors and quantify the duration of these simple conflict behaviors. The behaviors in the tube can be divided into the following five categories:

[0067] 2.2.3 Quantitative analysis of conflict behaviors within the device

[0068] like Figure 4 and Figure 5 The results show that the behaviors of the conflict winners and the conflict losers in the conflict zone are different, mainly including the following three situations: the first is that the conflict winner initiates the pushing first, and the worker bees of the conflict losing group retreat. Although the loser bees push back for a few seconds, they are eventually pushed out of the tube by the winner bees; the second is that the loser bees actively initiate the attack, and the worker bees of the conflict winning group push back and win the final victory. During this process, the loser bees stop more often; the third is that the worker bees of the conflict winning group initiate the pushing, and the loser bees only retreat without other behaviors.

[0069] By counting the number and time of the above behaviors, it was found that the workers in the conflict victory group were more inclined to initiate pushing, with 1.64±0.10 times and 7.56±0.44s in each test, while the workers in the conflict failure group had 0.56±0.10 times and 1.10±0.23s in each test. There were extremely significant differences in the number and time (P<0.001). There was no significant difference in the number and time of counter-pushing between the workers in the conflict victory group and the workers in the conflict failure group (P>0.05, Figure 5 b). The retreat behavior of the worker bees in the conflict failure group was significantly different from that in the conflict victory group (P<0.001) ( Figure 5 c). There was a difference in the number of immobility occurrences between the conflict victory group and the conflict failure group (P<0.05), but no difference in the duration (P>0.05, Figure 5 d) 2.2.2.4 Behavioral changes of the conflict winner after returning to the nest

[0070] As we all know, the queen bee is the only egg-laying bee and occupies the highest dominant position. Worker bees cannot lay eggs and can only engage in nest care such as raising and foraging, and are in a subordinate position (Duchateau and Verthuis, 1988; Cnaani et al., 2000).

[0071] The present invention determines the impact of conflict victory on the social status of bumblebee worker bees by evaluating the behavior of worker bees after returning to the nest after the conflict. It was observed that 10 worker bees in the blank group in the nest showed normal care behaviors such as incubation and wax cup drilling in the nest; 5 queen bees showed egg-laying behavior in the nest. After returning to the nest, 40 control bees that did not experience conflict showed normal egg-biting and egg-laying behaviors, 100% showed wax cup drilling, and 100% showed normal incubation; after returning to the nest, 20 worker bees in the conflict failure group had no egg-biting and egg-laying behaviors, 75% showed wax cup drilling, and 90% showed normal incubation; however, after returning to the nest, 20 conflict victory bees showed 40% biting egg bags, 25% showed egg laying, and no normal incubation and other care behaviors. These results show that the experience of victory in fighting conflicts has actually improved the social status of worker bees themselves, and they can lay eggs, similar to queen bees. However, worker bees with failure experiences still show care behaviors such as incubation, and are in a subordinate position. This is the first time we have found that the experience of conflict victory can improve an animal's status within the hive society (see Figure 6 ).

[0072] Compared with the prior art, the present invention has achieved beneficial effects:

[0073] 1) The present invention breaks through the limitations of existing methods and can conduct non-physical harm conflict tests between individuals in lower animal societies for the first time, which will promote research in related fields.

[0074] 2) The present invention can provide a paradigm or inspiration for inter-individual conflict testing of other lower animals.

[0075] 3) The present invention not only increases the incidence of conflicts, but also makes the intensity of conflicts controllable. The types of conflict behaviors become simpler and more objectively identifiable; conflict behaviors are easier to quantify. Therefore, the present invention provides a device and method that can quantify conflicts and easily determine the winners and losers.

[0076] , 4) Based on the conflict device developed by the inventor and the advantages of bumblebee social research discovered by the inventor, the inventor can not only use it to explore the impact of social conflict and its winner / loser effect at the individual level and small group level; more importantly, combined with the observability of bumblebee society, using the device developed by the inventor, it is also possible to explore the impact of individual conflict on its own social status and on the original society of the hive at a more macro level.

[0077] 5) This method is novel in design, simple in manufacturing, low in cost, easy in operation, and has good repeatability, which greatly improves the efficiency of experimental detection.

Claims

1. A device for detecting social conflicts between bumblebee individuals, characterized in that: The device comprises a large box, in which two small boxes (1) are arranged, and the two small boxes (1) are connected through a cylindrical pipe (2). The two small boxes (1) are free exploration areas, and the middle cylindrical pipe (2) is a conflict area. The small boxes (1) are all open on the top surface, and the top surface is covered by a transparent top cover (3) with a slot; a vertical cutout (4) is arranged on the upper middle part of the cylindrical pipe (2), and a partition (5) that can move up and down is installed at the vertical cutout (4); the lower end of the partition (5) is irregularly shaped and must maintain a gap of 1-2 mm with the inner side of the cylindrical pipe, and a thin rope (6) is attached to the partition (5).

2. The device for detecting social conflicts among bumblebee individuals according to claim 1, characterized in that: The bottom surface of the cylindrical pipe opening is maintained at the same level as the bottom surface of the small box.

3. The device for detecting social conflicts among bumblebee individuals according to claim 1, characterized in that: The side and bottom surfaces of the small box are made of opaque white material, and the bottom and sides are opaque; the top cover and cylindrical pipe of the small box are made of transparent material, which is transparent and visible; the bottom surface of the cylindrical pipe must be kept rough.

4. The device for detecting social conflicts among bumblebee individuals according to claim 1, characterized in that: The device also includes a camera, which is installed on the inner side of the large box, and the camera lens is kept at the same level as the cylindrical pipe.

5. The device for detecting social conflicts among bumblebee individuals according to claim 1, characterized in that: The small box is 5cm×5cm×5cm.

6. The device for detecting social conflicts among bumblebee individuals according to claim 1, characterized in that: The diameter of the cylindrical pipe must match the size of the bumblebee and only allow one bumblebee to pass through.

7. A method for detecting social conflicts between bumblebee individuals using the detection device according to any one of claims 1 to 6, comprising the following steps: S1. Sample and device preparation: Randomly select healthy, active, and uniform-sized bumblebees from the bumblebee social hive or the small colony established by the experimenter, mark them with colored stickers, and starve them individually in plastic cages for 30-60 minutes; remove the cylindrical pipes with vertical cuts in the device from the two small boxes, replace them with complete cylindrical pipes without vertical cuts, and after installation, disinfect them for use; S2. Adaptation stage For the worker bees to be tested, under dark conditions with the lights off, the experimenter gently moves a single worker bee to be tested into the free exploration area on one side of the device, and crawls and explores freely for 30 seconds. Usually, the bumblebees do not show any stressful behavior. Next, under lighting conditions, the bumblebees are adapted to the tube penetration. A drop of sugar water is added to the circular tube mouth of the free exploration area on the other side. A single worker bee autonomously and voluntarily shuttles back and forth in the tube 10 times under lighting conditions. Tube penetration adaptation generally takes 10 minutes. If the bumblebee fails to complete 10 tube penetrations within 10 minutes, it will be abandoned. After the scene adaptation is completed, the bumblebee is returned to the hive or returned to the small group established by the experimenter according to the experimenter's experimental purpose. S3, testing phase: The next day, the cylindrical tube with a vertical cutout is replaced, and the partition (5) is matched with the vertical cutout. Under dark conditions, the two randomly paired bumblebees are moved to the free exploration areas on both sides respectively, and allowed to adapt for another 1 minute. Then, under light conditions, the camera video recording is turned on. The two bumblebees being tested can autonomously, voluntarily, and relatively quickly enter the tube on their side. When the two bumblebees meet at the partition, the partition is gently lifted with a rope, and the video records the pushing behavior of the two bumblebees in the tube, the winning and losing results, and the fighting time at the end of the conflict. The bumblebee pushed out of the tube is the loser, and vice versa, the winner. The test phase is set to 5 minutes. After the test is completed each day, the bumblebees are returned to the hive or return to the small group established by the experimenter according to the experimenter's experimental purpose. The number of conflict tests and the number of test days within a day can be determined according to the needs of the experimenter. S4. Data collection and analysis: Video image data is transmitted to a computer in real time. Adobe Premiere software is used to analyze and define behaviors frame by frame, and to quantify the number of occurrences of various behaviors, their duration, and win / loss results. This behavioral data analysis is used to analyze the process of win / loss.

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