A device for long-term and quantitative monitoring of bumblebee autonomous feeding behavior and a manufacturing method thereof

Abstract

The present invention relates to a device for long-term and quantitative monitoring of the autonomous feeding behavior of bumblebees and a manufacturing method thereof, and belongs to the technical field of insect feeding behavior. The device is a box body, and the box body is composed of two boxes with the same bottom but different heights that can be detachably connected. The upper higher box body is a living chamber, and the lower shorter box body is a water chamber. A top cover is provided above the living chamber, and ventilation holes are provided on the top cover and the side of the living chamber. A humidification port is provided on the bottom of the living chamber, and a plurality of micro-feeding tube mounting holes are provided below the side of the living chamber. The micro-feeding tube mounting holes are installed with micro-feeding tubes. The device first solves the influence of restrictive stress and restrictive movement on the life span of insects under single breeding conditions by simulating the living environment of bumblebees, and lays a key technical foundation for the experiment; at the same time, the micro-feeding tubes that are horizontally installed and specially processed solve the problem that it is difficult to accurately quantify the autonomous feeding of bumblebees, which are large insects with large food intake. The present invention is novel in design, simple in manufacture, low in cost, and easy to operate.

Description

Technical Field

[0001] The invention relates to a device for long-term and quantitative monitoring of the autonomous feeding behavior of bumblebees and a manufacturing method thereof, belonging to the technical field of insect behavior science. Background Art

[0002] Bumblebees are important pollinators of many wild plants and crops, and play a very important role in maintaining biodiversity and global food security. At present, there are about 250 known species of bumblebees in the world. Given the important role of pollinating insects and the current sharp decline in the population of pollinating insects, bumblebees, it is very important to study the feeding behavior of pollinating insects, which is related to the health of species. At present, bumblebees have become an important research object in various ecological studies (Goulson, 2010). Among them, the European ground bumblebee (Bombus terrestris) is commercially applied worldwide, bringing huge economic benefits to global facility agriculture (Velthuis and Van Doorn, 2006). Bumblebees are also becoming a new model animal for exploring the neural mechanisms of complex cognitive behaviors (Sadd et al., 2015). At present, it has been proved through the European ground bumblebee that lower animals have a variety of advanced cognition. Dayman found that European ground bumblebees can spontaneously solve the rope-pulling problem (Dayman, 2020); 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-recognition (Ravi et al., 2020; Brebner and Chittka, 2021). Because bumblebees live in a gregarious society similar to humans and are large individuals, they are suitable for pharmacological or neurobiological research and have an advantage in the study of social mechanisms.

[0003] Previously, a device for quantitatively monitoring the autonomous feeding behavior of Drosophila was developed for Drosophila of the order Diptera. It can accurately measure food intake at the individual level and has been used in various Drosophila experiments, including the study of nutritional balance, taste discrimination, metabolism and addiction behavior of Drosophila. However, this standard device consists of a tiny finger-shaped tube, a tiny water tank and a micro-capacity micro-feeding tube (5μl). The micro-feeding tube must be installed vertically on the top surface of the tiny finger-shaped tube, with the feeding port facing vertically downward. A small amount of liquid is stored in the tube by the surface tension of the micro-feeding tube without dripping. When the fruit fly licks the liquid food, it moves down by its own gravity. The volume of the sucked liquid is accurately calculated by measuring the length of the liquid surface with a vernier caliper. The device is only suitable for insects such as Drosophila, which are tiny and eat a small amount of food every day. Our analysis found that since the insect needs to crawl down along the smooth and fine glass tube to the feeding port, this places high demands on the ability of the fruit fly to operate the smooth and fine glass tube. This vertically mounted glass microfeeding tube can be manipulated with relative ease by flies with normal locomotion. However, the operational challenges posed by this setup preclude the assessment of relevant feeding behaviors in flies with locomotion defects, either naturally occurring or genetically engineered.

[0004] In 2016, Katz et al. tried to make a quantitative feeding device for bees. However, after our experiments, we found that the micro-feeding tube feeding device for bees invented by Katz et al. has the following defects: 1) It does not take into account that bees are of medium size and usually have a large activity space. The very narrow device space will cause bees to face strong restrictive stress, which will seriously inhibit the normal flight, crawling and other motor activities of bees, thereby affecting their feeding motivation. 2) It is well known that restrictive stress will lead to high stress levels in animals. Animals are always in an unhealthy state such as stress, which is easy to induce death and completely cannot meet the requirements of long-term experiments. As the bumblebee feeding experiment published in the world's top magazine Nature recently showed, the narrow device affects the lifespan and survival rate of bumblebees (Kessler et al. Nature, 2015, Bees prefer foods containing neonicotinoid pesticides). 3) Katz et al. took into account that medium-sized bees have a large food intake, and they used a glass micro-feeding tube with a larger diameter to load a large volume of food at one time. In order to prevent the droplets from dripping naturally due to gravity, the designer ensured that the feeding port was "vertically upward" through the U-design of the pipe and the 120-degree bend design, thus ensuring that the liquid food would never drip due to gravity. However, experimental studies have shown that bees cannot grasp smooth surfaces as easily as fruit flies. Therefore, the difficult operation on the vertical, fine, and smooth micro-feeding tube will seriously interfere with their feeding behavior and also affect the accurate assessment of their food intake. Bees with movement defects cannot be evaluated using this device; in addition, although this design of the glass tube can hold a large volume of food and does not drip naturally, it directly causes a large amount of food to be deposited in the U tube. The bee's beak cannot touch the liquid surface of the food in this state, and it cannot be ingested. The bee cannot eat the entire tube of food. At the same time, this design greatly increases the difficulty, accuracy and workload of data measurement. Therefore, the device has defects in operational convenience and precision measurement. The difficulty of operating the device also causes bees in poor health or with movement defects to be unable to complete feeding behavior, resulting in experimental failure. It can be seen that the design of the device is a failure. In addition, bumblebees are large insects with large activity spaces and larger daily food intake. In addition, the inventor's laboratory research found that bumblebees cannot effectively grasp and cling to smooth surfaces like fruit flies, and manipulating glass micro-feeding tubes is a daunting challenge for bumblebees. This feature affects their feeding behavior and thus affects the accurate assessment of their food intake. Therefore, long-term and quantitative monitoring of bumblebee autonomous feeding behavior remains a big problem.

[0005] In summary, the devices developed by predecessors are only suitable for fruit flies or medium-sized bees with small individuals and small appetites, and are not suitable for large insects with large appetites. Long-term and precise quantitative devices involving large insects are still lacking. In order to monitor the autonomous feeding behavior of bumblebees in a long-term and quantitative manner, a device suitable for the autonomous feeding choices of large individuals such as bumblebees is needed. First, the problem of low insect activity and high mortality caused by restrictive stress needs to be solved. Then, the problem of the incompatibility between the existing precise quantitative capillary device and the operating characteristics of bumblebees needs to be solved, which in turn affects the normal feeding behavior and thus affects the reliability of the experimental results. At present, there is no device manufactured by a manufacturing method that can meet the above experimental requirements. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a device for long-term and quantitative monitoring of the autonomous feeding behavior of bumblebees and a manufacturing method thereof. The manufactured device can solve the above technical problems.

[0007] A device for long-term, quantitative monitoring of the autonomous feeding behavior of bumblebees, the device is a box, the box is composed of two boxes with the same bottom but different heights that can be detachably connected, the higher box on top is a living chamber, the lower shorter box on the bottom is a water chamber, a top cover is provided above the living chamber, ventilation holes are provided on the top cover and the side of the living chamber, a humidification port is provided on the bottom of the living chamber, a plurality of micro-feeding tube mounting holes are provided at the lower side of the living chamber, and micro-feeding tubes are installed in the micro-feeding tube mounting holes.

[0008] Further, the feeding port end of the micro-feeding tube is installed horizontally with the bottom of the living room. The installation angle of the micro-feeding tube should be close to horizontal installation, and cannot be installed vertically on the top surface of the living room, otherwise the liquid in the tube will drip due to gravity.

[0009] Furthermore, a first bending portion is formed by bending at 1-2 cm from the feeding mouth end, and the angle between the feeding mouth end and the first bending portion is 90°. A second bending portion is formed by bending at 1.3-1.5 cm from the first bending point, and the second bending portion forms an angle of 15° with the horizontal.

[0010] Further, the microfeeding tube is inserted into the microfeeding tube mounting hole and further fixed with tape or light clay.

[0011] Preferably, the box body is made of a transparent organic glass plate or other transparent materials to facilitate naked eye observation and video recording.

[0012] Furthermore, the bottom surface of the living room is frosted to keep it rough, and a honeycomb-like structure is placed on the bottom surface to facilitate insects to live and walk on it; the inner side of the living room is installed with cardboard with a nest-like color and a rough surface to simulate the scene inside the nest, ensuring that the insects can live, climb and fly in the living room, live comfortably, reduce the stress caused by walking inconvenience, and avoid affecting their exploration motivation. The water chamber provides humidity guarantee. The two inner sides of the living room are installed with rough cardboard to promote the upward crawling movement of bumblebees, improve the overall activity of bumblebees, help bumblebees stay healthy, and facilitate the acquisition of higher quality experimental results.

[0013] Preferably, the living room has a size of 12 cm long × 12 cm wide × 15 cm high, and the water room has a size of 12 cm long × 12 cm wide × 5 cm high.

[0014] Preferably, the micro-feeding tube installation hole is located at a position 0.4 cm from the bottom surface on the side of the living chamber, and the diameter of the micro-feeding tube installation hole is 1 mm-1.2 mm. The height is determined according to the body height of the bumblebee insect crawling horizontally, so as to avoid causing feeding obstacles to the bumblebee and allow the bumblebee to easily stretch its beak to feed.

[0015] Preferably, the micro-feeding tube is made of high temperature resistant glass (e.g. borosilicate glass). To facilitate quantitative measurement and operation, the volume of a single micro-feeding tube should be controlled at about 80 microliters. To this end, the glass tube is 150 mm long, with an inner diameter of 0.84 mm and an outer diameter of 1 mm.

[0016] Furthermore, the device also includes a camera, and the camera is installed on the side of the device.

[0017] The present invention also provides a method for manufacturing a device for long-term and quantitative monitoring of the autonomous feeding behavior of bumblebees, comprising the following steps:

[0018] S1: Manufacturing of living room and water room: Provide two detachably connected transparent organic glass plates or other transparent material boxes with the same bottom and different heights, the tall box is on the top and the short box is on the bottom, the taller box on the top is the living room, and the shorter box on the bottom is the water room, and a top cover is provided on the living room;

[0019] S2: Ventilation holes, humidification ports and micro-feeding tube mounting holes are arranged on the top cover and sides of the living room. A plurality of ventilating holes are arranged on the bottom surface of the living room. A plurality of micro-feeding tube mounting holes with a diameter of 1 mm to 1.2 mm are arranged at 0.4 cm below the bottom surface of the living room side.

[0020] S3: Preparation of microfeeding tube: Prepare a capillary glass tube with a length of 150 mm, an inner diameter of 0.84 mm, an outer diameter of 1 mm, and high temperature resistance, and bend the capillary glass tube at 1-2 cm at one end to form a first bending portion, the angle between the one end of the capillary glass tube and the first bending portion is 90°, and bend the capillary glass tube at 1.3-1.5 cm away from the first bending point to form a second bending portion, the second bending portion is at an angle of 15° with the horizontal, to obtain a microfeeding tube;

[0021] S4: Installation of components Insert the microfeeding tube filled with food into the microfeeding tube installation hole, and ensure that the feeding port end of the microfeeding tube is installed horizontally, and further fixed with tape or light clay.

[0022] Preferably, the bottom surface of the living room is frosted and kept rough, and a honeycomb-like structure is placed in the living room; cardboard with a nest-like color and rough surface is installed on the two inner sides of the living room to stimulate the bumblebees to climb upwards and bite.

[0023] Preferably, when the micro-feeding tube containing food in S4 is filled with food, petroleum jelly is dripped into the non-feeding mouth end to reduce evaporation.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The device manufactured using this method firstly simulates the living environment of bumblebees, thus solving the negative effects of restrictive stress and restricted exercise on the activity and even lifespan of bumblebees under single-rearing conditions, laying a key technical foundation for subsequent experiments; at the same time, the micro-feeding tube, which is horizontally installed and processed with special processes, not only reduces the difficulty of feeding operation to zero, but also solves the problem of the difficulty of accurate quantification of autonomous feeding of bumblebees with larger individuals and larger food intake. The details are as follows:

[0026] 1) Through nearly a thousand repeated experiments, the present invention has taken a unique approach and innovatively proposed a horizontal installation mode for capillary glass tubes for the first time. The advantages of this installation completely eliminate the difficulty for bumblebees to operate vertical, fine, and smooth glass micro-feeding tubes. Even bumblebees with impaired motor ability can easily obtain food, thereby achieving full coverage of feeding or nutritional assessment of insects in various states.

[0027] 2) The horizontal installation of the micro-feeding tube in the device and the combination of its process structure can not only use capillary action to hold the liquid and prevent it from dripping, but also ensure that the microgravity can push the food to the feeding port. At the same time, the process structure ensures that the liquid food does not settle in the micro-feeding tube. At the same time, paraffin oil is used to seal the non-feeding end of the micro-feeding tube to minimize the evaporation of food, effectively ensuring the accuracy of volume measurement.

[0028] 3) By utilizing the inventor's solid neurobiological background and combining it with his long-term and careful observation and analysis of various neural behaviors of bumblebees under different scene conditions, the inventor has fully explored the complex instinctive behaviors of bumblebees and designed a minimum tall living chamber to stimulate their take-off motivation and flight behavior; the bottom surface of the living chamber needs to be kept rough to stimulate them to walk horizontally on the rough surface and reduce the stress caused by slipping; inspired by ergonomics, an irregular curved surface structure like a honeycomb is placed on the bottom surface, which is conducive to bumblebees lying or standing according to the natural curvature of the bumblebee's body, stimulating them to comfortably live and crawl on it to explore; the two inner sides of the living chamber are installed with cardboard with a nest-like color and a rough surface, which stimulates the bumblebee's climbing motivation and the maintenance of climbing movements, and stimulates the bumblebee's dental (upper forehead) biting behavior. In short, these environments stimulate the maintenance of the bumblebees' various instincts, allowing the bumblebees to still exhibit various movement behaviors similar to those in the nest inside the device, effectively reducing the negative impact of various restrictive mental stresses on the insects' motivation, physiology, and lifespan, and greatly improving the insects' welfare, thereby ensuring that the experiment can run stably and long-term, and obtaining more reliable experimental data that is closer to the natural state.

[0029] 4). This device can ensure the short-term or long-term survival rate and activity of the observed bumblebees, which is the basis for ensuring that the experimental data is closer to the actual situation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a structural schematic diagram of the device of the present invention;

[0031] Figure 2 This is a diagram of the structure of the microfeeding tube;

[0032] Figure 3 To use this device, various behavior state diagrams of bumblebees are shown, among which, Figure 3 A represents a picture of a bumblebee perching on a nest-like structure. Figure 3 B represents a bumblebee climbing on a nest-like structure. Figure 3 C represents the flying state of bumblebees in the living room; Figure 3 D represents a picture of a bumblebee climbing on a rough cardboard in the color of a nest. Figure 3 E represents a bumblebee lying on a rough cardboard of a similar nest color to practice its teeth (biting the cardboard with its upper forehead). Figure 3 F means that the bumblebee can operate the smooth, fine micro-feeding tube without any obstacles and easily and complete the feeding state diagram;

[0033] Figure 4 A comparison diagram of the device in the literature and the device of the present invention; Figure 4 A is a graph showing the survival rate of bumblebees in a feeding experiment using the device of the present invention during a 2-day adaptation period and a 6-day experiment. Figure 4 A; Figure 4B is a graph showing the survival rate of bumblebees fed with thiamethoxam during the 2-day adaptation period and the 6-day experiment using the device of the present invention; Figure 4 C is a schematic diagram of the device usage status in the literature; Figure 4 D is for utilization Figure 4 Bumblebee survival rate after 24 hours in the device in C;

[0034] Figure 5 This is a diagram of the experimental results of using the device manufactured by the method to monitor whether bumblebees have a preference for neonicotinoid pesticides (imidacloprid), wherein: Figure 5 A represents the result diagram of bumblebees' preference when faced with pesticide-containing food and normal food in the device; Figure 5 B shows that based on the data in Figure A, the PI index of the experimental group and the control group, which was averaged over 6 days, was obtained, and both groups showed no preference. Figure 5 C represents the food intake results of bumblebees when faced with food containing pesticide (imidacloprid) and normal food in the device;

[0035] Figure 6 This is a diagram of the experimental results of using the device manufactured by the method to monitor whether bumblebees have a preference for neonicotinoid pesticides (thiamethoxam), wherein: Figure 6 A represents the result diagram of bumblebees' preference when faced with pesticide-containing food and normal food in the device; Figure 6 B represents the PI index of the experimental group and the control group obtained for 6 days based on the data in Figure A, both of which showed no preference formed; Figure 6 C represents the food intake results of bumblebees when faced with food containing pesticide (thiamethoxam) and normal food in the device;

[0036] Figure 7 This is a diagram of the experimental results of using the device manufactured by this method to monitor whether bumblebees have a preference for theanine, wherein: Figure 7 A means that when bumblebees are choosing between food containing high concentration of theanine and normal food in the device, it is observed whether the bumblebees can voluntarily and actively show immediate and long-term preference for food containing theanine; Figure 7 B represents the preference of bumblebees for theanine-containing food and normal food in the device;

[0037] Figure 8 The diagram is a survival rate diagram of bumblebees in a feeding experiment using the device of the present invention during a 2-day adaptation period and a 20-day theanine experiment.

[0038] Among them: 1-living chamber, 2-water chamber, 3-ventilation hole, 4-humidification port, 5-micro-feeding tube installation hole, 6-micro-feeding tube, 7-top cover, 61-feeding port end, 62-first bending part, 63-second bending part, 64-non-feeding port end, 8-honeycomb-like structure, 9-cardboard with a nest color and rough surface.

[0039] Example 1 A device for long-term, quantitative monitoring of bumblebee autonomous feeding behavior and its manufacturing method

[0040] Reference Figure 1 and Figure 2 As shown, a device for long-term and quantitative monitoring of the autonomous feeding behavior of bumblebees, the device is a box, the box is composed of two boxes with the same bottom but different heights that can be detachably connected, the higher box on the top is a living chamber 1, and the lower box on the bottom is a water chamber 2, a top cover 7 is provided on the top of the living chamber 1, and ventilation holes 3 are provided on the top cover and the side of the living chamber 1, a humidification port 4 is provided on the bottom of the living chamber, and a plurality of micro-feeding tube installation holes 5 are provided below the side of the living chamber, and the micro-feeding tube installation holes are installed with micro-feeding tubes 6. The feeding port end 61 of the micro-feeding tube 6 is installed horizontally. The installation angle of the micro-feeding tube 6 should be close to horizontal installation, and it cannot be installed vertically on the top surface of the living chamber, otherwise the liquid in the tube will drip due to gravity. Furthermore, a first bending portion 62 is formed by bending at 1-2 cm of the feeding port end 61, and the angle between the feeding port end and the first bending portion is 90°. A second bending portion 63 is formed by bending at 1.3-1.5 cm from the first bending point, and the second bending portion 63 forms an angle of 15° with the feeding port end 61. The micro-feeding tube is inserted into the micro-feeding tube installation hole and further fixed with tape or light clay. The box body is made of a transparent organic glass plate or other transparent materials, which is convenient for naked eye observation and video recording. The bottom surface of the living room is frosted to keep it rough, and a honeycomb-like structure 8 is placed on the bottom surface to facilitate insects to live and walk on it; the inner side of the living room is installed with a cardboard 9 with a nest-like color and a rough surface to ensure that it can live, climb and fly in the living room, live comfortably, reduce the stress caused by walking inconvenience, and avoid affecting its exploration motivation. The water chamber provides humidity and air circulation needs for bumblebees to the maximum extent. The two inner sides of the living chamber are installed with rough cardboard 9 to promote the upward crawling movement of bumblebees, improve the overall activity of bumblebees, facilitate the bumblebees to stay healthy, and facilitate the acquisition of higher quality experimental results. The size of the living chamber is 12cm long × 12cm wide × 15cm high, and the size of the water chamber is 12cm long × 12cm wide × 5cm high. The micro-feeding tube installation hole is located at a position 0.4cm close to the bottom surface on the side of the living chamber, and the diameter of the micro-feeding tube installation hole is 1mm-1.2mm. The height is determined according to the body height of the bumblebee insect crawling horizontally to avoid causing feeding obstacles to the bumblebee, allowing the bumblebee to easily stretch its beak to feed, and the insect is easier to grab the feeding tube for feeding. The material of the large-capacity feeding tube must be a glass material that can withstand high temperatures (such as borosilicate glass). In order to facilitate quantitative measurement and operation, the volume of a single large-capacity glass capillary should be controlled at about 80 microliters. For this purpose, the glass tube is 150mm long, 0.84mm inner diameter, and 1mm outer diameter. The device also includes a camera, which is installed on the side of the device where no cardboard is installed in the living room.

[0041] A method for manufacturing a device for long-term and quantitative monitoring of bumblebee autonomous feeding behavior comprises the following steps:

[0042] S1, providing two detachably connected boxes with the same bottom but different heights, the tall box is on the top and the short box is on the bottom, the taller box on the top is the living room 1, the shorter box on the bottom is the water room 2, and a top cover 7 is provided above the living room;

[0043] S2: A plurality of ventilation holes 3 are provided on the top cover and the side of the living room, a plurality of humidification ports 4 are provided on the bottom of the living room, and a plurality of micro-feeding tube mounting holes 5 with a diameter of 1 mm to 1.2 mm are provided below the side of the living room at a distance of 0.4 cm from the bottom;

[0044] S3: prepare a capillary glass tube with a length of 150 mm, an inner diameter of 0.84 mm, an outer diameter of 1 mm, and high temperature resistance, and bend the capillary glass tube at 1-2 cm at one end to form a first bending portion 62, the angle between the one end of the capillary glass tube and the first bending portion is 90°, and bend the capillary glass tube at 1.3-1.5 cm away from the first bending point to form a second bending portion 63, the second bending portion forms an angle of 15° with the horizontal, to obtain a microfeeding tube 6;

[0045] S4: Install the micro-feeding tube 6 horizontally in the micro-feeding tube mounting hole 5 and further secure it with tape or light clay.

[0046] Furthermore, the micro-feeding tube 6 is inserted into the micro-feeding tube installation hole 5, and the feeding port end of the micro-feeding tube 6 is installed horizontally. The box body is made of a transparent organic glass plate or other transparent materials. The bottom surface of the living room is frosted to keep it rough, and a honeycomb-like structure 8 is placed in the living room; the two inner sides of the living room are installed with cardboard 9 with a nest-like color and a rough surface to simulate the scene inside the nest. The size of the living room is 12 cm long × 12 cm wide × 15 cm high, and the size of the water chamber is 12 cm long × 12 cm wide × 5 cm high. A camera is installed on the side of the living room that is not the cardboard. When the micro-feeding tube filled with food is filled with food, lime oil is dripped into the non-feeding port end 64 to reduce evaporation.

[0047] The device manufactured by the above method is as follows Figure 1 and Figure 2 shown.

[0048] The box body is made of transparent organic glass plate or other transparent materials, which is convenient for naked eye observation and video recording. The bottom surface of the living room is frosted to keep it rough, and a honeycomb-like structure is placed on the bottom surface, which is convenient for insects to live and walk on it; the inner side of the living room is installed with cardboard of a nest-like color and rough surface to simulate the scene inside the nest, so as to ensure that the bumblebee can live, climb and fly in the living room, live comfortably, reduce the stress caused by walking inconvenience, and avoid affecting its exploration motivation. The water chamber provides humidity and air circulation for the bumblebee to the maximum extent. Rough cardboard is installed on the two inner sides of the living room to promote the upward crawling movement of the bumblebee, improve the overall activity of the bumblebee, facilitate the bumblebee to stay healthy, and facilitate the acquisition of higher quality experimental results. The height of the micro-feeding tube hole is determined according to the body height of the bumblebee insect crawling horizontally, so as to avoid causing feeding disorders of the bumblebee, allow the bumblebee to easily extend its beak to feed, and the insect can more easily grab the feeding tube to feed. The micro-feeding tube must be made of high temperature resistant glass (e.g. borosilicate glass). To facilitate quantitative measurement and operation and to ensure that food does not spill, the volume of a single micro-feeding tube should be controlled within 80 microliters. To this end, the glass tube is 150 mm long, 0.84 mm in inner diameter, and 1 mm in outer diameter.

[0049] 1) Through nearly a thousand repeated experiments, the present invention has taken a unique approach and innovatively proposed a horizontal installation mode for capillary glass tubes for the first time. The advantages of this installation completely eliminate the difficulty for bumblebees to operate vertical, fine, and smooth glass micro-feeding tubes. Even bumblebees with impaired motor ability can easily obtain food, thereby achieving full coverage of feeding and nutritional assessment of insects in various states.

[0050] 2) The horizontal installation of the micro-feeding tube in the device and the combination of its process structure can not only use capillary action to hold the liquid and prevent it from dripping, but also ensure that the microgravity can push the food to the feeding port. At the same time, the process structure ensures that there is no sedimentation of liquid food in the micro-feeding tube. At the same time, paraffin oil is used to seal the non-feeding end of the micro-feeding tube to minimize the evaporation of food, effectively ensuring the accuracy of volume measurement.

[0051] 3) By utilizing the inventor's solid neurobiological background and combining it with his long-term and careful observation and analysis of various neural behaviors of bumblebees under different scene conditions, the inventor has fully explored the complex instinctive behaviors of bumblebees and designed a minimum tall living chamber to stimulate their take-off motivation and flight behavior; the bottom surface of the living chamber needs to be kept rough to stimulate them to walk horizontally on the rough surface and reduce the stress caused by slipping; inspired by ergonomics, an irregular curved surface structure like a honeycomb is placed on the bottom surface, which is conducive to bumblebees lying or standing according to the natural curvature of the bumblebee's body, stimulating them to comfortably live and crawl on it to explore; the two inner sides of the living chamber are installed with cardboard with a nest-like color and a rough surface, which stimulates the bumblebee's climbing motivation and the maintenance of climbing movements, and stimulates the bumblebee's dental (upper forehead) biting behavior. In short, these environments stimulate the maintenance of the bumblebees' various instincts, allowing the bumblebees to still exhibit various movement behaviors similar to those in the nest inside the device, effectively reducing the negative impact of various restrictive mental stresses on the insects' motivation, physiology, and lifespan, and greatly improving the insects' welfare, thereby ensuring that the experiment can run stably and long-term, and obtaining more reliable experimental data that is closer to the natural state.

[0052] 4) This device can ensure the short-term or long-term survival rate and activity of the observed bumblebees, which is the basis for ensuring that the experimental data is closer to the actual situation.

[0053] The device has novel design, simple manufacture, low cost, easy operation and is reusable.

[0054] Example 2 Comparison between the feeding device in the literature and the present device

[0055] The device was used to compare with the device in the literature to study the feeding behavior of bumblebees. It was found that the state of bumblebees in the device was closer to the natural state, such as perching, climbing, flying, biting, etc. Figure 3 A. Figure 3 B. Figure 3 C. Figure 3 D. Figure 3 As shown in E, Figure 3 F It can be seen that the bumblebees can operate the smooth micro-feeding tube without obstacles and easily and complete feeding. The bumblebees have zero difficulty in operating the micro-feeding tube, which eliminates the occurrence of inhibition of feeding behavior due to the difficulty of feeding operation. In this state of the device, the bumblebees are less stressed and the experimental data are more accurate.

[0056] Comparison of survival rates of individual bumblebees in the experiment, such as Figure 4As shown in the figure, during the 2-day adaptation period and the 6-day experimental period, the survival rate of the bumblebees in the control group (control, all tubes were filled with sugar water food only) in the device was 100%, and the survival rate of the bumblebees in the drug experimental group (1nM IMD+Food, the drug tube was filled with sugar water food containing 1nM imidacloprid, and the control tube was still filled with sugar water) was 100%. Figure 4 A; During the 2-day adaptation period and the 6-day experimental period, the survival rate of bumblebees in the control group (control) was 100%, and the survival rate of bumblebees in the drug experimental group (1nM thiamethoxam TMX+food) was 100%. Figure 4 B.

[0057] Figure 4 C shows a diagram of the device used in the literature, specifically a single bumblebee transferred to a single container. The container contains three large-volume 3 ml centrifuge tubes. There are four 2 mm holes on the centrifuge tubes, and the bees can stand on the centrifuge tubes and suck sugar water from the large opening. Each feeding tube is filled with deionized water, 0.5 M sucrose, or 0.5 M sucrose containing neonicotinoid compounds. 24 hours after the start of the experiment, observe whether the bees survive. Bumblebees that did not eat from the centrifuge tubes were removed. Note: These test bumblebees always die, possibly from stress or other reasons (see Kessler et al., 2015, Nature methods). The device in the literature (160 length X 110 width X 45 mm height, see Kessler et al., 2015, Nature). Bumblebees can only walk or perch on smooth bottom surfaces. Figure 4 As shown in D, the proportion of bumblebees that survived 24 hours after exposure to different drug concentrations of 0, 1, 10, 100, and 1000 nM in the device described in the literature.

[0058] Based on this device, in the 6-day low-concentration neonicotinoid pesticide experiment, no bumblebee individuals in the control group or the experimental group died. Figure 8 ), and no bumblebee individuals died in the control group and the experimental group. None of the bumblebee individuals died during the experiment as previously reported (Kessler et al., 2015, Nature). The combined features of the horizontal installation of the capillary feeding tube and the special process treatment allow bumblebees to select and obtain food without obstacles, and enable people to accurately measure the bumblebee food consumption in real time. At the same time, this combined feature ensures that food will not be deposited in the feeding tube, indicating that the design of this experimental device is successful and fits the living habits and living environment of bumblebees.

[0059] Example 3: Study on whether bumblebees can selectively feed on sugar water and neonicotinoid pesticides using the present device

[0060] Animal husbandry: European ground bumblebees (Bombus terrestris) were purchased from Biot, Belgium, as experimental animals. After the arrival of the colony, the hive was moved into a 50 cm × 35 cm × 25 cm wooden box for breeding (nest box), and a 60 cm × 43 cm × 30 cm wooden box was used as a flight box to simulate the natural open environment for the bumblebees to fly and forage freely. The nest box and the flight box 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. The lights were turned on from 8:00 am to 20:00 pm, and each 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. To avoid the rhythm affecting the bumblebee's state, the experimental time was from 8:00am to 20:00pm every day.

[0061] The specific experiment includes the following steps:

[0062] S1. Preparation stage: including the preparation of the device and the preparation of the bumblebees

[0063] Before the experiment, the device was cleaned with detergent and 75% alcohol to remove impurities and odors. After cleaning, nest-like structures and pollen balls were placed at the bottom of the living room; a certain volume of drinking water was filled into the water room;

[0064] Healthy and active bumblebees were randomly selected from the hive, taken out with tweezers, and cold anesthetized before being weighed, measured for chest width and body length, and other pretreatments; all bumblebees in the experimental and control groups came from three bee colonies.

[0065] S2, Adaptation stage:

[0066] Adaptation phase: At 7:30 in the morning, in a dark environment, cover a bumblebee with a black lid and carefully move it into the living room in the device. Fill the glass capillary with normal food, and drop a drop of paraffin oil at the far end (to reduce the evaporation of the liquid), insert it horizontally into the mounting hole of the living room and fix it (experimental group). At the same time, there is no bumblebee in the other device, but the glass capillary with food and paraffin oil is installed, which is set as the evaporation control group. Turn on the lights on time at 8:00 to conduct the experiment. At 8 o'clock in the evening, the experiment ends, the volume of food reduction in the experimental group is measured with a micrometer, and then the evaporation volume is subtracted. The food consumption of the bumblebee is obtained by calculation, and the data is recorded. The death of the bumblebees in the device is counted, and all microfeeding tubes are removed. All microfeeding tubes are cleaned and dried with alcohol and used again the next day. The adaptation period is 2 days; the calculation is consistent with the following.

[0067] S4. Experimental phase: The experimental period was set to last for 6 days. At 7:30 in the morning, under the condition of turning on the red light in the dark, the insects were covered with a black lid. In the living room, 4 micro-feeding tubes containing neonicotinoid pesticides were installed on one side, and 4 micro-feeding tubes containing control food were installed on the other side. At the same time, two control groups were set up: one group was a blank control group with ordinary food on both sides of the device, and the other group was an evaporation control group with the same conditions as the experimental group but without bumblebees. After the installation was completed, the lights were turned on on time at 8:00 for the experiment. The consumption of liquid was checked every 4 hours. When the liquid consumption exceeded 2 / 3 of the micro-feeding tube, the reduced volume of food was recorded and the micro-feeding tube was replaced; the above micro-feeding tubes all needed to be dripped with paraffin oil to reduce evaporation. After the last measurement at 8 o'clock in the evening, the reduced volume of food in the experimental group was subtracted from the evaporation volume, and the food consumption of the bumblebees was obtained by calculation, and the data was recorded; then all micro-feeding tubes were removed, cleaned and dried with alcohol for use the next day. In order to avoid spatial position preference and memory of experimental animals, the positions of the drug tube and the control food tube were swapped during the experiment the next day.

[0068] S5. Data statistics and analysis

[0069] The consumption of different foods and the total consumption of the experimental group were summed up after the end of each day's experiment, and the preference index (PI) of bumblebees for food containing neonicotinoid pesticides was calculated, that is, the preference index PI = (consumption of food containing neonicotinoid pesticides - ordinary food consumption) / total consumption, and the total food intake and PI of the blank control group were also counted (the PI of the blank control group is: the consumption of ordinary sugar water on the same side as the experimental group with neonicotinoid pesticides added - the consumption of sugar water on the opposite side) / total consumption). PI varies between -1 and +1, positive values ​​indicate preference, and negative values ​​indicate rejection. After the original data was entered into Excel, SPASS 26 data analysis was performed to obtain the proportion of the first choice of the drug tube and the average value, preference index, standard and P value of each food consumption, etc., and origin2021 was used to draw the graph. When the data of each group were compared within the same group, one-way repeated measures ANOVA was used to analyze the differences of the data of each group. P<0.05 indicated a significant difference, P<0.01 indicated an extremely significant difference, and 0.01 was extremely significant, respectively, indicated by "*" and "**".

[0070] Results Analysis

[0071] like Figure 5As shown in the figure, when the experimental group of bumblebees (1nM IMD+food group) were faced with the choice of pesticide-containing food and normal food in the device, it was observed whether the bumblebees could voluntarily and actively show immediate and long-term preference for food containing field-dose neonicotinoid pesticide imidacloprid (e.g. Figure 5 A). From the results of the PI index from 1 to 6 days, bumblebees cannot develop a preference for the neonicotinoid pesticide imidacloprid in the short or long term, but fluctuate around the "zero line" to show randomness. This result is almost consistent with the results of the control group. When the bumblebees in the control group are faced with normal food in all the tubes in the device, they naturally cannot form a preference for food intake. From the results of the PI index from 1 to 6 days, they generally fluctuate around the "zero line" to show randomness; Figure 5 B shows the PI index of the experimental group and the control group averaged over 6 days based on the data in Figure A, and both groups also showed no preference formed; Figure 5 C means that when the experimental group of bumblebees (1nM IMD+food group) were faced with the choice of pesticide-containing food and normal food in the device, there was no significant difference in the daily food intake of bumblebees in the two tubes (P>0.05). This data also shows from another perspective that bumblebees have no preference for the food containing the neonicotinoid pesticide imidacloprid. Note: Based on this device, through long-term, precise quantitative monitoring, it was found that bumblebees could not choose between sugar water food and food containing the neonicotinoid pesticide imidacloprid, that is, it was found that bumblebees could not have an immediate short-term preference or a long-term preference for the field dose of neonicotinoid pesticide imidacloprid food.

[0072] like Figure 6 As shown in the figure, when the bumblebees in the experimental group (1nM TMX+food group) were faced with the choice of food containing thiamethoxam and normal food in the device, it was observed whether the bumblebees could voluntarily and actively show immediate and long-term preference for food containing field doses of the neonicotinoid pesticide thiamethoxam (e.g. Figure 6 A). From the results of the PI index from 1 to 6 days, bumblebees cannot develop a preference for the intake of the neonicotinoid pesticide thiamethoxam in the short or long term, but only fluctuate around the "zero line" and show randomness. This result is almost consistent with the results of the control group. When the bumblebees in the control group are faced with normal food in all tubes in the device, they naturally cannot form a food intake preference. From the results of the PI index from 1 to 6 days, it generally fluctuates around the "zero line" and shows randomness. Based on the data in Figure A, the 6-day average PI index of the experimental group and the control group also showed that no preference was formed (see Figure 6B). When the experimental group of bumblebees (1nM IMD+food group) were faced with the choice of food containing thiamethoxam and normal food in the device, there was no significant difference in the amount of food consumed by the bumblebees in the two tubes per day (P>0.05). This data also shows from another perspective that bumblebees have no preference for food containing the neonicotinoid pesticide thiamethoxam (e.g. Figure 6 C). Note: Based on this device, through long-term, precise quantitative monitoring, it was found that bumblebees could not choose between sugar water food and food containing the neonicotinoid pesticide thiamethoxam, that is, it was found that bumblebees could not have an immediate short-term preference or a long-term preference for field doses of the neonicotinoid pesticide thiamethoxam.

[0073] Therefore, based on this device, through long-term, precise quantitative monitoring, it was found that bumblebees could not choose between sugar water food and food containing the neonicotinoid pesticides imidacloprid and thiamethoxam, that is, bumblebees could neither produce immediate short-term preferences nor long-term preferences for field doses of the neonicotinoid pesticides imidacloprid and thiamethoxam.

[0074] Example 4: Study on whether bumblebees can selectively feed between sugar water and theanine using the present device

[0075] The method of this embodiment is the same as that of embodiment 3, except that the low concentration of neonicotinoid pesticide imidacloprid or thiamethoxam is replaced with high concentration of theanine, and the experimental period is 2 days of adaptation and 20 days of testing. According to statistics, the survival rate of the tested bumblebees is still 100% (such as Figure 8 ).

[0076] The results are as follows Figure 7 As shown in the figure, when the bumblebees in the experimental group (1mM L-theanine+food) were faced with a choice between food containing high concentration of theanine and normal food in the device, it was observed whether the bumblebees could voluntarily and actively show immediate and long-term preference for food containing theanine. From the results of the PI index from 1 to 20 days, the bumblebees showed a strong preference for food containing theanine, and this result was significantly different from the results of the control group (such as Figure 7 A); When bumblebees in the experimental group (1mM L-theanine+food) were faced with the choice between food containing theanine and normal food in the device, it can be seen that in most cases, the amount of food containing amino acid produced by bumblebees was much greater than the amount of normal food consumed (e.g. Figure 7 B).

[0077] Based on this device, through long-term, precise quantitative monitoring, it was found that bumblebees can choose between sugar water food and sugar water food containing theanine, and can produce both immediate short-term preferences and long-term preferences.

Claims

1. A device for long-term, quantitative monitoring of bumblebee autonomous feeding behavior, characterized in that: The device is a box body, and the box body is composed of two boxes with the same bottom but different heights that are detachably connected. The higher box body on the top is a living chamber (1), and the shorter box body on the bottom is a water chamber (2). A top cover (7) is provided on the top cover and the side of the living chamber. A humidification port (4) is provided on the bottom of the living chamber. A plurality of micro-feeding tube mounting holes (5) are provided below the side of the living chamber. The micro-feeding tube mounting holes (5) are used to mount micro-feeding tubes (6), and the feeding port end (61) of the micro-feeding tube (6) is installed horizontally; the feeding port end (61) is bent at 1-2 cm from the feeding port end (61) to form a first bent portion (62), and the angle between the feeding port end and the first bent portion is 90°; the feeding port end (61) is bent at 1.3-1.5 cm from the first bent portion to form a second bent portion (63), and the second bent portion (63) forms an angle of 15° with the horizontal; The micro-feeding tube installation hole (5) is located on the side of the living chamber at a position 0.4 cm close to the bottom surface. The diameter of the micro-feeding tube installation hole (5) is 1 mm-1.2 mm. The micro-feeding tube is made of a high-temperature resistant capillary glass tube. The capillary glass tube has a length of 150 mm, an inner diameter of 0.84 mm, and an outer diameter of 1 mm.

2. The device for long-term and quantitative monitoring of autonomous feeding behavior of bumblebees according to claim 1, characterized in that: The bottom surface of the living room is frosted to keep it rough, and a honeycomb-like structure (8) is placed in the living room; two inner sides of the living room are installed with cardboard (9) with a similar color to the nest and a rough surface; and the water room is filled with drinking water.

3. The device for long-term and quantitative monitoring of autonomous feeding behavior of bumblebees according to claim 1, characterized in that: The living room has a size of 12 cm long×12 cm wide×15 cm high, and the water room has a size of 12 cm long×12 cm wide×5 cm high. The living room and the water room are made of transparent organic glass plates or other transparent materials.

4. The device for long-term and quantitative monitoring of autonomous feeding behavior of bumblebees according to claim 1, characterized in that: The device also includes a camera, which is installed on the side of the living room where no cardboard is installed.

5. A method for manufacturing a device according to any one of claims 1 to 4, comprising the following steps: S1: Fabrication of living and water chambers Two detachably connected transparent boxes made of organic glass or other transparent materials with the same bottom and different heights are provided, the tall box is on the top and the short box is on the bottom, the taller box on the top is the living room (1) and the shorter box on the bottom is the water room (2), and a top cover (7) is provided above the living room; S2: Ventilation holes, humidification ports, and microfeeding tube installation holes A plurality of ventilation holes (3) are arranged on the top cover and the side of the living room, a plurality of humidification ports (4) are arranged on the bottom of the living room, and a plurality of micro-feeding tube mounting holes (5) with a diameter of 1 mm to 1.2 mm are arranged below the side of the living room at a distance of 0.4 cm from the bottom; S3: Preparation of microfeeding tubes Prepare a high temperature resistant capillary glass tube with a length of 150 mm, an inner diameter of 0.84 mm, an outer diameter of 1 mm, and high temperature bending at 1-2 cm from one end of the capillary glass tube to form a first bending portion (62), the angle between the one end of the capillary glass tube and the first bending portion is 90°, and at a distance of 1.3-1.5 cm from the first bending portion, form a second bending portion (63), the second bending portion forms an angle of 15° with the horizontal, to obtain a microfeeding tube (6); S4: Installation of components Insert the micro-feeding tube (6) filled with food into the micro-feeding tube mounting hole (5), and ensure that the feeding port end of the micro-feeding tube (6) is installed horizontally, and further fixed with tape or light clay.

6. The method for manufacturing the device according to claim 5, characterized in that: The bottom surface of the living room is frosted to keep it rough; a honeycomb-like structure (8) is placed in the living room; and cardboard (9) with a honeycomb-like color and a rough surface is installed on the two inner sides of the living room.

7. The method for manufacturing the device according to claim 5, characterized in that: After the micro-feeding tube containing food described in S4 is filled with food, paraffin oil is dripped into the non-feeding port end (64).

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