Application of Theanine

Abstract

The present invention belongs to the technical field of insect protection, and relates to the application of theanine, and specifically to the use of theanine in preparing pollinating insect health products, and preventing or controlling pollinating insect pesticide poisoning products. The present invention first discovered that long-term theanine intake has no toxic side effects on pollinating insects, plays a health care role for normal pollinating insects, and can prevent and improve various damages of pesticides to pollinating insects, including movement disorders, olfactory learning and memory disorders, and social behavior disorders caused by neonicotinoid pesticides. The theanine provided by the present invention is highly safe and green, and does not show toxicity to humans, mice, and pollinating insects. It can not only promote the health of normal insects, but also improve the drugs for insect neonicotinoid pesticide poisoning. It provides a safe raw material for preparing drugs or feeds for pollinating agricultural insects damaged by neonicotinoid pesticides, and can induce insects to have a strong feeding preference, which is of great significance to the survival and reproduction of insects, and provides a new scientific basis and protection strategy for solving the global food crisis, the pollinating insect crisis, and the safety protection of biodiversity.

Description

Technical Field

[0001] The invention belongs to the technical field of insect protection, relates to the application of theanine, and specifically relates to the use of theanine in preparing products for insect health care, prevention or control of insect pesticide poisoning. Background Art

[0002] As we all know, 90% of wild plants and 75% of food crops depend on insect pollination. Pollinating insects are not only the guarantor of global food security, but also the maintainer of global biodiversity. However, the harm of pesticides is shocking, but it is a quick-acting method to control pests, a key tool to fight off insects for food, and a powerful safeguard for global food security. In the 17th century, humans used tobacco extracts as insecticides. In 1828, the active ingredient of the extract was determined to be nicotine. In 1904, nicotine was successfully synthesized and used to control pests such as fruit trees, vegetables and rice (Tomizawa & Casida, 2005). However, nicotine is more toxic to mammals than to insects, and has high environmental risks. In the 1970s, people began to optimize the structure of nicotine and develop new insecticides that are less toxic to mammals and do not have resistance to other insecticides (such as organophosphates and pyrethroids) (Tomizawa & Casida, 2001). In 1991, imidacloprid was first introduced to the market (Jeschke & Nauen, 2008). Imidacloprid is a systemic insecticide. The product has good rapid effect, with high protection effect one day after application, and a residual period of about 25 days. The successful development of this neonicotinoid insecticide, which is highly toxic to insects (both contact and stomach poison, highly selective), low toxicity to mammals (environmentally safe), and has no cross-resistance with other pesticides, immediately triggered a global boom in the research and development of compounds with similar structures. In 1993, in order to distinguish it from previous nicotine insecticides, people called the imidacloprid insecticides based on nicotine structure optimization as neonicotinoid insecticides. Neonicotinoid insecticides are a milestone in human insecticide research and modern agriculture. They occupy a major position in the global insecticide market, play a pivotal role in global food and agricultural security, and become the world's most successful, most effective and best-selling insecticides. Neonicotinoid insecticides are widely used around the world and in China.

[0003] Neonicotinoid insecticides have unique physical and chemical properties, which make them broad-spectrum and highly effective in killing insects. First, neonicotinoid insecticides are extremely non-volatile at room temperature, which facilitates the application of field spraying. Second, as polar insecticides, neonicotinoid insecticides have higher solubility and lower lipophilicity compared with other non-polar insecticides. Neonicotinoid insecticides are systemic and can be gradually distributed to the whole body tissues of the plant, thereby quickly killing pests that invade any part of the plant. The mechanism of action of neonicotinoid insecticides is that they selectively bind to acetylcholine receptors as nicotinic acetylcholine receptor (nAChRs) agonists, compete with insect acetylcholine (ACh), block the normal conduction of the central nervous system, and then cause insect paralysis and death. Because this type of insecticide has a unique mechanism of action, there is no cross-resistance with conventional insecticides.

[0004] Neonicotinoid insecticides have been on the market for nearly 30 years, and have undergone four iterations in the process of development, depending on the different substituents. The first generation of neonicotinoid drugs are chloronicotinoids, including imidacloprid, acetamiprid, and thiacloprid; the second generation of neonicotinoid insecticides are thiononicotinoids, including thiamethoxam and clothianidin; the third generation of neonicotinoid insecticides are furonicotinoids, including flubiprofen; the fourth generation of neonicotinoid insecticides are sulfenimines, including sulfoxaflor.

[0005] Sub-health of pollinators caused by the sub-lethal effects of pesticides will lead to behavioral and physiological changes, and their ability to adapt to changing environments will be reduced. Sub-health will inevitably lead to a decrease in the immunity of pollinators, which will cause insects to be infected with various diseases, including mass infectious diseases in insect nests, and finally lead to the collapse of individual pollinators and the entire bee colony.

[0006] The decline in the survival and reproduction capacity of pollinating insect populations will inevitably have a fatal impact on human safety. Therefore, neonicotinoid pesticides have become one of the major threats to pollinating insects. Under the threat of the urgent crisis of pollinating bees, food crisis, and plant diversity maintenance crisis, the European Union and other countries have begun to announce restrictions on the use of neonicotinoid pesticides. Opponents are still protesting against the ban and restrictive use of neonicotinoid pesticides. In 2013, the European Union issued a ban on the three neonicotinoid pesticides, imidacloprid, thiamethoxam and clothianidin; in November 2016, Canada proposed to eliminate all agricultural uses and most outdoor uses of imidacloprid, and put 19 products containing thiamethoxam and 16 products containing clothianidin under special review; in April 2018, the European Union banned the outdoor use of the three neonicotinoid pesticides, imidacloprid, thiamethoxam and clothianidin; in December 2018, France included five neonicotinoid pesticides, imidacloprid, thiamethoxam, clothianidin, thiamethoxam and acetamiprid, on the banned list; the U.S. Environmental Protection Agency canceled the registration of 12 neonicotinoid pesticide products that harm bees, which will take effect on May 20, 2019, including products from Syngenta and Bayer. The 12 canceled product registrations include 7 seed dressing agents, including clothianidin and thiamethoxam. Large chemical companies in Europe and the United States hope to develop alternative chemical insecticides (third-generation neonicotinoid insecticides) that are "harmless" to pollinating insects. However, more and more studies have shown that the newly developed third-generation and fourth-generation neonicotinoid insecticides with low toxicity can still be toxic to pollinating insects.

[0007] Combining the principles of zoology, evolutionary biology, neuroreceptor pharmacology, and neurobehavior, we know that the sub-lethal effects of pesticides will lead to sub-health of pollinators, which will bring about behavioral and physiological changes, and reduce their ability to adapt to changing environments. Sub-health will inevitably lead to a decrease in the immunity of pollinators, which will cause insects to be infected with various diseases, including group infectious diseases in the hive, and finally lead to the collapse of individual pollinators and the entire bee colony. The decline in the survival and reproduction ability of pollinator populations will inevitably have a fatal impact on human safety.

[0008] In summary, in this complex dilemma, we must find another new protection technology route for pollinating insects, so as to prevent and eliminate the toxicity of pesticides, protect pollinating insects, and find new protection ideas and protection technology routes.

[0009] The inventors conducted research on drug screening for insect resistance to field-dose neonicotinoid pesticide toxicity and designed a route for isolation and screening from plants. Fortunately, they discovered that theanine has an unexpected effect.

[0010] Theanine was first isolated from green tea by Japanese scholar Yajiro Sakato in 1950. Studies have found that theanine is very stable, resistant to high temperatures, acids, and alkalis, and can remain intact and non-decomposed. Theanine brings a fresh flavor. At the same time, based on experiments on higher mammals, it was found that it also has many physiological functions. For example, it can break through the blood-brain barrier and directly affect brain activity, thereby affecting people's emotions. This effect may also have a positive effect on human health physiologically. In 1964, Japan approved the use of theanine as a food additive. Theanine has the characteristics of sweet and fresh flavor. Theanine has excellent processing characteristics and stability, and can be widely used in snacks, candies and jellies, beverages, chewing gum, chocolate and other foods. Theanine also appears as a key ingredient in functional foods and beverages and dietary supplements, designed to relax the human body without causing drowsiness. The US FDA gave theanine the GRAS classification in 1985. GRAS (Generally Recognized as Safe) means "generally recognized safety", which means that theanine can be used freely and without limit in various foods. Various scientific experiments have fully proved that theanine has the characteristics of food raw materials, meets the necessary nutritional requirements, is non-toxic and harmless, and does not cause any acute, subacute, chronic or other potential harm to human health. It is particularly worth mentioning that a large number of experiments on higher mammals have shown that theanine has the effects of anti-cancer, anti-tumor, lowering blood pressure, calming the nerves, promoting brain function and nerve growth.

[0011] Currently, theanine has become a multifunctional plant health product that is popular in the international market for humans, pigs, and poultry. However, there are no reports on whether it can promote the health of lower invertebrate insects and whether it has an anti-insecticide toxicity effect. Summary of the invention

[0012] In view of the above technical problems, the present invention provides the use of theanine in preparing products for insect health care, prevention or control of insect pesticide poisoning.

[0013] The application of the protection of theanine of the present invention includes the following two aspects:

[0014] (1) Use in the preparation of pollinating insect health care;

[0015] (2) Use in the preparation of products for preventing or controlling insect pesticide poisoning in pollinating insects.

[0016] Furthermore, the product is a health product for pollinating insects, a medicine or feed for preventing pesticide poisoning of pollinating insects or improving pesticide damage.

[0017] Furthermore, the product contains theanine.

[0018] Furthermore, the product is a solid or liquid containing theanine.

[0019] In addition, theanine can induce insects to have a strong feeding preference, which means that insects will actively ingest a large amount of theanine. This scientific discovery ensures that insects can actively ingest a large amount of theanine in the field, solving the key technical obstacle that the insects may not be able to alleviate the poisoning effect in the field due to insufficient drug intake. At the same time, the present invention also experiments whether a large amount of theanine intake will cause cumulative toxicity, and it is found that it will not cause cumulative toxicity.

[0020] The present invention systematically studies whether theanine has health-promoting effects on normal insects and whether it can resist the damage of insecticides to insects: the preventive and protective effects of theanine drugs on social dysplasia, movement disorders, olfactory learning and memory disorders induced by insecticides in micro-nests are studied, and whether long-term exposure to theanine drugs is toxic to insects is also studied.

[0021] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0022] 1. The present invention discovers for the first time that long-term intake of theanine has no toxic side effects on pollinating insects, has a health-care effect on normal pollinating insects, and can improve various damages caused by pesticides to pollinating insects, including movement disorders, olfactory learning and memory disorders, and social behavior disorders caused by pesticides. It is the first time to confirm that theanine can effectively prevent the damage caused by pesticides, which is of great significance to the survival and reproduction of pollinating insects. It will also provide a technical basis for providing a solution to ensure food and biodiversity security for the world.

[0023] 2. The theanine provided by the present invention is highly safe, green, and has no side effects on pollinating insects. It provides a safe raw material for preparing medicines or feeds for treating pesticide damage to pollinating insects, and can induce pollinating insects to have a strong feeding preference, providing an application basis for the application of theanine in the fields, and providing a new scientific basis and protection strategy for solving the global food and agricultural crisis and biodiversity security protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Normal bumblebees can take theanine (CJ-33) on their own for a long time, show a preference for intake, and do not suffer from poisoning. Figure 1 a. Total daily food intake of bumblebees in each group; Figure 1 b. Trend of total food intake of bumblebees in the 1mM group; Figure 1 c. Daily preference index of bumblebees for different concentrations of CJ-33 sugar water; Figure 1 d. The mean of the preference index of bees for different concentrations of CJ-33 sugar water over 20 days; Figure 1 e. Changes in body weight of bumblebees in each group; Figure 1 f. Mortality rate of bumblebees in each group.

[0025] Figure 2 Theanine prevents the developmental impairment of bumblebee micro-nests caused by imidacloprid, Figure 2 a represents the developmental progress of micronests; Figure 2 b indicates microcolony mortality; Figure 2 c represents the nest start time; Figure 2 d represents the change in nest weight; Figure 2 e indicates the time of first spawning; Figure 2 f represents the number of eggs laid.

[0026] Figure 3 Theanine prevents the bumblebee dyskinesia (crawling) caused by imidacloprid; Figure 3 a represents the motion trajectory diagram; Figure 3 b represents the total crawling distance; Figure 3 c represents the average crawling speed; Figure 3 d represents the number of times you pass through the "door"

[0027] Figure 4 Theanine prevents imidacloprid-induced dysmotility (flight) in bumblebees; Figure 4 a represents the proportion of bumblebees that have lost the ability to fly; Figure 4 b represents the average flight speed; Figure 4 c represents the flight duration; Figure 4 d represents the flight distance;

[0028] Figure 5 Theanine prevented the bumblebee's insensitivity to sugar solutions caused by imidacloprid;

[0029] Figure 6 Theanine prevented the olfactory learning and memory impairment of bumblebees caused by imidacloprid; Figure 6 a represents the learning curve of the bumblebee, Figure 6 b indicates the memory test of bumblebees; DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention, but the present invention is not limited to other types of pesticide poisoning. Based on the dual effects of theanine on lower animals, insects, and prevention and control of poisoning, and based on the health effects of theanine on higher animals, humans, pigs, and poultry in the prior art, the present invention speculates that mammals such as humans and pigs can also prevent and control pesticide poisoning by taking theanine.

[0031] Example Effect of Theanine on Preventing or Controlling Insect Resistance to Pesticide Poisoning

[0032] Bumblebees belong to the genus Bombus, superfamily Apoidea, order Hymenoptera. Bumblebees are polyphagous social insects, and their evolution is at the intermediate stage between advanced social bees and primitive solitary bees. Bumblebees belong to the phylum Arthropoda, class Insecta, order Hymenoptera, superfamily Apoidea, family Apidae, genus Bombus (Williams et al., 2008). There are about 250 species of bumblebees, which are one of the few pollinating insects that can be distributed in colder areas. They are important pollinators of many wild plants and even endangered plants in high latitudes, high altitudes, and plateaus, and play a very important role in maintaining the balance of natural ecosystems. Bumblebees are important pollinators of many insect-pollinated plants, especially Leguminosae, Solanaceae and some endangered plants. Bumblebees are widely distributed in Yunnan.

[0033] In addition, some bumblebees have been commercialized both internationally and domestically, bringing huge economic benefits to global facility agriculture (Velthuis and Van Doorn, 2006). Bumblebee pollination can reduce the use of antibiotics and chemical hormones in facility crops and improve the safety of facility agricultural products; bumblebees have the characteristics of strong moisture tolerance, poor phototropism, and underdeveloped information exchange, making them ideal pollinating insects for facility agriculture. Using bumblebees as pollinators in facility agriculture can increase yields, improve fruit quality, reduce the rate of deformed fruits, and shorten the fruit ripening period. Bumblebees are commercially used for pollination of a variety of crops, including low-bush blueberries, greenhouse tomatoes, and peppers. In many cases, bumblebees are more effective at pollinating than honeybees due to differences in body size, morphological characteristics, pollination methods, and foraging habits. For example, bumblebees can obtain pollen through sound waves and can forage in harsher conditions than honeybees. At present, the prevention and control of pests and diseases in facility agriculture is still mainly based on chemical control. After crops are sprayed with pesticides, contaminated nectar and pollen can easily cause bumblebees to be poisoned and die.

[0034] 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 freely and forage. 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 30% sucrose solution per day. The experimental subjects were mainly experimental bumblebees that were normally raised in the nest.

[0035] The main research contents of this invention include the following four aspects:

[0036] 1. Research on the autonomous preference of bumblebees to ingest CJ-33. The device and method used in this experiment can be found in the patent application number CN202210432776.5 filed by the inventor, which is a device for long-term, quantitative monitoring of bumblebee autonomous feeding behavior and its manufacturing method. 2. Research on the improvement effect of CJ-33 on the poor development of bumblebee nests caused by neonicotinoid insecticides, mainly directly evaluating the colony level.

[0037] 3. Study on the improvement effect of CJ-33 on movement disorders in bumblebees caused by neonicotinoid insecticides, evaluated at the individual level. 4. Study on the improvement effect of CJ-33 on olfactory learning and memory damage in bumblebees caused by neonicotinoid insecticides, evaluated at the individual level.

[0038] 1. Bumblebees prefer to consume CJ-33

[0039] Experimental methods

[0040] 1.1 Preparation stage (preparation of equipment, preparation of bumblebees)

[0041] Use detergent and 75% alcohol to clean the device in turn to remove impurities and odors. After cleaning, place nest-like structures and pollen at the bottom of the living room, install cardboard with a rough surface similar to the color of the nest on the side of the living room; fill the water chamber with a certain volume of clean water. Before the formal experiment begins, there is a two-day adaptation phase, the purpose of which is to adapt the bumblebees to feeding through capillaries to avoid the impact of feeding disorders on the results of the previous experiments. The specific operation is to use chopsticks to move a randomly selected bumblebee into the living room of the patented CAFE device invented by this laboratory at 8:30 in the morning under dark conditions. Three capillaries with special structures are fixed on the left and right sides of each living room, and the tubes contain unlimited sugar water that is continuously added by humans for the bumblebees to feed freely. The capillaries are removed at 8:30 in the evening, cleaned and dried with alcohol, and ready for use the next day. All bumblebees in the experimental and control groups come from three colonies.

[0042] 1.2 Experimental Phase

[0043] The formal experimental cycle lasted for 20 consecutive days. At 7:30 am on the first day after the adaptation, under the condition of dark red light, bumblebees were taken out of the living room of the CAFE device, put into centrifuge tubes, weighed and put back. After that, feeding tubes were installed under dark conditions. The three feeding tubes on one side contained 30% sucrose food containing a certain concentration of CJ-33, and the three tubes on the other side contained 30% sucrose food; at the same time, two control groups were set up: one group was a blank control group with normal 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, the lights were turned on at 8:00 to start the feeding experiment until 8:00 in the evening. During this period, when the food consumption in a single capillary exceeded 2 / 3, the liquid reduction was measured with a vernier caliper and recorded, and then the same solution was added to the capillary. At the end of the experiment, the total amount of the same liquid reduction in the experimental group and the amount of sugar water reduction on the same side of the evaporation group were counted, and the difference between the two was the daily consumption of a certain liquid. After the experiment, all feeding capillaries were removed, cleaned and dried with alcohol for use the next day. To avoid the spatial position preference and memory of the experimental animals, the positions of the drug tube and the control food tube were swapped during the experiment the next day. The fasting bumblebees were weighed at 7:30 am the next day after the formal experiment and then immediately frozen and killed.

[0044] 1.3 Data Statistics and Analysis

[0045] Referring to the experimental statistical method of the fruit fly CAFE device (Devineni and Heberlein, 2009), we first measured the weight changes and food consumption of bumblebees during the experiment, and then calculated whether the bumblebees in the experimental group showed a preference for food containing CJ-33 every day, and the bumblebees in the blank group showed a preference for normal food every day through the preference index (PI) = (food consumption containing CJ-33 - normal food consumption) / total consumption. The PI value varies between -1 and +1, with positive values ​​indicating feeding preference and negative values ​​indicating feeding rejection. The original data were entered into Excel, and data analysis was performed using IBM SPSS Statistics 26, and graphs were drawn using GraphPad Prism 8.3.0. One-Way Repeated Measures ANOVA was used to compare the trend of data changes within the group, and the independent sample T / K test was used to compare the differences in data between groups. When the data of each group were compared, there was a significant difference when p<0.05, and there was an extremely significant difference when p<0.01, which were represented by "a", "b" and "c" respectively.

[0046] 1. Results: Bumblebees autonomously and preferentially ingested CJ-33 for a long time without any poisoning

[0047] like Figure 1 As shown in the figure, the changes in the total amount of food intake of bumblebees in the 0mM CJ-33 group, 0.5mM CJ-33 group, 1mM CJ-33 group, 1.5mM CJ-33 group, and 2mM CJ-33 group during the entire 20-day experimental period. It can be seen that the total amount of food intake of each group is generally stable, among which the growth rate of the total amount of food intake of bumblebees in the 1mM group is the largest. For the convenience of observation, 20 days are divided into four stages with 5 days as one stage, and the changes in the total amount of food intake of bumblebees in the 1mM group in the four stages are counted ( Figure 1 b). The average daily food intake of bumblebees in the last stage was significantly higher than that in the first three stages (p=0.005). The above results show that 1 mM concentration of CJ-33 can increase the food intake of bumblebees.

[0048] The present invention uses the preference index to further analyze the difference between bumblebee responses to food containing CJ-33 and normal food. Figure 1 c It can be seen that the preference index of the 0mM group has been fluctuating in a small range around 0. Figure 1The average preference index of 20 days was counted in d. The average preference index of the 0mM group within 20 days was 0.0071±0.0075. This proves that bumblebees take food randomly and without preference. Based on the same method, the preference indexes of the 0.5mM, 1mM, 1.5mM, and 2mM groups were 0.2113±0.0158, 0.2749±0.0275, 0.1388±0.064, and 0.0291±0.0194, respectively. Further statistics showed that the preference index of the 0.5mM group was significantly higher than that of the 0mM group (p=0.000); the preference index of the 1mM group was not significantly different from that of the 0.5mM group (p=0.054), but significantly higher than that of the 0mM group (p=0.000); the preference index of the 1.5mM group was significantly higher than that of the 0mM group (p=0.000), but significantly lower than that of the 1mM group (p=0.006); the preference index of the 2mM group was not significantly different from that of the 0mM group (p=0.099), but significantly lower than that of the 1.5mM group (p=0.013). The above results show that compared with normal food, bumblebees show a preference for food containing a certain concentration of CJ-33, and this preference value increases with the increase of CJ-33 concentration, and then decreases after exceeding a certain concentration, showing an inverted U-shaped relationship. This shows that the preference of bumblebees for sugar water containing CJ-33 is dose-dependent.

[0049] The study continues to evaluate whether the long-term preference of bumblebees for CJ-33 affects their health. Figure 1 e showed that within 20 days, the weight changes of bumblebees in the 0, 0.5, 1, 1.5, and 2mM groups were 0.0013±0.003g, -0.001±0.0027g, 0.0006±0.0022g, 0.0001±0.0027g, and -0.0008±0.0033g, respectively. Statistics showed that there was no significant difference between the groups (p=0.983). At the same time, the mortality rate of bumblebees in each group was 0 ( Figure 1 f). These results suggest that long-term preferential intake of CJ-33 does not harm the health of bumblebees.

[0050] The above results indicate that bumblebees voluntarily and autonomously ingest CJ-33 with preference, and that long-term ingestion of theanine by bumblebees is non-toxic.

[0051] 2. Study on the effect of CJ-33 on the poor development of bumblebee colonies caused by neonicotinoid insecticides

[0052] 2.1 Experimental methods:

[0053] Types and concentrations of neonicotinoid insecticides: Imidacloprid is the first generation of iconic neonicotinoid pesticides and a representative of high toxicity. It has the most target pests for prevention and control, and it has always been the most widely used and most widely used insecticide. Since 2012, it has been restricted for use by the European Union and other countries, but it is still widely used in other parts of the world. Therefore, imidacloprid was selected. The field concentration of imidacloprid ranges from 0.39 to 86 nM (Byrne et al., 2014, Zioga et al., 2020). A test concentration of 100 nM, which is higher than the field concentration, was used to explore whether CJ-33 can resist the damage caused to bumblebees by the highest concentration of imidacloprid in the field.

[0054] S1 Preparation stage: including the preparation of the device and the preparation of bumblebees

[0055] Before the experiment, clean the small group rearing device, disinfect it with alcohol, and then dry it. Before moving the bees, cover the bottom of the device with cat litter and sawdust to absorb the bee excrement and ensure that the device remains dry and warm. Select newly emerged bumblebees from the original nest and move them into the small group isolation device. This type of bee has white bristles on its body and its wings are not fully unfolded, which is obviously different from other bumblebees. There are 12 bumblebees in each small group.

[0056] S2 drug administration / micro-nest establishment and drug administration phase:

[0057] Establishment of bumblebee micro-nests: Previous studies have reported that 12 days after emergence is a sensitive critical period for bumblebee brain development. During this period, the brain develops rapidly. If neonicotinoid pesticides attack the insect nervous system during the critical period of brain development, it will inevitably damage the insect nervous system and the various behaviors regulated by it to the greatest extent. If the bumblebee is exposed to drugs during this time window (similar to the infant period after birth), the brain is easily damaged, and once damaged, it is difficult to improve later. Therefore, in order to explore the maximum protective power of the CJ-33 drug, the experimental task was carried out during this period. Newly emerged bumblebees were selected from the original nest and placed in a micro-nest breeding device. A micro-nest group consists of 10 bees and is raised for 12 days. According to different treatments, four micro-nest groups were set; blank control group, imidacloprid group, imidacloprid + CJ-33 group, CJ-33 group. The food of the bumblebees in the blank control group is normal sugar solution; the food of the bumblebees in the CJ-33 group is normal sugar solution containing CJ-33. Since CJ-33 may increase the amount of sugar water consumed by bumblebees, which will cause the amount of imidacloprid ingested by individual bumblebees to be different, the feeding method was adjusted to ensure that the amount of imidacloprid ingested by bumblebees in the imidacloprid group and the imidacloprid + CJ-33 group is the same. Only in this way can the experimental results be compared. Specifically: At 8 a.m., a fixed 4 ml of imidacloprid sugar solution was provided to the micro-nests in the imidacloprid group for them to take. After the imidacloprid sugar solution was consumed, a normal sugar solution was provided; similarly, a fixed 4 ml of imidacloprid + CJ-33 sugar solution was provided to each micro-nest in the imidacloprid + CJ-33 group, and after the imidacloprid + CJ-33 sugar solution was consumed, a normal sugar solution was provided. The bumblebee breeding conditions were set at a temperature of 25 ± 2 ° C, a relative humidity of 50 ± 5%, and a light and dark rhythm of 12:12 h.

[0058] S3 data statistics and analysis

[0059] The data recorded during the experiment were entered into Excel and then analyzed using IBM SPSS Statistics 26, and graphed using GraphPad Prism 8.30. Nonparametric tests were used to compare the mortality and number of offspring among different groups; multiple repeated measures analysis of variance was used to compare the nest weight gain among different groups.

[0060] 2.2 Results:

[0061] First, the development of micro-nests was recorded visually by taking photos every three days. Then, the changes in the development status of micro-nests under different treatments were analyzed, and further quantitative analysis of mortality, nesting conditions, and reproduction conditions was conducted.

[0062] like Figure 2As shown in Figure b, the mortality rate of the blank group was 7.75±3.07%, and the mortality rate of the imidacloprid group was 26.87±2.33%. The mortality rate of the imidacloprid group was significantly higher than that of the blank group (P=0.002); while the mortality rate of the imidacloprid+CJ-33 group was 10.87±1.95%, which was not significantly different from the blank control group (p=0.442), but was significantly lower than that of the imidacloprid group (p=0.000). The above results show that the intake of CJ-33 prevents the increase in the mortality rate of the micro-nest caused by imidacloprid. Further, the mortality rate of the CJ-33 group was not significantly different from that of the blank group (p=1). It can be seen that CJ-33 itself does not damage the survival of individuals in the micro-nest.

[0063] At the time of nest startup, such as Figure 2 As shown in Figure 3, the blank group started nesting on the 3rd ± 0.35th day, the imidacloprid group did not start nesting until the end of 12 days of feeding, and the imidacloprid + CJ-33 group started nesting on the 2.75 ± 0.34th day, which was not significantly different from the blank group (p = 0.645). The above results show that CJ-33 prevented the disappearance of the nesting behavior caused by imidacloprid. Further, the nesting start time of the CJ-33 group was not significantly different from that of the blank group (p = 0.161). It can be seen that CJ-33 itself does not delay the start time of the bumblebee's nest.

[0064] In terms of the changes in nest weight over time, Figure 2 As shown in Figure d, at the beginning of the test, the nest weight gain rate of the blank group was faster, and although it increased in the later period, it gradually slowed down. The imidacloprid group had almost no nesting, so there was a significant difference compared with the blank group (p = 0.01). After CJ-33 was ingested, there was no significant difference in the nest weight gain of the imidacloprid + CJ-33 group compared with the blank control group (p = 0.332). This result shows that the addition of CJ-33 not only prevents the disappearance of the nesting behavior caused by imidacloprid, but can even make the nesting behavior consistent with the level of the blank group. Further, when CJ-33 was ingested by normal bumblebees, the nest weight of the CJ-33 group increased by 77.2% (p = 0.025) compared with the nest weight of the blank control group. It can be seen that it can greatly promote the nesting behavior of normal bumblebees, which can be seen to have a health care effect on normal insects.

[0065] In terms of the first spawning time, Figure 2As shown in Figure 5, during the micro-nest rearing period, the blank group laid eggs for the first time on the 6th ± 0.9th day, the imidacloprid group did not lay eggs during the twelve-day rearing period, and when CJ-33 was ingested, the bumblebees in the imidacloprid + CJ-33 group laid eggs for the first time on the 6th ± 0.88th day, which was not significantly different from the blank group (p = 0.558). This shows that CJ-33 prevented the bumblebees from not laying eggs due to imidacloprid. When CJ-33 was ingested by normal bumblebees, the CJ-33 group laid eggs for the first time on the 4th ± 0.41th day, which was not significantly different from the blank group (p = 0.574). This shows that CJ-33 will not delay the first egg-laying time of bumblebees.

[0066] Then, in terms of the number of eggs laid, Figure 2 f, the number of eggs laid in the blank group was 17.25 ± 3.02, and the number of eggs laid in the imidacloprid + CJ-33 group was 15.125 ± 2.41. Statistics showed that there was no significant difference between the two groups (p = 0.328). This shows that CJ-33 can not only prevent the disappearance of the egg-laying behavior caused by imidacloprid, but also make the number of eggs laid no different from the normal level. Further, when CJ-33 was ingested by normal bumblebees, the number of eggs laid in the CJ-33 group was 32.375 ± 4.36, which exceeded the number of eggs laid in the blank group, and the excess ratio was as high as 90.57% (p = 0.05). This shows that CJ-33 can greatly promote the egg-laying of normal bumblebees, further illustrating that CJ-33 has a health-care effect on normal pollinating insects.

[0067] 3. Study on the effect of CJ-33 on the improvement of bumblebee movement disorders caused by neonicotinoid insecticides (individual level)

[0068] 3.1 Experimental methods

[0069] S1. Rearing and establishment of micro-nest colonies: has been described in detail above.

[0070] S2. Crawling ability test

[0071] At 9:00 a.m. on the 13th day of micro-nest rearing, the bumblebee climbing ability test began. Under red light, the target bumblebee was lifted up with a wooden chopstick and transferred to the playground, taking advantage of the bumblebee's love of climbing. After being placed in the device, the bumblebee first adapted to dark conditions for 15 seconds; then, the light was turned on and the camera was turned on. Recording was performed for 10 minutes. Behavior trajectory data was collected using Noldus software.

[0072] S3. Flight capability test

[0073] At 9:00 a.m. on the 13th day of micro-nest rearing, the flight ability of bumblebees was tested. Under dark conditions, the bees were gently clamped into a small box, and then the small box was buried in crushed ice for 20 minutes of cryoanaesthesia. To prevent the bumblebees from waking up during the subsequent operation, the cryoanaesthetized bees were placed on a small plastic plate on ice. A bee tag was attached to the dorsal side of the bumblebee thorax using propolis, and a copper sheet with a diameter of about 2 mm made of copper wire was attached to the bee tag, and a 2 cm copper wire was connected to the copper sheet. After completing this operation, the bumblebees were transferred to an independent small compartment for recovery for 3 hours, during which sufficient sugar water and pollen were provided. The bumblebees that recovered from anesthesia were fixed to the flight grinding arm using the copper wire on their backs. The other side of the flight grinding arm was balanced with space mud according to the weight of the bumblebees. After placing the flight grinding arm on the flight grinding arm, the copper wire was adjusted to make the bumblebees fly in the correct direction. In order to prevent the bumblebees from consuming stored energy by flapping their wings, a foam body was placed under the bumblebees' feet. This operation can greatly reduce the possibility of bumblebees flapping their wings. When the bumblebee is suspended on the flight grinding arm and keeps rolling the ball, it means that it is in a non-stress state and the flight test can begin. Carefully remove the ball with tweezers, and the bumblebee will start flying immediately. If the bumblebee still does not fly after removing the ball, put the ball under the bumblebee's feet again and remove it after 5 minutes. If the bumblebee still fails to fly after 5 operations, this bee is defined as a bee that "lost the ability to fly" and is eliminated. After the experiment, the proportion of such bees is counted. In order to provide certain visual feedback to the flying bumblebees, a paper cylinder with 2.5 cm wide vertical alternating black and white stripes printed by laser on its inner wall is placed outside the flight area (Tosi et al., 2017). The test was carried out in the same room to ensure uniform lighting, temperature (25±2℃), and humidity (50±5%) to avoid the influence of test conditions on its flight ability.

[0074] S4 Data Statistics and Analysis

[0075] The original data were entered into Excel, analyzed using IBM SPSS Statistics 26, and plotted using GraphPad Prism 8.30. Nonparametric tests were used to compare the crawling distance, crawling speed, and number of door-passing times of bumblebees under different treatments within a certain period of time, as well as the duration, distance, average speed, and proportion of bumblebees that lost their ability to fly of a single flight under different treatments.

[0076] 3.2 Results:

[0077] 3.2.1 Theanine improves bumblebee crawling disorder caused by imidacloprid

[0078] In the total crawling distance, Figure 3As shown in a and 3b, the total crawling distance of the blank group was 1703.58±209.45cm. The total crawling distance of the imidacloprid group was 636.43±98.1cm, and the total crawling distance of the imidacloprid+CJ-33 group was 1185.04±113.78cm. The total crawling distance of the imidacloprid group was significantly lower than that of the blank group (p=0.001). The total crawling distance of the imidacloprid+CJ-33 group was significantly higher than that of the imidacloprid group (p=0.001), but there was no significant difference compared with the blank group (p=0.128). This shows that CJ-33 can prevent the decrease in the total crawling distance of bumblebees caused by imidacloprid. Further, when CJ-33 was ingested by normal bumblebees, the total crawling distance of the CJ-33 group (1844.67±202.52cm) was not significantly different from that of the blank group (p=0.589). This indicates that CJ-33 does not affect the total crawling distance of normal bees.

[0079] At crawling speed, Figure 3 As shown in Figure c, the average crawling speed of the blank group was 2.84±0.34cm / s. The average crawling speed of the imidacloprid group was 1.07±0.16cm / s. The average crawling speed of the imidacloprid + CJ-33 group was 1.94±0.19cm / s, and the crawling speed of the imidacloprid group was significantly lower than that of the blank group (p=0.001). The crawling speed of the imidacloprid + CJ-33 group was significantly higher than that of the imidacloprid group (p=0.001), but there was no significant difference with the blank group (p=0.128). This shows that CJ-33 can prevent the decrease in the crawling speed of bumblebees caused by imidacloprid. Further, when CJ-33 was ingested by normal bumblebees, the average speed of the CJ-33 group (93.08±0.33cm / s) was not significantly different from the blank group (p=0.554), which shows that CJ-33 does not affect the crawling speed of normal bees.

[0080] In terms of the number of door penetrations, Figure 3 As shown in Figure d, the number of bumblebee penetrations in the blank group was 20.28±2.17 times. The number of bumblebee penetrations in the imidacloprid group was 6.57±1.20 times. The number of bumblebee penetrations in the imidacloprid+CJ33 group was 14.38±1.87 times. The number of penetrations in the imidacloprid group was significantly lower than that in the blank group (P=0.000). The number of penetrations in the imidacloprid+CJ33 group was significantly higher than that in the imidacloprid group (p=0.002), but there was no significant difference with the blank group (p=0.057). This shows that CJ-33 improved the reduction in the number of bumblebee penetrations caused by imidacloprid. Furthermore, when CJ-33 was ingested by normal bumblebees, the number of penetrations in the CJ-33 group (21.52±2.76 times) was not significantly different from that in the blank group (p=0.792), which shows that CJ-33 did not affect the number of penetrations of normal bees.

[0081] 3.2.2 CJ-33 prevents bumblebee flight disorders caused by imidacloprid

[0082] like Figure 4 As shown in a, only the imidacloprid group had bees that were unable to start flying after multiple stimulations, and the proportion of bees that could not fly was 13.69±4.86%. Statistics showed that it was significantly lower than that of the blank group and the imidacloprid + CJ-33 group (p=0.04), which shows that CJ-33 can reduce the number of bumblebees that have lost their ability to fly due to imidacloprid.

[0083] In terms of flight speed, Figure 4 As shown in Fig. 2a, the average speed of bumblebees in the blank group was 3.27±0.19km / h, the average speed of bumblebees in the imidacloprid group was 4.20±0.47km / h, and the average speed of the imidacloprid+CJ-33 group was 5.27±0.37km / h. Statistics show that the average speed of the imidacloprid group was not significantly different from that of the blank group (p=0.178), and the average flight speed of the imidacloprid+CJ-33 group was not significantly different from that of the imidacloprid group (p=0.128), but was significantly higher than that of the blank group (p=0.000). This shows that the combined effect of imidacloprid and CJ-33 can increase the speed of movement. At the same time, although there was no significant difference between the imidacloprid group and the blank group, by observing the data, it was found that the distribution range of the two groups of data was quite different, so the Levane test was further used to compare the homogeneity of variance of the data. The test results showed that the variance of the imidacloprid group was significantly greater than that of the blank group (p = 0.004), and the variance of the imidacloprid + CJ-33 group was not significantly different from that of the imidacloprid group (p = 0.631). This result shows that CJ-33 causes the average flight speed of bumblebees to be more discrete, while CJ-33 has no effect on the discrete average flight speed caused by imidacloprid of bumblebees. Further, when CJ-33 was ingested by normal bumblebees, the average single flight speed of the CJ-33 group (5.08 ± 0.35 km / h) was significantly higher than that of the blank group (p = 0.001). This result shows that CJ-33 can enhance the flight ability of normal bees.

[0084] In terms of flight time, Figure 4 As shown in Figure c, the flight time of the blank group was 0.39 ± 0.05h, the flight time of the imidacloprid group was 0.07 ± 0.02h, and the flight time of the imidacloprid + CJ-33 group was 0.29 ± 0.06h. Statistics show that the flight time of the imidacloprid group was significantly lower than that of the blank group (p = 0.000). There was no significant difference in the flight time of the imidacloprid + CJ-33 group with the blank group (p = 0.195), but it was significantly higher than the imidacloprid group (p = 0.000). This shows that CJ-33 can prevent the shortening of flight time caused by imidacloprid. Further, when CJ-33 was ingested by normal bees, the flight time of CJ-33 (0.39 ± 0.05h) was not significantly different from that of the blank group (p = 0.811). This shows that CJ-33 does not affect the flight time of normal bees.

[0085] In terms of flight distance, Figure 4 As shown in Figure d, the flight distance of the blank group was 1.17±0.16km, the flight distance of the imidacloprid group was 0.26±0.1km, and the flight distance of the imidacloprid+CJ-33 group was 1.26±0.25km. Statistics show that the flight distance of the imidacloprid group was significantly lower than that of the blank group (p=0.000). The flight distance of the imidacloprid+CJ-33 group was significantly higher than that of the imidacloprid group (p=0.000), but there was no significant difference with the blank group (p=0.94). This shows that CJ-33 can prevent the shortening of the flight distance of bumblebees caused by imidacloprid. Further, when CJ-33 was ingested by bumblebees of normal bees, the flight distance of the CJ-33 group (1.83±0.19km) was significantly higher than that of the blank group (p=0.028). This shows that CJ-33 can make normal bees fly farther, further indicating that CJ-33 can promote the movement ability of normal pollinating insects.

[0086] 4. Study on the improvement effect of CJ-33 on olfactory learning and memory impairment of bumblebees caused by neonicotinoids (individual level)

[0087] Experimental methods

[0088] S1. Establishment and rearing of micro-nests: has been described previously.

[0089] S2. Preparation of bumblebees before the experiment (including capture, fixation and recovery)

[0090] After 12 days of micro-nest rearing, on the morning of the 13th day, the bumblebees were transferred to a plastic box with a lid using chopsticks and buried under crushed ice for 20 minutes of freezing anesthesia until the bumblebees were motionless. The anesthetized bumblebees were transferred to a metal tube and fixed with two tapes, one fixed to the back of the abdomen and the other fixed between the thorax and the head. Be careful not to tighten it too much to ensure the activity of the bumblebees and to ensure that other parts of the bumblebee body, except the mouthparts and antennae, cannot come into contact with sucrose stimulation. After fixation, each bumblebee was numbered to facilitate identification in subsequent experiments. The fixed bees needed to recover at 28°C for 4 hours before the next experiment. During the recovery period, 2ul of 30% sucrose solution was fed every 1 hour to ensure that the bumblebees survived the waiting time and maintained sufficient hunger, so as to have the motivation to carry out associative learning. 10 minutes before the start of the experimental training, the antennae of the bumblebees were touched with 40% sucrose solution to test the motivation of the bumblebees to extend their beaks. If the bumblebee shows a proboscis extension reflex (PER) after its antennae touch sucrose, it is considered to have sufficient motivation for associative learning, and such bumblebees can be used in subsequent experiments. These bees with a proboscis extension reflex are defined as trainable bees, and their proportion is counted after the experiment. Conversely, bumblebees that do not extend their proboscis after touching the sucrose solution are eliminated.

[0091] S3. Experimental Phase

[0092] After 4 hours of fixation, training began. A 40% sucrose solution was used as an unconditioned stimulus (US). Low-concentration feeding and high-concentration training were beneficial to increase the sensitivity of bumblebees to sucrose solution during training and optimize learning performance. Diluted citral gas was used as a conditioned stimulus (CS). With reference to the experimental method of Brian H. Smith, conditioned olfactory PER training was carried out: each bumblebee was trained 12 times, with an interval of 10 minutes each time. During each training, the bumblebee was moved to the training field and allowed to adapt to the environment for 15 seconds to reduce the interference of sudden changes in the visual environment. After the adaptation, the olfactory stimulus (citral odor) was first blown at a uniform speed to the bumblebee antennae. After the odor was presented for 4 seconds, the bumblebee was given 2μl of 40% sucrose solution as a reward. The sucrose solution was first touched lightly on the bumblebee antennae. After the bumblebee stretched its beak, the sucrose solution was moved to the front of its beak and waited for it to eat. This process lasted for 3 seconds before the odor source was removed. The bumblebee continued to eat the sucrose solution for 3 seconds before the sucrose solution was removed. After removing the sugar solution, quickly move the exhaust fan to 10 cm behind the bumblebee's body to remove the surrounding citral odor. After each training session, let the bumblebee continue to rest in the training field for 15 seconds before removing it, which is conducive to the formation of memory in the bumblebee. The learning situation is assessed by the beak extension response to the citral airflow. Only the bees that successfully extend their beaks in the interval between the provision of the airflow and the provision of the sucrose solution are considered to have learned to associate the conditioned stimulus (CS: citral odor) with the unconditioned stimulus (US: sucrose solution). These bumblebees are recorded as "learned" and the other bees are recorded as "not learned". After the training, the bees are placed in a dark room, maintained at a temperature of 28°C and a humidity of 50±5%, and fed 2ul of 30% sugar water every 1h to prevent the bumblebees from excessive starvation and death.

[0093] Memory retrieval test: Non-reinforcement experiments were performed on the test bumblebees 1h, 6h, and 24h after the conditioning training to evaluate the memory retention of the bumblebees.

[0094] S4 Data Statistics and Analysis

[0095] The data of the olfactory conditioned proboscis reflex experiment were statistically analyzed using a dichotomous method (correct PER: 1, incorrect or no PER: 0). The original data were entered into Ecel, and the data were analyzed using IBM SPSS Statistics 26, and the graphs were drawn using GraphPad Prism 8.3.0. The chi-square test of the four-table data was used to compare the percentage of trainable bees; the Cochran Q test was used for a single learning curve to determine the changes caused by the number of learning times; the repeated measures analysis of variance was used to compare the learning curves between groups; the chi-square test in the non-parametric test was used to evaluate the differences in component memory retention. When the data of each group were compared, p<0.05 showed significant differences, and p<0.01 showed extremely significant differences, which were indicated by "a", "b", "c" or "*", "**".

[0096] Results: CJ-33 enhanced the sucrose sensitivity of bumblebees and improved the olfactory learning and memory impairment of bumblebees caused by neonicotinoids

[0097] like Figure 5 As shown in the figure, imidacloprid caused a significant decrease in bumblebee sensitivity to sucrose, and fewer bees were able to enter PER training. However, adding theanine at the same time significantly improved this situation. The addition of theanine restored the sucrose sensitivity that was reduced by imidacloprid, and was even better than the control group. Comparing the addition of theanine alone with the control group, it can be seen that theanine can enhance the sucrose sensitivity of bumblebees.

[0098] like Figure 6 As shown in Figure B, after 12 training sessions, 57.14% of the bumblebees in the control group were able to correctly respond to the olfactory conditioned stimulus, while 80% of the bumblebees in the sucrose water with theanine added were able to correctly respond to the olfactory conditioned stimulus after 12 training sessions, and there was a very significant difference (U=26.5, P=0.008), indicating that 1 mM theanine significantly improved the olfactory learning effect of bumblebees; when consuming imidacloprid, only 25% of the bumblebees were able to correctly respond to the olfactory conditioned stimulus, and there was a very significant difference between this learning curve and the learning curve of the control group (U=19.5, P=0.002), indicating that 10 0nM imidacloprid significantly reduced the olfactory learning effect of bumblebees; when theanine and imidacloprid were added at the same time, 81.8% of the bumblebees could correctly respond to the olfactory conditioned stimulus after 12 training sessions. This learning curve was not only significantly different from the learning curve of the pesticide stress group (U=10.5, P=0.000), but also significantly different from the blank control group (U=36.5, P=0.038), indicating that 1mM theanine can not only restore the olfactory learning ability damage caused by 100nM imidacloprid, but can even make the olfactory learning ability of bumblebees under pesticide stress better than that of the blank control bees.

[0099] like Figure 6As shown in Figure 2, in order to compare the differences in learning rates, a statistical analysis of the changes caused by the number of learning times within a single learning curve was performed. The results are shown in Figure 2. Figure 6 As shown in a, after the first four trainings, the proportion of bumblebees in the blank group that acquired olfactory conditioned responses increased significantly (p=0.000), the proportion of bumblebees in the imidacloprid group that acquired olfactory conditioned responses did not change significantly (p=0.25), but the proportion of bumblebees in the imidacloprid+CJ-33 group that acquired olfactory conditioned responses increased significantly (p=0.000); after twelve trainings, 53.33% of the bumblebees in the blank group (n=30) formed olfactory conditioned responses (p=0.000), 25% of the bumblebees in the imidacloprid group (n=24) formed olfactory conditioned responses (p=0.016), and 70% of the bumblebees in the imidacloprid+CJ-33 group (n=30) formed olfactory conditioned responses (p=0.000). This result shows that the bumblebees in the blank group, imidacloprid group, and imidacloprid+CJ-33 group all eventually acquired olfactory conditioned responses. However, the learning speed of bumblebees in the imidacloprid group was slower than that of the blank group, while CJ-33 avoided the problem of slow olfactory learning speed caused by imidacloprid. Furthermore, when CJ-33 was ingested by normal bumblebees, after the first four training sessions, the proportion of the CJ-33 group that learned olfactory conditioned responses increased significantly (p=0.000); after twelve training sessions, 76.67% of the bees in the CJ-33 group (n=30) formed olfactory conditioned responses (P=0.000). This result shows that CJ-33 has no effect on the olfactory learning speed of normal bees.

[0100] In order to compare the differences in learning effects, the learning curves were compared pairwise. The results are as follows Figure 6 As shown in a, after 12 learning sessions, the rate of bumblebees in the imidacloprid group that formed an olfactory conditioned response was significantly lower than that in the control group (P = 0.023), while the rate of bumblebees in the imidacloprid + CJ-33 group that formed an olfactory conditioned response was not significantly different from that in the blank group (P = 0.184), but was significantly higher than that in the imidacloprid group (p = 0.006). This indicates that CJ-33 can prevent the decline in olfactory learning effect caused by imidacloprid. Furthermore, when CJ-33 was ingested by normal bumblebees, the rate of bumblebees in the CJ-33 group that formed an olfactory conditioned response was significantly higher than that in the blank group (P = 0.027). This result indicates that CJ-33 can improve the learning effect of normal bees.

[0101] The results of the memory retrieval test were as follows Figure 6As shown in b, 1h, 6h, and 24h after the training, 50%, 43.75%, and 21.05% of the bumblebees in the blank group were able to extend their beaks in response to the conditioned stimulus, respectively; only 12.5% ​​and 10% of the bumblebees in the imidacloprid group were able to extend their beaks in response to the conditioned stimulus at the first two time points, and all individuals died at 24h; 60%, 55.85%, and 41.67% of the bumblebees in the imidacloprid+CJ-33 group were able to respond correctly to the conditioned stimulus at the three time points. The chi-square test showed that the short-term memory of the bumblebees in the imidacloprid group at 1 and 6h was significantly lower than that in the blank group (p=0.033; p=0.022), while the short-term memory of the bumblebees in the imidacloprid+CJ-33 group at 1 and 6h and the long-term memory at 24h were no different from those in the blank group (p=0.588, p=0.571, P=0.489). This result shows that CJ-33 can prevent the decline of olfactory memory ability of bumblebees caused by imidacloprid. Furthermore, when CJ-33 was ingested by normal bumblebees, the number of bumblebees in the CJ-33 group that extended their beaks in response to conditioned stimuli at 1 and 6 hours (66.67%, 58.82%) was not significantly different from that in the blank group (p=0.147; p=0.214), while the number of bumblebees that extended their beaks in response to conditioned stimuli at 24 hours (55.55%) was significantly higher than that in the blank group (p=0.046), which shows that CJ-33 can enhance the long-term memory of normal bees.

Claims

1. Application of theanine, Features: The applications described include: 1) The use of theanine as the sole active ingredient in the preparation of products that promote normal bumblebee nesting and egg laying, and improve flight ability, olfactory learning and memory abilities; 2) Use of theanine as the sole active ingredient in the preparation of a product for improving the damage of imidacloprid to bumblebees.

2. The use according to claim 1, Features: The damages of imidacloprid to bumblebees include poor development of bumblebee nests, movement disorders, and olfactory learning and memory damage caused by imidacloprid.

3. The use according to claim 1, Features: The product is a solid or liquid containing theanine.

4. The use according to claim 1, Features: The product is medicine or feed.

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