A method for controlling reproduction of the tea small hole by using sex pheromone

Abstract

The application develops a method capable of utilizing sex pheromone to control the reproduction of tea timber bark beetle, which can not only obtain tea timber bark beetle sex pheromone quickly and highly, but also effectively utilize the obtained sex pheromone to control the reproduction of tea timber bark beetle. The method adopts the way of electric shock to collect tea timber bark beetle from rotten wood. The method selects low temperature 16 DEG C, high humidity 88% or so, low concentration alcohol stimulation and blue light irradiation, realizes the rapid hatching of tea timber bark beetle eggs. The method selects low temperature 18 DEG C, high humidity 86% or so, 1200 Lux strong light stimulation, realizes the rapid emergence of tea timber bark beetle pupae. The method selects high temperature 32 DEG C, long light cycle 20L:4D, 120 mg / L sodium hydroxide stimulation, and adds nutrition enhancer brine shrimp eggs, and improves the sex pheromone yield.

Description

Technical Field

[0001] This invention belongs to the field of insect control, specifically relating to a method for controlling the reproduction of the tea bark beetle using sex pheromones. Background Technology

[0002] The tea branch beetle (Bark beetle) belongs to the order Coleoptera, family Weevilidae, subfamily Bark beetleinae, and genus Bark beetle. It has a very wide host range, with over 100 species belonging to 39 families in Asia alone. Adults: Females are 2.4-2.6 mm long, while males are yellowish-brown, about 1.3 mm long, and cylindrical. Eggs: About 0.6 mm long, oblong, gradually deepening in color from white to pale yellow when first laid. Larvae (mature stage): Milky white, about 2.4 mm long, smaller at the front and slightly larger at the back, plump with wrinkled appearance; the head is yellowish-brown at this stage. Pupae: Female pupae are initially milky white, gradually deepening to pale yellow or yellowish-brown, about 2.4 mm long; male pupae are similar to female pupae but smaller, about 1.3 mm long. The tea wood beetle can have 6 generations a year in Guangdong. The insect goes through morphological changes from egg to larva to pupa to adult. The egg stage lasts about 6-7 days, the larval stage about 30-35 days, the pupal stage about 4-6 days, and the pre-oviposition stage about 8-10 days. The generations overlap, with each generation lasting 36-50 days. The overwintering adults of this insect begin to emerge during sunny days in mid-to-late February when the average daily temperature is 20℃-22℃, seeking new host plants. The time from emergence to invasion is only 10-180 minutes. They then mate, lay eggs, and emerge as adults in the new tunnels. After emergence, they remain in the tunnels for 6-8 days before emerging again. Egg-laying peaks in early April, with two population peaks in mid-June and early-to-mid October. The population then declines until reaching its lowest point in winter. The insect primarily overwinters as adults in the same tunnels, but a small number also overwinter as larvae and pupae. Rearing studies show that the incidence of the tea wood beetle varies depending on the season, weather conditions, and plant growth, indicating a predictable pattern in its occurrence. The lowest population size occurs in the cold winter, naturally reducing the insect's harmful activity, with most remaining in a dormant state. The adult and larval tea bark beetles bore into the branches and trunks of host plants, damaging the internal structure of the branches and trunks, leading to insufficient water and nutrient supply to the plants and their gradual death. The entry point (boring hole) of the tea bark beetle is very small, only about 2 mm, and is generally difficult to detect.

[0003] Control measures for the tea wood beetle mainly include chemical control, biological control, manual cleaning and trapping, and the use of pheromones or attractants. Chemical control includes spraying insecticides, injecting pesticides into the trunk, fumigating to kill insects, and spraying lime sulfur or 50% sulfur suspension on the surface of the trunk. Biological control mainly utilizes the natural enemies of the tea wood beetle. Manual cleaning and trapping mainly involves cutting open the insect holes and tunnels with a knife to kill the eggs, pupae, and adults hidden inside. Pheromones or attractants are chemical substances that can simulate or induce the natural pheromones of insects, thereby guiding the insect's behavior, such as gathering, reproduction, and foraging. Therefore, pheromones or attractants can induce the tea wood beetle to gather in specific areas, making it convenient for targeted control. Using chemical pesticides to control pests can easily pollute the environment and harm other non-target organisms; biological control is difficult to implement and its effects are often unsatisfactory; manual cleaning is time-consuming, labor-intensive, and costly; currently, using pheromones or attractants to control tea bark beetles has become a more environmentally friendly method.

[0004] Insect sex pheromones, also known as sex pheromones, are trace chemical substances secreted by specific organs of a particular sex within the same insect species. These pheromones can be detected by receptors in opposite-sex individuals of the same species, triggering behavioral responses or physiological effects (such as courtship, directional mating, and mating). They ensure sexual relations between males and females within the same species and maintain orderly reproduction. In most insect species, females release these pheromones to attract males, while in some species, males release them to attract females. Methods of controlling insect populations using sex pheromones mainly include mating interference and mass trapping. Mating interference involves releasing large quantities of sex pheromones into the environment during the insect's courtship period, causing olfactory desensitization in insects. This reduces mating behavior in adult females, and prevents males from finding mates, thus delaying or eliminating mating and reducing population size and the degree of damage. Mass trapping involves placing numerous sex pheromone traps in fields or forests to lure and kill male adults, preventing females from mating and reproducing. This results in a severe imbalance in the male-to-female ratio, minimizing the population of pests and their offspring. However, current research on the sex pheromones of the tea bark beetle is scarce, with even less research on their extraction, identification, composition, and applications. Summary of the Invention

[0005] This invention aims to solve the technical problems of difficulty in controlling tea leaf beetle and the difficulty in implementing control using sex pheromones. It develops a method that can use sex pheromones to induce and control the reproduction of tea leaf beetle. This method can not only obtain tea leaf beetle sex pheromones quickly and efficiently, but also effectively use the obtained sex pheromones to induce and control the reproduction of tea leaf beetle, thereby achieving the effect of effectively controlling tea leaf beetle.

[0006] In the process of controlling the reproduction of the tea wood beetle using sex pheromones, this invention is the first to employ an electric shock method to collect the beetle from decaying wood. The current applied to the moist, decaying wood is controlled at 1-1.2 mA, achieving rapid and effective separation of the beetle from the decaying wood. This invention also improves the hatching conditions for the tea wood beetle eggs, selecting a low temperature of 16℃, high humidity of approximately 88%, low-concentration alcohol stimulation, and blue light irradiation to achieve rapid hatching of the eggs; larvae hatch in 3-4 days. Furthermore, this invention optimizes the emergence conditions of the pupae, selecting a low temperature of 18℃, high humidity of approximately 86%, and strong light stimulation of 1200 Lux to achieve rapid emergence of the tea wood beetle pupae; adults emerge in 2-3 days. This invention provides nutritional enhancement for newly emerged adult tea bark beetles. Alkaline stimulation with high temperature (32°C), long photoperiod (20L:4D), and 120 mg / L sodium hydroxide, along with the addition of Artemia eggs as a nutritional enhancer, stimulates and promotes the synthesis of sex pheromones in adult tea bark beetles, increasing the production of sex pheromones and providing abundant resources for subsequent use of sex pheromones.

[0007] This invention is achieved through the following technical solution:

[0008] A method for controlling the reproduction of tea bark beetle using sex pheromones includes the following steps:

[0009] (1) Harvesting of adult tea bark beetles:

[0010] Moist, decaying wood infested with adult tea leaf beetles was collected. The beetles were then extracted from the wood using an electric shock method. The electric shock device consisted of a controller, a 12V low-voltage DC power supply, an ammeter, wires, electrode A, and electrode B. Electrode A and electrode B were the positive and negative terminals, respectively, and were inserted into the two ends of the decaying wood. The current was controlled at 1-1.2 mA. If the wood was not sufficiently moist, a suitable amount of salt water could be sprayed onto it. After 5 minutes of electric shock, all the adult tea leaf beetles escaped from the decaying wood and were quickly collected in a rearing box.

[0011] (2) The rearing and egg-laying of the tea leaf beetle:

[0012] Place the rearing box inside the rearing box, with a rearing temperature of 25-27℃, relative humidity of 60-65%, and a photoperiod of 14L:10D. The feed used is a mixture of bark powder and water, which should be free of lumps, able to be formed into a ball by hand, and slightly loose when released. After the adult tea wood beetles in the rearing box mate and lay eggs, collect the egg masses.

[0013] (3) Rapid hatching of tea bark beetle eggs:

[0014] The collected egg masses were placed in an incubator, and the temperature inside the incubator was controlled at 16°C. The egg masses were periodically sprayed with a low concentration of alcohol (30% by volume) to maintain a relative humidity of about 88%. At the same time, the blue light in the incubator was turned on. The blue light produced a wavelength of 480~500 nm and a light intensity of 500~600 Lux. The blue light was turned on for 1 hour every morning during the incubation period. Under these conditions, the eggs will hatch into larvae in 3-4 days.

[0015] (4) Larval rearing of the tea bark beetle:

[0016] The hatched larvae are placed in a larval rearing box, which is then placed in the rearing box. The rearing temperature is 25-27℃, the relative humidity is 60-65%, and the photoperiod is 14L:10D. The feed used is a mixture of bark powder and water. The feed should be free of lumps, can be formed into a ball by hand, and should crumble slightly when loosened. After the larvae pupate, the pupae are collected for later use.

[0017] (5) Rapid emergence of the tea leaf beetle pupa:

[0018] The collected pupae are placed in a hatching box, and the temperature inside the hatching box is controlled at 18℃ and the relative humidity at about 86%. At the same time, a white high-intensity light is turned on inside the hatching box, and the light intensity of the light is 1200 Lux. Under these conditions, the pupae will hatch into adults in 2-3 days.

[0019] (6) Nutritional fortification of adult tea bark beetles:

[0020] The newly emerged adult insects were placed in an adult rearing box, maintaining a temperature of 32°C and a photoperiod of 20L:4D. A 120 mg / L sodium hydroxide solution was sprayed into the rearing box three times a day, maintaining a humidity of 60-65%. The feed used was a mixture of bark powder and Artemia eggs as a nutritional fortifier, with a weight ratio of bark powder to Artemia eggs of 10:1. The feed was mixed with water to form a clump-free mixture that could be formed into a ball by hand but crumble slightly when loosened. The nutritional fortification was continued for 3 days.

[0021] (7) Extraction of sex pheromones:

[0022] Unmated female tea bark beetles, after being fortified with nutrients for 3 days, were placed on an experimental table, and their gonads were removed. Sex pheromones were extracted from the gonads using a dichloromethane immersion extraction method. The gonads were immersed in dichloromethane at 4°C for 60 minutes, with 30 μL of dichloromethane used for each gonad. The extracted sex pheromones were then identified and analyzed using gas chromatography-antennae potential coupling technology and two-dimensional gas chromatography-time-of-flight mass spectrometry.

[0023] (8) Preparation and use of lure cores:

[0024] The extracted and identified sex pheromones are made into attractants, and the attractants are made into lures and placed in trapping equipment. The trapping equipment is placed in the forest, and each lure contains a dose of 200 μg of sex pheromones.

[0025] The technical solution of the present invention has the following advantages:

[0026] 1. This invention utilizes electrocution to collect tea wood beetles from decaying wood. Research indicates that tea wood beetles are sensitive to a certain degree of electrocution. Through extensive experimentation, the inventors confirmed that a current of 1-1.2 mA has a significant impact on tea wood beetles. This application employs a 1-1.2 mA current to repel and collect tea wood beetles from moist, decaying wood, achieving efficient and thorough harvesting. Currently, most methods for removing live tea wood beetles involve mechanical means, such as prying or even splitting the tree, which can cause mechanical damage or even death to the beetles, resulting in poor separation. The tea wood beetle collection method used in this invention demonstrates significantly improved separation efficiency.

[0027] 2. This invention improves the incubation conditions for tea bark beetle eggs, shortening the incubation time and providing conditions for rapid acquisition of sex pheromones. The typical incubation time for tea bark beetle eggs is 6-7 days. This invention shortens the incubation time to 3-4 days by selecting low temperature and high humidity, low-concentration alcohol stimulation, and blue light irradiation. The inventors found in experiments that maintaining low temperature and high humidity during egg incubation can stimulate the secretion of incubation enzymes to a certain extent, prompting the embryo to hatch earlier. Low-concentration alcohol stimulation can stimulate peristalsis or convulsions within the egg, thereby generating mechanical force to assist in breaking the eggshell. Blue light irradiation may indirectly affect the embryonic development process by regulating the biological clock or hormone signals through photosensitive proteins. Through extensive experiments, this invention screened a low temperature of 16℃, high humidity of 88%, low-concentration alcohol stimulation of 30%, and blue light irradiation, achieving rapid hatching of tea bark beetle eggs.

[0028] 3. This invention optimizes the eclosion conditions of the pupae, shortening the eclosion time of *Bretschneidera sinensis* pupae, and also providing conditions for the subsequent rapid acquisition of sex pheromones. The general eclosion time for *Bretschneidera sinensis* pupae is 4-6 days. This invention shortens the eclosion time to 2-3 days by selecting low temperature, high humidity, and strong light stimulation. Experiments show that maintaining low temperature and high humidity during pupal eclosion can indirectly promote chitinase-mediated pupal shell degradation by maintaining pupal body water balance and the ecdysone (20E) signaling pathway. Strong light stimulation can promote the secretion and release of eclosion hormone (EH) by brain nerve cells, triggering pre-eclosion behaviors such as pupal peristalsis and searching for eclosion support points. Through extensive experimentation, this invention screened for a low temperature of 18℃, high humidity above 86%, and strong light stimulation of 1200 Lux, achieving rapid eclosion of *Bretschneidera sinensis* pupae.

[0029] To rapidly obtain sex pheromones from the tea wood beetle, we focused on shortening its life cycle. The larval and adult development stages of the tea wood beetle are relatively stable, making cycle regulation ineffective. Therefore, our research concentrated on the egg hatching and pupal emergence processes, aiming to shorten these two cycles. After numerous adjustments and optimizations to hatching and emergence conditions, we finally identified the conditions that influence egg hatching and pupal emergence times, successfully shortening both the egg hatching and pupal emergence cycles. This significantly reduces the overall life cycle of the tea wood beetle, creating favorable conditions for rapid sex pheromone acquisition.

[0030] 4. This invention provides nutritional fortification for newly emerged adult tea leaf beetles, significantly increasing the secretion of sex pheromones. Using the nutritional fortification conditions of this invention, a significant amount of sex pheromones can be extracted from the gonads within 3 days. High temperatures accelerate the lipid metabolism pathway of adult tea leaf beetles, promoting the synthesis of sex pheromone precursors. Long photoperiods regulate the synthesis of cytochrome P450 monooxygenase, thereby regulating oxidation reactions and promoting the synthesis of tea leaf beetle sex pheromones. A certain degree of alkaline stimulation can promote the synthesis of fatty acid reductase in adult tea leaf beetles, catalyzing the reduction of fatty acids to alcohols, further forming pheromone components. Supplementing with the nutritional fortifier Artemia eggs can, to some extent, induce the synthesis of juvenile hormones in tea leaf beetles, thereby promoting pheromone gland activity and regulating sex pheromone synthesis. Through extensive experimentation, this invention screened alkaline stimulation using high temperature (32°C), long photoperiod (20L:4D), and 120 mg / L sodium hydroxide, as well as the addition of the nutrient fortifier Artemia eggs. The inventors combined these techniques to greatly enhance the synergistic effect of inducing pheromone synthesis, significantly increasing the pheromone production of adult tea bark beetles and providing abundant resources for subsequent pheromone use. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to examples.

[0032] Example 1: Separation and Harvesting of Tea Leaf Beetles

[0033] Determination of the energizing current: Adult tea bark beetles were placed in a conductive rearing dish. Electrodes were connected to both sides of the dish, and different currents were applied, starting from 0.1 mA and gradually increasing in strength. The current at which the adults exhibited a significant reaction was measured. This significant reaction was mainly manifested as slight twitching or trembling of the insect's body, restlessness, and erratic movement. The test results showed that a current of 1-1.2 mA was required for the adult tea bark beetles to begin exhibiting a significant reaction.

[0034] Electrostatic treatment time on damp, rotten wood: Damp, rotten wood infested with tea leaf beetles was collected and divided into five equal portions, which were placed on an insulated experimental platform. An electric shock device was connected to the rotten wood, comprising a controller, a 12V low-voltage DC power supply, an ammeter, wires, electrode A, and electrode B; electrode A and electrode B were the positive and negative terminals, respectively, inserted into the two ends of the rotten wood. The current was controlled at 1-1.2 mA. The electrostatic treatment times for the five portions of rotten wood were 1 minute, 3 minutes, 5 minutes, 7 minutes, and 9 minutes, respectively. After electrostatic treatment, the tea leaf beetle repulsion rate of the five groups of rotten wood was manually measured, with three replicates per group. The repulsion rates were 60%, 80%, 100%, 100%, and 100%, respectively. Therefore, a current of 1-1.2 mA and an electrostatic treatment time of 5 minutes were selected.

[0035] Example 2: Rapid Hatching of Tea Bark Beetle Eggs

[0036] (1) Determination of low temperature and high humidity conditions: Previous studies have shown that the suitable incubation conditions for tea bark beetle eggs are a temperature of 25-30℃ and a humidity of 60-70%. During the experimental testing of incubation temperature and humidity for tea bark beetle eggs, the inventors found that the incubation time under low temperature and high humidity conditions was shorter than other temperature and humidity combinations. To verify the effect of low temperature and high humidity, the inventors conducted a comparative experiment on the same batch of eggs that had just been laid in a rearing box, designing different temperature and humidity cross-combination experiments. The temperature gradients were 15℃, 20℃, 25℃, 30℃, and 35℃, and the humidity gradients were 50%, 60%, 70%, 80%, 90%, and 100%, respectively. That is, the incubation conditions for tea bark beetle eggs were: 15℃, 50% humidity; 15℃, 60% humidity; 15℃, 70% humidity; 15℃, 80% humidity; 15℃, 90% humidity; and 15℃, 100% humidity. Temperature 20℃, humidity 50%; Temperature 20℃, humidity 60%; Temperature 20℃, humidity 70%; Temperature 20℃, humidity 80%; Temperature 20℃, humidity 90%; Temperature 20℃, humidity 100%. Temperature 25℃, humidity 50%; Temperature 25℃, humidity 60%; Temperature 25℃, humidity 70%; Temperature 25℃, humidity 80%; Temperature 25℃, humidity 90%; Temperature 25℃, humidity 100%. Temperature 30℃, humidity 50%; Temperature 30℃, humidity 60%; Temperature 30℃, humidity 70%; Temperature 30℃, humidity 80%; Temperature 30℃, humidity 90%; Temperature 30℃, humidity 100%. Temperature 35℃, humidity 50%; Temperature 35℃, humidity 60%; Temperature 35℃, humidity 70%; Temperature 35℃, humidity 80%; Temperature 35℃, humidity 90%; Temperature 35℃, humidity 100%. Incubation time is 3-4 days. All other conditions in the rearing box are the same. Three replicates per group. The average hatching rate of tea bark beetle eggs after 3-4 days under different temperature and humidity conditions is shown in the table below.

[0037]

[0038] Table 1. Average hatching rate of tea bark beetle eggs over 3 days under different temperature and humidity conditions.

[0039] Table 2. Average hatching rate of tea bark beetle eggs over 4 days under different temperature and humidity conditions.

[0040] Experiments have shown that low-temperature and high-humidity incubation conditions of 15-20℃ and 80-90% can shorten the incubation time of tea bark beetle eggs to 3-4 days.

[0041] To further refine the incubation conditions under low-temperature and high-humidity conditions of 15-20℃ and 80-90%, the inventors conducted further experiments. Temperature gradients of 15℃, 16℃, 17℃, 18℃, 19℃, and 20℃, and humidity gradients of 80%, 82%, 84%, 86%, 88%, and 90%, were established. The incubation period was 4 days, with three replicates per group. The final experimental results showed that the average hatching rate was highest under the low-temperature and high-humidity conditions of 16℃ and 88%, consistently reaching 94-96%.

[0042] (2) Determination of low-concentration alcohol: In their research on the hatching of tea bark beetle eggs, the inventors discovered that low-concentration alcohol stimulation can stimulate the embryo inside the egg to move or twitch, thereby generating mechanical force to assist in breaking the eggshell. Freshly laid eggs from the same batch were placed in a rearing box and divided into six groups, including five experimental groups and one blank control group. The rearing temperature was 25-27℃, the relative humidity was 60-65%, and the photoperiod was 14L:10D. The eggs in the five experimental groups were sprayed with alcohol at volume fractions of 10%, 30%, 50%, 70%, and 90%, respectively, twice a day. The blank control group was sprayed with distilled water. After 4 days, the average hatching rate of the eggs in each group was calculated, with three replicates per group. The five experimental groups had hatching rates of 65%, 90%, 80%, 66%, and 52%, respectively, while the blank control group had a hatching rate of 65%.

[0043] (3) Determination of rapid incubation conditions:

[0044] Freshly laid tea bark beetle eggs were divided into eight groups and placed in eight rearing boxes, including seven experimental groups and one control group. The incubation conditions for the seven experimental groups were as follows: low temperature and high humidity (16℃, 88%) group, 30% alcohol spraying group, blue light irradiation group, low temperature and high humidity (16℃, 88%) group + 30% alcohol spraying group, 30% alcohol spraying group + blue light irradiation group, low temperature and high humidity (16℃, 88%) group + blue light irradiation group, and low temperature and high humidity (16℃, 88%) group + 30% alcohol spraying group + blue light irradiation group. Other conditions were the same as those for the blank control group. The blank control group had a temperature of 25-27℃, a relative humidity of 60-65%, a photoperiod of 14L:10D, and did not receive alcohol spraying or blue light irradiation treatment. The blue light irradiation wavelength was 480~500 nm, and the light intensity was 500~600 Lux. Under the same photoperiod as the control group, the blue light lamp was turned on for an extra hour every morning.

[0045] Three days after incubation, the average hatching rate of each group was measured, with three replicates per group, in the following order: experimental group 94%, 90%, 91%, 97%, 96%, 97%, 100%. The blank control group had a hatching rate of 66%. Therefore, the low temperature and high humidity (16℃, 88%) group + 30% alcohol spraying group + blue light irradiation group were determined to be the environmental conditions for rapid hatching of tea bark beetle eggs.

[0046] Example 3: Rapid Emergence of Tea Tree Bark Beetle Pupae

[0047] (1) Determination of low temperature and high humidity conditions: During the experimental testing of the eclosion temperature and humidity of the tea bark beetle pupa, the inventors found that the eclosion time under low temperature and high humidity was shorter than other temperature and humidity combinations. In order to verify the effect of low temperature and high humidity, the inventors conducted a comparative experiment on the same batch of pupae in a rearing box and designed different temperature and humidity cross-combination experiments (two-factor random combination). The temperature gradients were 15℃, 20℃, 25℃, 30℃, and 35℃, and the humidity gradients were 50%, 60%, 70%, 80%, 90%, and 100%, respectively. That is, the eclosion conditions of the tea bark beetle pupa were: temperature 15℃, humidity 50%; temperature 15℃, humidity 60%; temperature 15℃, humidity 70%; temperature 15℃, humidity 80%; temperature 15℃, humidity 90%; temperature 15℃, humidity 100%. Temperature 20℃, humidity 50%; Temperature 20℃, humidity 60%; Temperature 20℃, humidity 70%; Temperature 20℃, humidity 80%; Temperature 20℃, humidity 90%; Temperature 20℃, humidity 100%. Temperature 25℃, humidity 50%; Temperature 25℃, humidity 60%; Temperature 25℃, humidity 70%; Temperature 25℃, humidity 80%; Temperature 25℃, humidity 90%; Temperature 25℃, humidity 100%. Temperature 30℃, humidity 50%; Temperature 30℃, humidity 60%; Temperature 30℃, humidity 70%; Temperature 30℃, humidity 80%; Temperature 30℃, humidity 90%; Temperature 30℃, humidity 100%. Temperature 35℃, humidity 50%; Temperature 35℃, humidity 60%; Temperature 35℃, humidity 70%; Temperature 35℃, humidity 80%; Temperature 35℃, humidity 90%; Temperature 35℃, humidity 100%. Emergence time is 2-3 days. All other conditions in the rearing terrarium are the same. Three replicates are used per group. The average emergence rate of the tea wood bark beetle pupa over 2-3 days under different temperature and humidity conditions is shown in the table below.

[0048]

[0049] Table 3. Average emergence rate of tea wood bark pupa under different temperature and humidity conditions over 2 days.

[0050] Table 4. Average emergence rate of tea wood bark pupa under different temperature and humidity conditions over 3 days.

[0051] Experiments have shown that low temperature and high humidity conditions of 15-20℃ and 80-90% can shorten the emergence time of tea bark beetle pupae to 2-3 days.

[0052] To further refine the elongation process under low-temperature, high-humidity conditions of 15-20℃ and 80-90% humidity, the inventors conducted detailed experiments. Temperature gradients of 15℃, 16℃, 17℃, 18℃, 19℃, and 20℃, and humidity gradients of 80%, 82%, 84%, 86%, 88%, and 90%, were set, with an elongation time of 3 days and three replicates per group. The final experimental results showed that the average elongation rate was highest under the low-temperature, high-humidity conditions of 18℃ and 86%, consistently reaching 93-96%.

[0053] (2) Determination of light intensity: Pupae from the same batch were placed in a rearing box and divided into six groups, including five experimental groups and one blank control group. The rearing temperature was 25-27℃ and the relative humidity was 60-65%. The light intensities of white high-intensity lamps in the five experimental groups were 800 Lux, 1000 Lux, 1200 Lux, 1400 Lux and 1600 Lux, respectively, while the blank control group was under normal light. The experimental groups and the blank control group maintained the same 14L:10D photocycle. After 3 days, the average hatching rate of eggs in each group was calculated, with three replicates per group. The hatching rates for the five experimental groups were 67%, 76%, 91%, 74% and 55%, respectively, while the rate for the blank control group was 68%.

[0054] (3) Determination of rapid feathering conditions:

[0055] Freshly hatched tea wood pupae from the same batch were divided into four groups and placed in four rearing boxes: three experimental groups and one control group. The incubation conditions for the three experimental groups were as follows: low temperature and high humidity (18℃, 86%) group, strong light irradiation group (1200 Lux), and low temperature and high humidity (18℃, 86%) group + strong light irradiation group (1200 Lux). Other conditions were the same as those for the blank control group. The blank control group had a temperature of 25-27℃, a relative humidity of 60-65%, a photoperiod of 14L:10D, and no strong light irradiation treatment.

[0056] Three days after emergence, the average emergence rate of each group was measured, with three replicates per group, in the order described above: experimental group 94%, 92%, 100%. The blank control group was 67%. Therefore, the low temperature and high humidity (18℃, 86%) group + strong light irradiation group (1200 Lux) was determined to be the environmental conditions for rapid emergence of the tea bark beetle pupa.

[0057] Example 4: Nutritional fortification of adult tea bark beetles

[0058] (1) Determination of temperature during the nutritional fortification stage: A certain degree of high-temperature stimulation can induce the secretion of sex pheromones by adult tea bark beetles. Newly emerged adult tea bark beetles from the same batch were divided into five groups and placed in rearing boxes at 26℃, 28℃, 30℃, 32℃, and 34℃ respectively. Other rearing conditions were the same for all five groups: relative humidity 60-65% and photoperiod 14L:10D. After 3 days, sex pheromones were extracted from the gonads of each group for quantitative analysis, with three replicates per group. The sex pheromones were extracted using the dichloromethane immersion extraction method, and quantitative analysis was performed using gas chromatography-antennae potential coupling technology and two-dimensional gas chromatography-time-of-flight mass spectrometry. The results showed that the 32℃ group had the highest sex pheromone content, significantly higher than the other four groups, while the 26℃ group had the lowest sex pheromone content, slightly lower than the 28℃ group.

[0059] (2) Determination of photoperiod during the nutrient fortification stage:

[0060] Newly emerged adult tea bark beetles from the same batch were divided into six groups and placed in six rearing boxes. The photoperiods of each group were adjusted to 12L:12D, 14L:10D, 16L:8D, 18L:6D, 20L:4D, and 22L:2D, respectively. All other rearing conditions were the same for all six groups: temperature 25-27℃, relative humidity 60-65%. After three days, sex pheromones were extracted from the gonads of each group for quantitative analysis, with three replicates per group. The sex pheromone extraction method was used, and the quantitative analysis was performed using gas chromatography-antennae potential coupling and two-dimensional gas chromatography-time-of-flight mass spectrometry. The results showed that the 20L:4D group had the highest sex pheromone content, significantly higher than the other five groups, while the 12L:12D group had the lowest relative sex pheromone content.

[0061] (3) Determination of the alkaline environment during the nutrient fortification stage:

[0062] Newly emerged adult tea bark beetles from the same batch were divided into six groups and placed in six rearing boxes, with five experimental groups and one control group. Sodium hydroxide solution was sprayed into the rearing boxes of the five experimental groups three times a day, maintaining a humidity of 60-65%. The concentrations of sodium hydroxide solution sprayed into the five groups were 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L, and 160 mg / L, respectively. The control group was not sprayed. The temperature was 25-27℃, the relative humidity was 60-65%, and the photoperiod was 14L:10D. Other conditions for the experimental groups were the same as those for the control group. After 3 days, sex pheromones were extracted from the gonads of each group for quantitative analysis, with three replicates per group. The sex pheromone extraction method was used for dichloromethane immersion extraction, and the quantitative analysis was performed using gas chromatography-antennae potential coupling technology and two-dimensional gas chromatography-time-of-flight mass spectrometry. The results showed that the 120 mg / L sodium hydroxide group had the highest sex pheromone content, significantly higher than the other five groups, while the control group had the lowest relative sex pheromone content.

[0063] (4) Determination of nutritional fortifiers:

[0064] Newly emerged adult tea wood beetles from the same batch were divided into six groups and placed in six rearing boxes, with five experimental groups and one control group. The experimental groups were fed a mixture of bark powder and a nutrient fortifier at a weight ratio of 10:1, with fortification lasting for three days. The nutrient fortifiers used in the five experimental groups were: Artemia eggs, tea powder, chlorella, soybean powder, and yeast powder, respectively. The control group received only bark powder and no nutrient fortifier. All feeds were mixed with water to a state free of lumps, able to be formed into a ball by hand, and slightly loose when released. All other rearing conditions were the same for all six groups: temperature 25-27℃, relative humidity 60-65%, and photoperiod 14L:10D. After three days, sex pheromones were extracted from the gonads of each group for quantitative analysis, with three replicates per group. The sex pheromone extraction method was used, and quantitative analysis was performed using gas chromatography-antennae potential coupling and two-dimensional gas chromatography-time-of-flight mass spectrometry. The results showed that the sex pheromone content was the highest in the Artemia egg group, significantly higher than the other five groups, while the sex pheromone content was the lowest in the control group.

[0065] (5) Determination of nutritional fortification conditions:

[0066] Newly emerged tea bark beetle adults were divided into sixteen groups and placed in sixteen rearing boxes, with fifteen experimental groups and one control group. The rearing conditions for the fifteen experimental groups were as follows: high temperature 32℃, photoperiod 20L:4D, spraying with 120 mg / L sodium hydroxide solution, supplemented with Artemia eggs for nutritional enhancement; high temperature 32℃ + photoperiod 20L:4D, spraying with 120 mg / L sodium hydroxide solution + supplemented with Artemia eggs for nutritional enhancement; high temperature 32℃ + spraying with 120 mg / L sodium hydroxide solution, supplemented with Artemia eggs for nutritional enhancement + photoperiod 20L:4D; high temperature 32℃ + supplemented with Artemia eggs for nutritional enhancement; photoperiod 20L:4D + spraying with 120 mg / L sodium hydroxide solution; high temperature 32℃ + photoperiod 20L:4D + spraying with 120 mg / L sodium hydroxide solution + supplemented with Artemia eggs for nutritional enhancement; high temperature 32℃ + photoperiod 20L:4D + supplemented with Artemia eggs for nutritional enhancement; high temperature 32℃ + spraying with 120 mg / L sodium hydroxide solution, spraying with 120 mg / L sodium hydroxide solution, supplemented ... The experimental group was treated with a 120 mg / L sodium hydroxide solution plus Artemia salina egg fortification, at a high temperature of 32℃ and a photoperiod of 20L:4D, and was sprayed with 120 mg / L sodium hydroxide solution plus Artemia salina egg fortification. The control group was treated with a temperature of 25-27℃, relative humidity of 60-65%, a photoperiod of 14L:10D, without sodium hydroxide solution spraying or nutrient fortification, and was only fed bark powder. All feeds were mixed with water to a state free of lumps, able to be formed into a ball by hand, and slightly loose when released. Other conditions in the experimental group were the same as those in the control group. Adding Artemia salina egg fortification refers to a bark powder to Artemia salina egg weight ratio of 10:1 in the feed. After 3 days, sex pheromones were extracted from the gonads of each group for quantitative analysis, with three replicates per group. Sex pheromones were extracted using the dichloromethane immersion extraction method, and quantitative analysis was performed using gas chromatography-antennae potential coupling technology and full two-dimensional gas chromatography-time-of-flight mass spectrometry. The results showed that the group with high temperature of 32℃ + photoperiod of 20L:4D + spraying with 120 mg / L sodium hydroxide solution + supplementation with Artemia eggs had the highest sex pheromone content, which was significantly higher than the other fifteen groups. The control group had the lowest sex pheromone content.

[0067] Example 5: Extraction and Use of Sex Pheromone

[0068] Unmated female tea bark beetles, after being nutritionally fortified for 3 days, were placed on an experimental platform. Their gonads were harvested, and sex pheromones were extracted using a dichloromethane extraction method. The gonads were immersed in dichloromethane at 4°C for 60 minutes, with 30 μL of dichloromethane used per gonad. The extracted sex pheromones were then identified and analyzed using gas chromatography-antennae potentiometric chromatography (GC-ETC) and two-dimensional gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS). The extracted and identified sex pheromones were used to prepare attractants, which were then placed into baits in trapping equipment. The trapping equipment was placed in the experimental forest, with each bait containing 200 μg of sex pheromones.

[0069] Example 6: Controlling the Reproduction of Tea Tree Bark Beetle Using Sex Pheromone Induction

[0070] (1) Harvesting of adult tea bark beetles:

[0071] Moist, decaying wood infested with adult tea leaf beetles was collected. The beetles were then extracted from the wood using an electric shock method. The electric shock device consisted of a controller, a 12V low-voltage DC power supply, an ammeter, wires, electrode A, and electrode B. Electrode A and electrode B were the positive and negative terminals, respectively, and were inserted into the two ends of the decaying wood. The current was controlled at 1-1.2 mA. If the wood was not sufficiently moist, a suitable amount of salt water could be sprayed onto it. After 5 minutes of electric shock, all the adult tea leaf beetles escaped from the decaying wood and were quickly collected in a rearing box.

[0072] (2) The rearing and egg-laying of the tea leaf beetle:

[0073] Place the rearing box inside the rearing box, with a rearing temperature of 25-27℃, relative humidity of 60-65%, and a photoperiod of 14L:10D. The feed used is a mixture of bark powder and water, which should be free of lumps, able to be formed into a ball by hand, and slightly loose when released. After the adult tea wood beetles in the rearing box mate and lay eggs, collect the egg masses.

[0074] (3) Rapid hatching of tea bark beetle eggs:

[0075] The collected egg masses were placed in an incubator, and the temperature inside the incubator was controlled at 16°C. The egg masses were periodically sprayed with a low concentration of 30% alcohol by volume, and the relative humidity inside the incubator was maintained at about 88%. At the same time, the blue light in the incubator was turned on. The wavelength of the blue light produced by the blue light was 480~500 nm and the light intensity was 500~600 Lux. During the incubation period, the blue light was turned on for an additional hour every morning under the basic photoperiod (14L:10D). Under these conditions, the eggs will hatch into larvae in 3-4 days.

[0076] (4) Larval rearing of the tea bark beetle:

[0077] The hatched larvae are placed in a larval rearing box, which is then placed in the rearing box. The rearing temperature is 25-27℃, the relative humidity is 60-65%, and the photoperiod is 14L:10D. The feed used is a mixture of bark powder and water. The feed should be free of lumps, can be formed into a ball by hand, and should crumble slightly when loosened. After the larvae pupate, the pupae are collected for later use.

[0078] (5) Rapid emergence of the tea leaf beetle pupa:

[0079] The collected pupae were placed in a hatching box, and the temperature inside the hatching box was controlled at 18°C ​​and the relative humidity at about 86%. At the same time, a white high-intensity light was turned on inside the hatching box. The light intensity of the light was 1200 Lux and the photoperiod was 14L:10D. Under these conditions, the pupae will emerge as adults in 2-3 days.

[0080] (6) Nutritional fortification of adult tea bark beetles:

[0081] The newly emerged adult insects were placed in an adult rearing box, maintaining a temperature of 32°C and a photoperiod of 20L:4D. A 120 mg / L sodium hydroxide solution was sprayed into the rearing box three times a day, maintaining a humidity of 60-65%. The feed used was a mixture of bark powder and Artemia eggs as a nutritional fortifier, with a weight ratio of bark powder to Artemia eggs of 10:1. The feed was mixed with water to form a clump-free mixture that could be formed into a ball by hand but crumble slightly when loosened. The nutritional fortification was continued for 3 days.

[0082] (7) Extraction of sex pheromones:

[0083] Unmated female tea bark beetles, after being fortified with nutrients for 3 days, were placed on an experimental table, and their gonads were removed. Sex pheromones were extracted from the gonads using a dichloromethane immersion extraction method. The gonads were immersed in dichloromethane at 4°C for 60 minutes, with 30 μL of dichloromethane used for each gonad. The extracted sex pheromones were then identified and analyzed using gas chromatography-antennae potential coupling technology and two-dimensional gas chromatography-time-of-flight mass spectrometry.

[0084] (8) Preparation and use of lure cores:

[0085] The extracted and identified sex pheromones are made into attractants, and the attractants are made into lures and placed in trapping equipment. The trapping equipment is placed in the forest, and each lure contains a dose of 200 μg of sex pheromones.

Claims

1. A method for controlling the reproduction of the tea bark beetle using sex pheromones, characterized in that, Includes the following steps: (1) Harvesting of adult tea bark beetles: Moist, decaying wood infested with adult tea leaf beetles was collected, and the beetles were extracted from the wood using an electric shock method. The electric shock device included a controller, a 12V low-voltage DC power supply, an ammeter, wires, electrode A, and electrode B. Electrode A and electrode B were the positive and negative terminals, respectively, and were inserted into the two ends of the decaying wood. The current was controlled to be 1-1.2 mA. If the decaying wood was not sufficiently moist, a suitable amount of salt water could be sprayed onto it. After 5 minutes of electric shock, all the adult tea leaf beetles escaped from the decaying wood and were quickly collected in a rearing box. (2) The rearing and egg-laying of the tea bark beetle; (3) Rapid hatching of tea bark beetle eggs: The collected egg masses were placed in an incubator, and the temperature inside the incubator was controlled at 16°C. The egg masses were periodically sprayed with a low concentration of 30% alcohol by volume, and the relative humidity inside the incubator was maintained at about 88%. At the same time, the blue light in the incubator was turned on. The wavelength of the blue light produced by the blue light was 480~500 nm and the light intensity was 500~600 Lux. The blue light was turned on for 1 hour every morning during the incubation period. Under these conditions, the eggs will hatch into larvae in 3-4 days. (4) Larval rearing of tea bark beetle; (5) Rapid emergence of the tea leaf beetle pupa: The collected pupae were placed in a hatching box, and the temperature inside the hatching box was controlled at 18℃ and the relative humidity at about 86%. At the same time, a white high-intensity light in the hatching box was turned on, and the light intensity of the light was 1200 Lux. Under these conditions, the pupae will emerge as adults in 2-3 days. (6) Nutritional fortification of adult tea bark beetles: The newly emerged adult insects were placed in an adult rearing box, maintaining a temperature of 32°C and a photoperiod of 20L:4D. A 120 mg / L sodium hydroxide solution was sprayed into the rearing box three times a day, maintaining a humidity of 60-65%. The feed consisted of a mixture of bark powder and Artemia eggs as a nutritional fortifier, with a weight ratio of bark powder to Artemia eggs of 10:

1. The feed was prepared with water to a consistency that was free of lumps, could be formed into a ball by hand, and crumbled slightly when loosened. The nutritional fortification was continued for 3 days. (7) Extraction of sex pheromones; (8) The production and use of lure cores.

2. The method for controlling the reproduction of the tea bark beetle using sex pheromones as described in claim 1, characterized in that, The rearing and egg-laying process of the tea wood beetle includes: placing the rearing box in the rearing chamber, maintaining a rearing temperature of 25-27℃, a relative humidity of 60-65%, and a photoperiod of 14L:10D; using a feed made of bark powder and water, which is free of lumps, can be formed into a ball by hand, and slightly crumbles when loosened; and collecting the egg masses after the adult tea wood beetles in the rearing box mate and lay eggs.

3. The method for controlling the reproduction of the tea bark beetle using sex pheromones as described in claim 2, characterized in that, The larval rearing of the tea bark beetle includes: placing the hatched larvae in a larval rearing box, which is then placed in the rearing box. The rearing temperature is 25-27℃, the relative humidity is 60-65%, and the photoperiod is 14L:10D. The feed used is a mixture of bark powder and water, which is free of lumps, can be formed into a ball by hand, and crumbles slightly when loosened. After the larvae pupate, the pupae are collected for later use.

4. The method for controlling the reproduction of the tea bark beetle using sex pheromones as described in claim 1, characterized in that, The extraction of sex pheromones involved placing unmated female tea bark beetles, after 3 days of nutritional fortification, on an experimental table, removing their gonads, and extracting the sex pheromones from the gonads using a dichloromethane immersion extraction method. The gonads were immersed in dichloromethane at 4°C for 60 minutes, with 30 μL of dichloromethane used for each gonad. The extracted sex pheromones were then identified and analyzed using gas chromatography-antennae potential coupling technology and two-dimensional gas chromatography-time-of-flight mass spectrometry.

5. The method for controlling the reproduction of the tea bark beetle using sex pheromones as described in claim 1, characterized in that, Preparation and use of the lure core: The extracted and identified sex pheromones are made into an attractant, the attractant is made into a lure core and placed in the trapping equipment, and the trapping equipment is placed in the forest. The sex pheromone dose contained in a single lure core is 200 μg.

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