A method for studying the feedback of empoasca flavescens to light and sound stimuli

Abstract

The application discloses a method for researching on the feedback of Erythroneura japonica to light and sound stimulation, and relates to the technical field of Erythroneura japonica research. The method comprises the following steps: step S1, preparation of test insects and plants; step S2, research on the reaction of multiple groups of control tests; step S3, research on the reaction of Erythroneura japonica to light and sound; step S4, recording of the reaction data of Erythroneura japonica; and step S5, statistical analysis of data results. The method for researching on the influence of Erythroneura japonica on light and sound breaks through the combination of light and sound factors, systematically researches the independent regulation and interaction of the two factors on the behavior of Erythroneura japonica, and fills the blank of the sound factor in the existing research. By strictly controlling the feeding conditions of test insects, test environment parameters and behavior determination standards, a standardized research process is constructed, and the repeatability of the test and the reliability of the results are ensured.

Description

Technical Field

[0001] This invention relates to the field of research technology on the small green leafhopper, specifically a method for studying the small green leafhopper's response to light and sound stimuli. Background Technology

[0002] The adult *Lepidoptera spp.* is quite active and agile. However, in most cases, the adult leafhoppers that inhabit tea trees remain in the same position for a long time. This is related to the leafhoppers' feeding habits. Most leafhoppers will quickly fly away or hide in other areas to avoid light stimulation after being exposed to light. Leafhoppers are one of the earliest groups of insects to be proven to use vibration signals for sexual communication. The courtship signals of *Lepidoptera spp.* rely on plants for transmission, and its courtship behavior is very conservative.

[0003] Patent publication number CN115500323A discloses a method for rapidly obtaining a large number of tea green leafhopper nymphs. It uses blue light monitoring to directly screen tea branches carrying tea green leafhopper eggs, eliminating the mating, fertilization, and egg-laying stages, shortening the indoor rearing cycle, and rapidly and in large quantities obtaining nymphs of all instars of the tea green leafhopper. It eliminates the need for manual insect capture, avoiding the inefficient and labor-intensive insect rearing problems of time-consuming and labor-intensive manual capture and low survival rates of captured adults.

[0004] Patent publication number CN106550933A discloses a method for controlling the small green leafhopper. Based on the needs of food safety and ecological environment protection, the development and utilization of environmentally safe substances to replace traditional pesticides to control plant diseases and pests has received widespread attention. Using certain environmentally safe substances, such as plant volatiles, to control pests is an important research direction in this field. This method achieves the goal of efficiently and selectively aggregating the small green leafhopper while minimizing damage to natural enemies.

[0005] While the methods disclosed in the aforementioned patents provide ways to quickly obtain large numbers of tea green leafhopper nymphs for control, they do not involve a systematic study of the behavioral responses of the tea green leafhopper to light and sound stimuli. Furthermore, the light studies lack research on different wavelengths of light and specific sound frequencies, and there is a lack of targeted behavioral interference methods for the research subjects. Moreover, the research approach is too simplistic and cannot effectively study the responses of the tea green leafhopper to light and sound stimuli.

[0006] To address the aforementioned issues, there is an urgent need for innovative designs based on existing methods. Summary of the Invention

[0007] The purpose of this invention is to provide a method for studying the response of the small green leafhopper to light and sound stimuli, in order to solve the problems mentioned in the background art that the existing methods do not involve a systematic study of the behavioral response of the small green leafhopper to light and sound stimuli, lack research on different wavelengths of light and specific sound frequencies in light studies, lack specific behavioral interference methods for the research object, and have an overly simplistic research approach, which cannot effectively study the response of the small green leafhopper to light and sound stimuli.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for studying the response of the small green leafhopper to light and sound stimuli, comprising the following steps:

[0009] Step S1: Preparation of test insects and plants:

[0010] Rearing of the small green leafhopper, construction of experimental equipment and grouping of test insects;

[0011] Step S2, Response Study of Multiple Controlled Trials:

[0012] Single-factor influence test: The effects of single factors such as light and sound on the behavior of the small green leafhopper were tested respectively.

[0013] Step S3: Study on the leafhopper's response to light and sound:

[0014] The influence of light and sound interaction factors was tested to explore the effects of light and sound alone and in combination on the behavior of the small green leafhopper.

[0015] Step S4: Recording of leafhopper response data:

[0016] Behavioral observation and indicator recording, clarifying behavioral judgment criteria, and statistically analyzing the phototaxis, photophobia, sound-attracting, sound-avoiding rates, and behavioral response time of test insects;

[0017] Step S5: Statistical analysis of data results:

[0018] Statistical methods were used to analyze the experimental data to clarify the significance and mechanism of each factor on the behavior of the small green leafhopper.

[0019] Preferably, in step S1, the rearing of the small green leafhopper is divided into wild-type and indoor-type test insects. The wild-type test insects are adult insects collected from tea gardens and reared for 1 day, while the indoor-type test insects are second-generation 6-7 day old adult insects that have been continuously reared indoors. The rearing environment is controlled with a photoperiod of L14:D10, a temperature of 25±1℃, and a humidity of 75%±5%, and disease-free tea branches are used for rearing.

[0020] Preferably, in step S1, the test equipment setup includes a light-shielding and sound-insulating test chamber, which is equipped with a light adjustment module, a sound adjustment module, a behavior observation module, and an environmental control module. The light adjustment module uses LED lights with adjustable wavelength and intensity, the sound adjustment module uses a signal generator with adjustable frequency, intensity, and type, and a directional speaker, the behavior observation module uses a high-definition camera, and the environmental control module uses a temperature and humidity controller.

[0021] Preferably, in step S1, the test insects are grouped into resting and searching states according to sex and host status. The resting test insects are those that are at rest after being introduced into the tea branch, and the searching test insects are those that are active in the absence of tea branches. Each group contains no less than 30 test insects, and each test insect participates in only one test.

[0022] Preferably, in step S2, the single-factor effect test of illumination includes the combined effect test of illumination wavelength, illumination intensity, photoperiod, and temperature. The illumination wavelengths are selected as narrow-wavelength light of 421.6 nm, 475.2 nm, 498 nm, 592.6 nm, and 626.8 nm; the illumination intensity is set with a gradient of 0.01–10 μW·cm⁻¹s⁻¹; the photoperiod is set with a gradient of 8 h, 10 h, 12 h, 14 h, and 16 h; and the temperature is set with a gradient of 10–35 °C.

[0023] Preferably, in step S2, the single-factor sound influence test includes the influence tests of sound frequency, sound intensity, and sound type; the sound frequency is set with a gradient of 100Hz to 5000Hz, the sound intensity is set with a gradient of 30dB to 90dB, and the sound type includes pure tone, simulated tea garden wind sound, simulated tea picking machine operation sound, and simulated small green leaf cicada chirping sound.

[0024] Preferably, in step S3, the test of the influence of light and sound interaction factors is based on the significant influence parameters screened in step S2, setting standard light and different sound parameter combinations, standard sound and different light parameter combinations, and exploring the regulatory effect of temperature on the interaction.

[0025] Preferably, in step S4, the behavioral criteria are as follows: For insects in a searching state, moving towards the light source within 3 seconds indicates a phototactic response; for insects in a resting state, moving away from the light source under light stimulation indicates a photoavoidant response. For insects in a perch state, moving towards the speaker within 3 seconds indicates a sound-attracting response; moving away from the speaker indicates a sound-avoidant response. No of the above behaviors within 10 minutes indicates no response.

[0026] Preferably, in step S5, the data analysis uses SPSS 25.0 software, including two independent samples t-test, chi-square analysis, linear regression analysis and analysis of variance, to clarify the significance and linear relationship of the influence of each factor on the behavior of the small green leafhopper.

[0027] Preferably, the within-group factor for repeated measures ANOVA is time, white noise stress group, control group, analysis time and the interaction between treatment groups and the effect of noise stress on leafhopper mating behavior. All experiments are repeated at least 3 times, and experimental environmental parameters are strictly controlled to ensure experimental repeatability and reliability of results.

[0028] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention innovatively combines light and sound factors to systematically study the independent regulation and interaction of the two on the behavior of the small green leafhopper, filling the gap in existing research on sound factors. By strictly controlling the breeding conditions of test insects, experimental environmental parameters, and behavioral judgment criteria, a standardized research process is constructed to ensure the repeatability of the experiment and the reliability of the results. The study fully considers synergistic factors such as the sex, origin, host status, and environmental temperature of the test insects, making the results closer to the actual situation in tea gardens. It provides a new approach to overcome the limitations of single light regulation. The equipment of this invention is simple to set up, easy to operate, and has flexible parameter adjustment. It is highly versatile and suitable for precise laboratory research and field pilot testing. It can be widely used in the fields of insect behavior and green control of tea garden pests and diseases, and has important practical application value and broad prospects for promotion.

[0029] 1. This invention is the first to combine light and sound factors, breaking through the technical limitations of existing research on the behavior of the small green leafhopper, which focuses only on the single environmental factor of light. Existing research mainly revolves around the effects of light wavelength, intensity, and photoperiod on the phototaxis of leafhoppers, completely ignoring the regulatory role of sound, which is prevalent in tea garden environments, on leafhopper behavior. It also fails to explain the interaction between light and sound, resulting in an incomplete understanding of the intrinsic mechanisms of leafhopper behavioral changes and hindering the development of multi-factor synergistic regulation technologies. This invention, by systematically constructing a research system for single and interactive factors of light and sound, not only clarifies the independent regulatory mechanisms of light and sound on the phototaxis / photophobia and sound-attracting / sound-avoidance behaviors of the small green leafhopper, but also elucidates their synergistic or antagonistic relationships. It fills the gap in research on sound factors in the behavior of the small green leafhopper, providing a more comprehensive research perspective and more scientific conclusions. This provides core research evidence for subsequent multi-environmental factor synergistic regulation of leafhopper behavior and offers new research ideas to overcome the limitations of single light regulation.

[0030] 2. This invention strictly controls the rearing conditions and experimental environmental parameters of the test insects, minimizing the interference of irrelevant factors on the experimental results. Targeting the growth habits and behavioral characteristics of the *Spodoptera exigua*, this invention precisely sets core environmental conditions such as temperature, humidity, and photoperiod within the rearing room and experimental chamber. It clarifies the rearing standards and acclimatization cycles for wild and indoor test insects, refines the grouping criteria for test insects, and standardizes behavioral judgment criteria and data analysis methods. This effectively avoids experimental errors caused by fluctuations in rearing conditions, chaotic grouping, ambiguous judgment criteria, and non-standard data analysis, ensuring the repeatability and reliability of the results. The standardized research process constructed by this invention can provide a unified methodological reference for subsequent behavioral research on *Spodoptera exigua*, reducing the experimental design costs of subsequent research, minimizing deviations in research results due to methodological differences, ensuring the comparability of experimental results between different research teams, and promoting the rapid improvement and technological transformation of the *Spodoptera exigua* behavioral regulation theory.

[0031] 3. Furthermore, this invention fully considers multiple synergistic factors such as insect sex, origin, host status, and ambient temperature, breaking through the limitations of existing studies that neglect individual differences and environmental synergistic effects. The research scenario is closer to the actual tea garden field environment. In the field environment, the leafhopper *Spodoptera litura* naturally exhibits characteristics such as mixed male and female populations, dynamic changes in host status, and seasonal temperature fluctuations. These factors directly affect the leafhopper's behavioral response to light and sound. If these synergistic factors are ignored, the research results will be disconnected from actual field applications and will be difficult to provide practical guidance. This invention incorporates these synergistic factors into the research scope, clarifying the effects of light and sound on leafhopper behavior under different synergistic conditions. To understand the influence of this research, the results not only refine the theory of behavioral regulation of the small green leafhopper, but also provide targeted scientific support for optimizing the control technology of the small green leafhopper in tea gardens. Addressing the practical problem of reduced light-attracting effectiveness due to low temperatures in late autumn, this invention utilizes the sound-light interaction mechanism to enhance the leafhopper's phototaxis rate and improve the light-attracting control effect by applying sound stimuli of specific frequencies and intensities. Simultaneously, it provides a technical basis for the development of novel behavioral regulation products, which can be directly applied to tea garden production practices, reducing the use of chemical pesticides, minimizing pesticide residues, and improving tea quality and safety. This has significant practical application value and broad prospects for promotion.

[0032] 4. Furthermore, the experimental equipment of this invention is simple to set up, easy to operate, and flexible in parameter adjustment. It is highly versatile and practical, requiring no complex and expensive precision instruments, thus lowering the research threshold. The customized LED lights, signal generators, directional loudspeakers, and light-shielding and sound-insulating test chambers used in this invention are all commonly used equipment in the field of agricultural research. They are easy to obtain and assemble, and do not require professional high-end operating skills. Ordinary researchers can complete the equipment debugging and experimental operation after simple training. Core experimental parameters such as light wavelength, light intensity, sound frequency, sound intensity and type, and ambient temperature can all be flexibly adjusted according to research needs, and can be quickly adapted to different research objectives—such as exploring the behavioral characteristics of leafhoppers in specific tea-growing areas, the behavioral response of leafhoppers under specific environmental conditions, and the differences in the effects of different sound types on leafhoppers. There is no need to rebuild the experimental platform, which effectively improves experimental efficiency and reduces experimental costs.

[0033] 5. Furthermore, the research method of this invention is applicable to the study of the behavioral effects of the small green leafhopper in different scenarios. It can be used for precise laboratory research as well as for small-scale field pilot tests. At the same time, it can meet the behavioral research needs of the small green leafhopper in different tea-growing areas and at different growth stages. It is highly versatile and widely applicable, and can be widely used in research and practice in related fields such as insect behavior and green control of tea garden pests and diseases. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the workflow of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1: In a specific embodiment, the present invention provides the following technical solution: a method for studying the response of the small green leafhopper to light and sound stimuli, such as... Figure 1 As shown, the preparation process for studying the response of the small green leafhopper using this method is disclosed.

[0037] Step S1: Preparation of test insects and plants:

[0038] The rearing of the small green leafhopper, the construction of experimental equipment, and the grouping of test insects.

[0039] In step S1, the rearing of the small green leafhopper was divided into wild and indoor test insects. The wild test insects were adults collected from tea gardens and reared for 1 day, while the indoor test insects were second-generation adults aged 6-7 days who were continuously reared indoors. The rearing environment was controlled with a photoperiod of L14:D10, a temperature of 25±1℃, and a humidity of 75%±5%, using disease-free tea branches grown hydroponically.

[0040] In step S1, the test equipment setup includes a light-shielding and sound-insulating test chamber, which contains a light adjustment module, a sound adjustment module, a behavior observation module, and an environmental control module. The light adjustment module uses LED lights with adjustable wavelength and intensity, the sound adjustment module uses a signal generator with adjustable frequency, intensity, and type, and a directional speaker, the behavior observation module uses a high-definition camera, and the environmental control module uses a temperature and humidity controller.

[0041] In step S1, the test insects are grouped into resting and searching states according to sex and host status. The resting test insects are those that are at rest after being introduced into the tea branch, and the searching test insects are those that are active in the absence of tea branch. Each group contains no less than 30 test insects, and each test insect participates in only one experiment.

[0042] Step S2, Response Study of Multiple Controlled Trials:

[0043] Single-factor influence tests were conducted to examine the effects of single factors, namely light and sound, on the behavior of the small green leafhopper.

[0044] In step S2, the single-factor effect test of illumination includes the combined effects of illumination wavelength, illumination intensity, photoperiod, and temperature. Narrow-wavelength light of 421.6 nm, 475.2 nm, 498 nm, 592.6 nm, and 626.8 nm is selected; illumination intensity is set with a gradient of 0.01–10 μW·cm⁻¹s⁻¹; photoperiod length is set with gradients of 8 h, 10 h, 12 h, 14 h, and 16 h; and temperature is set with a gradient of 10–35 °C.

[0045] In step S2, the single-factor sound influence test includes the influence tests of sound frequency, sound intensity, and sound type. The sound frequency is set with a gradient of 100Hz to 5000Hz, the sound intensity is set with a gradient of 30dB to 90dB, and the sound types include pure tones, simulated tea garden wind sounds, simulated tea picking machine operation sounds, and simulated cicada chirping sounds.

[0046] Healthy adult *Spodoptera litura* leafhoppers were collected from the tea garden of the Tea Research Institute of the Chinese Academy of Agricultural Sciences and reared as both wild-type and indoor test insects.

[0047] Wild-type test insects: The collected adult insects were placed in insect cages of 60×60×60cm and raised on hydroponic Longjing 43 tea branches. The environmental conditions of the insect rearing room were controlled as follows: photoperiod L14:D10, temperature 25±1℃, humidity 75%±5%. After rearing for 1 day, they were used as wild-type test insects for later use. Each group of test insects had 40 insects.

[0048] Indoor test insects: Adults collected in the wild were continuously reared indoors. Starting from the 4th instar nymphs, they were transferred to individual fruit fly tubes and placed in RTOP-500Y artificial incubators. The rearing conditions were the same as in the insect rearing room. 6-7 day old second-generation adults in the indoor mating peak period were selected as indoor test insects for backup. 40 test insects were selected for each group of backup test insects.

[0049] Replace the hydroponic tea branches daily with fresh ones, ensuring that there are at least 8 leaves under each tea bud, and remove any diseased, weak, or dead tea leaves.

[0050] Experimental equipment setup. A 50×50×50cm light-shielding and sound-insulating test chamber was constructed. The chamber was equipped with modules for light adjustment, sound adjustment, behavior observation, and environmental control.

[0051] Lighting adjustment module: It uses LED lights customized by Hangzhou Yihao Agricultural Technology Co., Ltd. as the light source, which can emit narrow wavelength light with a half bandwidth of 20nm and a light spot diameter of 3.5cm. It is equipped with neutral density filters from Beijing Zhongjiao Jinyuan Technology Co., Ltd. and Uni-Trend UTP3305-Ⅱ power supply transformers to adjust the light wavelength and light intensity.

[0052] Sound adjustment module: It adopts a signal generator, power amplifier and directional speaker. The speaker is fixed on the right side wall of the test chamber and 10cm away from the activity area of ​​the test insect. It is used to adjust the sound frequency, sound intensity and sound type. The sound frequency range is 100Hz~5000Hz and the sound intensity range is 30dB~90dB.

[0053] Behavioral observation module: A high-definition camera (1080P resolution) is installed on the top of the test chamber, which is connected to an external monitor and recording equipment to observe and record the behavioral responses of the test insects in real time. The shooting angle covers the entire activity area of ​​the test insects.

[0054] Environmental control module: The temperature and humidity controller is used to control the temperature in the test chamber at 10-35℃ and the humidity at 75%±5%, and the temperature gradient can be adjusted.

[0055] Insect grouping: The prepared field- and indoor-type test insects were divided into 8 groups according to sex (female and male) and host status (inhabiting and searching), with 30 insects in each group. Each insect participated in only one experiment.

[0056] Insects in a resting state: Insects were introduced into hydroponically grown Longjing 43 tea branches and placed in a test chamber for dark adaptation for 10 minutes. Once the insects had settled on the stems or leaves of the tea branches, they were considered to be insects in a resting state.

[0057] Search-state test insects: Place the test insects in a test area without tea branches and allow them to adapt to the dark for 30 minutes. Once the test insects exhibit crawling or flying activities, they are considered search-state test insects.

[0058] Step S2: Single Factor Influence Test

[0059] The effects of single factors such as light and sound on the behavior of the small green leafhopper were tested separately, with each group of experiments repeated three times.

[0060] Single-factor effect test of light: The test chamber was kept free of sound interference (sound intensity ≤20dB) and the temperature was 25±1℃.

[0061] Light wavelength effect test: Five narrow wavelengths of light (421.6nm, 475.2nm, 498nm, 592.6nm, 626.8nm) were selected, with white light as a control. The light intensity was uniformly 50μW·cm-1s-1. Each group of test insects was irradiated, and the behavioral response of the test insects within 10 minutes was observed and recorded.

[0062] Light intensity effect test: 421.6nm violet light was selected as the stimulus light, and 12 light intensity gradients (0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 7, 8, 9, 10μW·cm-1s-1) were set to irradiate each group of test insects, and the behavioral response of the test insects within 10 minutes was observed and recorded.

[0063] Photoperiod effect test: The test insects were placed under five photoperiod conditions (photoperiod length 8h, 10h, 12h, 14h, 16h) for 3 days in advance. 421.6nm violet light was selected as the stimulus light and the light intensity was 2.83μW·cm-1s-1. The test insects in each group were irradiated respectively, and the behavioral response of the test insects within 10 minutes was observed and recorded.

[0064] Temperature-induced light synergy test: 421.6nm violet light was selected as the stimulus light, with a light intensity of 2.83μW·cm-1s-1. Six temperature gradients (10℃, 15℃, 20℃, 25℃, 30℃, 35℃) were set to irradiate each group of test insects. The behavioral responses of the test insects within 10 minutes were observed and recorded.

[0065] Light as a single factor: The small green leafhopper is most sensitive to 421.6nm violet light, and its phototaxis and photophobia rates increase significantly with increasing light intensity. The phototaxis rate initially increases and then decreases with increasing temperature within the range of 10–35℃, reaching its maximum at 30℃. The phototaxis rate of males increases with increasing photoperiod length, while there is no significant change in females.

[0066] Example 2: In one specific embodiment, such as Figure 1 As shown, this study investigates the effects of noise stress on the behavior of resting leafhoppers.

[0067] Step S3: Study on the leafhopper's response to light and sound:

[0068] The influence of light and sound interaction factors was tested to explore the effects of light and sound, both individually and in combination, on the behavior of the small green leafhopper.

[0069] In step S3, the test of the influence of light and sound interaction factors is based on the significant influence parameters screened in step S2. Standard light and different sound parameter combinations, and standard sound and different light parameter combinations are set up. At the same time, the regulatory effect of temperature on the interaction is explored.

[0070] The test chamber was kept free from light interference (light intensity ≤ 0.01 μW·cm⁻¹s⁻¹) and the temperature was 25 ± 1℃.

[0071] Sound frequency influence test: Three sound types were selected (pure tone, simulated tea garden wind sound, and simulated tea picking machine operation sound). Five frequency gradients were set for each sound type (100Hz, 500Hz, 1000Hz, 3000Hz, and 5000Hz). The sound intensity was uniformly set at 60dB. The test insects in each group were stimulated with sound, and their behavioral responses were observed and recorded within 10 minutes.

[0072] Sound intensity effect test: The most sensitive sound frequency (1000Hz pure tone) was selected for the test insects. Five sound intensity gradients (30dB, 45dB, 60dB, 75dB, 90dB) were set. The test insects in each group were stimulated with sound, and their behavioral response was observed and recorded within 10 minutes.

[0073] Sound type influence test: The sound intensity was set to 60dB, and four sound types were set (pure tone, tea garden wind simulation sound, tea picking machine operation simulation sound, and small green leaf cicada chirping simulation sound). The sound stimulation was applied to each group of test insects, and the behavioral response of the test insects within 10 minutes was observed and recorded.

[0074] Single sound factor: The small green leafhopper is most sensitive to a pure tone of 1000Hz. Its attraction to sound increases with increasing sound intensity, while its avoidance of sound decreases with increasing sound intensity. The simulated sound of a tea-harvesting machine significantly triggers the insect's avoidance response, while the simulated sound of the small green leafhopper's chirping slightly increases its attraction to sound.

[0075] Example 3: Based on the above examples, such as... Figure 1 As shown, the overall effect of this method on the result processing is disclosed.

[0076] Based on the test results of step S2, parameters that significantly affect the behavior of the test insects were selected: light parameters (421.6nm violet light, light intensity 2.83μW·cm⁻¹s⁻¹, photoperiod L14:D10) and sound parameters (1000Hz pure tone, sound intensity 60dB). A light-sound interaction test group was set up, with the temperature inside the test chamber controlled at 25±1℃. Each test group was repeated three times.

[0077] Interactive parameter settings: Standard illumination + different sound parameters: 421.6nm violet light, illumination intensity 2.83μW·cm⁻¹s⁻¹, photoperiod L14:D10, combined with 1000Hz pure tone (30dB, 60dB, 90dB), 500Hz pure tone (60dB), and tea-picking machine operation simulation sound (60dB), respectively. Standard sound + different illumination parameters: 1000Hz pure tone, sound intensity 60dB, combined with 421.6nm violet light (0.5, 2.83, 10μW·cm⁻¹s⁻¹), 475.2nm blue light (2.83μW·cm⁻¹s⁻¹), and photoperiod L10:D14 (421.6nm violet light, 2.83μW·cm⁻¹s⁻¹).

[0078] Behavioral observation: Each group of test insects was placed in the test chamber and allowed to adapt to darkness for 30 minutes (searching state) or 10 minutes (resting state). Then, light and sound stimulation were applied simultaneously, and the behavioral response of the test insects was observed and recorded within 10 minutes.

[0079] Temperature-coordinated interaction test: Standard interaction conditions (421.6nm violet light + 1000Hz pure tone) were selected, and 6 temperature gradients (10℃, 15℃, 20℃, 25℃, 30℃, 35℃) were set to stimulate each group of test insects. The behavioral response of the test insects within 10 minutes was observed and recorded.

[0080] Step S4: Recording of leafhopper response data:

[0081] Behavioral observation and indicator recording were conducted to clarify the criteria for behavioral judgment and to statistically analyze the phototaxis, photophobia, sound-attracting, sound-avoiding rates, and behavioral response time of the test insects.

[0082] In step S4, the behavioral criteria are as follows: For insects in the search state, moving towards the light source within 3 seconds indicates a phototactic response; for insects in the resting state, moving away from the light source under light stimulation indicates a photoavoidant response. For insects in the speaker state, moving towards the speaker within 3 seconds indicates a sound-attracting response; moving away from the speaker indicates a sound-avoidant response; and the absence of any of the above behaviors within 10 minutes indicates no response.

[0083] Step S4: Behavioral observation and indicator recording.

[0084] The behavioral responses of the test insects were observed and recorded in real time using a high-definition camera, and the results were judged and statistically analyzed according to the following criteria:

[0085] Light-related behaviors: In search-state test insects moving towards the light source within 3 seconds is a phototactic response; in resting-state test insects moving away from the light source under light stimulation is a photoavoidance response; no of the above behaviors within 10 minutes is considered no response.

[0086] Sound-related behaviors: If the test insect moves toward the speaker within 3 seconds, it is a sound-attracting response; if it moves away from the speaker, it is a sound-avoiding response; if it does not exhibit the above behaviors within 10 minutes, it is considered a no-response response.

[0087] The phototaxis, photophobia, sound-attracting, sound-avoiding rates, and behavioral response times of each group of test insects were statistically analyzed.

[0088] Step S5: Data Analysis. Statistical analysis was performed using SPSS 25.0 software.

[0089] The differences in insect behavior indicators under different light and sound parameters were analyzed using a two-independent-samples t-test.

[0090] Chi-square analysis was used to analyze the significance of the effects of light, sound, temperature and interaction on the behavior of test insects. Fisher's exact probability method was used when the expected count was <5.

[0091] Linear regression analysis was used to explore the linear relationship between light intensity, sound intensity and insect behavior indicators.

[0092] Analysis of variance was used to analyze the differences in the effects of insect sex, origin, and host status on behavioral responses.

[0093] Interaction between light and sound: When 421.6nm violet light and 1000Hz pure tone acted synergistically, the phototaxis rate of the test insects was significantly higher than that of light stimulation alone. Under low temperature conditions (<15℃), sound stimulation could slightly increase the phototaxis rate of the test insects, which could alleviate the problem of decreased phototaxis caused by low temperature.

[0094] Differences in test insects: There were no significant differences in the light and sound behavior responses of male and female test insects, and between wild and indoor test insects. Test insects in the habitat mainly showed light and sound avoidance responses, while test insects in the search state mainly showed light and sound attraction responses.

[0095] The above results indicate that the method of the present invention can effectively explore the effects of light and sound on the behavior of the small green leafhopper, providing a scientific basis for optimizing the light-trapping control effect of the small green leafhopper (such as combining specific sound stimulation in late autumn to enhance phototaxis rate).

[0096] Step S5: Statistical analysis of data results:

[0097] Statistical methods were used to analyze the experimental data to clarify the significance and mechanism of each factor on the behavior of the small green leafhopper.

[0098] In step S5, data analysis was performed using SPSS 25.0 software, including two independent samples t-test, chi-square analysis, linear regression analysis, and analysis of variance, to clarify the significance and linear relationship of each factor on the behavior of the small green leafhopper.

[0099] The within-group factor for repeated measures ANOVA was time, white noise stress group, and control group. The interaction between time and treatment group and the effect of noise stress on leafhopper mating behavior were analyzed. All experiments were repeated at least three times, and the experimental environmental parameters were strictly controlled to ensure the repeatability and reliability of the results.

[0100] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0101] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for studying the response of the small green leafhopper to light and sound stimuli, characterized in that, Includes the following steps: Step S1: Preparation of test insects and plants: Rearing of the small green leafhopper, construction of experimental equipment and grouping of test insects; Step S2, Response Study of Multiple Controlled Trials: Single-factor influence test: The effects of single factors such as light and sound on the behavior of the small green leafhopper were tested respectively. Step S3: Study on the leafhopper's response to light and sound: The influence of light and sound interaction factors was tested to explore the effects of light and sound alone and in combination on the behavior of the small green leafhopper. Step S4: Recording of leafhopper response data: Behavioral observation and indicator recording, clarifying behavioral judgment criteria, and statistically analyzing the phototaxis, photophobia, sound-attracting, sound-avoiding rates, and behavioral response time of test insects; Step S5: Statistical analysis of data results: Statistical methods were used to analyze the experimental data to clarify the significance and mechanism of each factor on the behavior of the small green leafhopper.

2. The method for studying the response of the small green leafhopper to light and sound stimuli according to claim 1, characterized in that: In step S1, the rearing of the small green leafhopper is divided into wild and indoor test insects. The wild test insects are adult insects collected from tea gardens and reared for 1 day. The indoor test insects are second-generation 6-7 day old adult insects that are continuously reared indoors. The rearing environment conditions are controlled as photoperiod L14:D10, temperature 25±1℃, humidity 75%±5%, and the tea branches without diseases and pests are used for rearing.

3. The method for studying the response of the small green leafhopper to light and sound stimuli according to claim 1, characterized in that: In step S1, the test equipment setup includes a light-shielding and sound-insulating test chamber. Inside the test chamber, there are a light adjustment module, a sound adjustment module, a behavior observation module, and an environmental control module. The light adjustment module uses LED lights with adjustable wavelength and intensity. The sound adjustment module uses a signal generator with adjustable frequency, intensity, and type, and a directional speaker. The behavior observation module uses a high-definition camera. The environmental control module uses a temperature and humidity controller.

4. The method for studying the response of the small green leafhopper to light and sound stimuli according to claim 1, characterized in that: In step S1, the test insects are grouped into resting and searching states according to sex and host status. The resting test insects are those that are at rest after being introduced into the tea branch, and the searching test insects are those that are active in the absence of tea branch. Each group contains no less than 30 test insects, and each test insect participates in only one test.

5. A method for studying the response of the small green leafhopper to light and sound stimuli according to claim 1, characterized in that: In step S2, the single-factor effect test of illumination includes the combined effect test of illumination wavelength, illumination intensity, light period, and temperature. The illumination wavelength is selected as narrow-wavelength light of 421.6nm, 475.2nm, 498nm, 592.6nm, and 626.8nm. The illumination intensity is set with a gradient of 0.01 to 10μW·cm-1s-1. The light period is set with a gradient of 8h, 10h, 12h, 14h, and 16h. The temperature is set with a gradient of 10 to 35℃.

6. The method for studying the response of the small green leafhopper to light and sound stimuli according to claim 1, characterized in that: In step S2, the single-factor sound influence test includes the influence tests of sound frequency, sound intensity, and sound type; the sound frequency is set with a gradient of 100Hz to 5000Hz, the sound intensity is set with a gradient of 30dB to 90dB, and the sound type includes pure tone, simulated tea garden wind sound, simulated tea picking machine operation sound, and simulated small green leaf cicada chirping sound.

7. A method for studying the response of the small green leafhopper to light and sound stimuli according to claim 1, characterized in that: In step S3, the test of the influence of light and sound interaction factors is based on the significant influence parameters screened in step S2. Standard light and different sound parameter combinations, and standard sound and different light parameter combinations are set, while the regulatory effect of temperature on the interaction is explored.

8. A method for studying the response of the small green leafhopper to light and sound stimuli according to claim 1, characterized in that: In step S4, the behavioral judgment criteria are as follows: the insect in the search state moves towards the light source within 3 seconds as a phototactic response; the insect in the resting state moves away from the light source under light stimulation as a photoavoiding response; the insect moves towards the speaker within 3 seconds as a sound-attracting response; and moves away from the speaker as a sound-avoiding response. If no of the above behaviors are observed within 10 minutes, it is considered no response.

9. A method for studying the response of the small green leafhopper to light and sound stimuli according to claim 1, characterized in that: In step S5, data analysis was performed using SPSS 25.0 software, including two independent samples t-test, chi-square analysis, linear regression analysis, and analysis of variance, to clarify the significance and linear relationship of each factor on the behavior of the small green leafhopper.

10. A method for studying the response of the small green leafhopper to light and sound stimuli according to any one of claims 1-9, characterized in that: The within-group factor for repeated measures ANOVA was time, white noise stress group, and control group. The interaction between time and treatment group and the effect of noise stress on leafhopper mating behavior were analyzed. All experiments were repeated at least three times, and the experimental environmental parameters were strictly controlled to ensure the repeatability and reliability of the results.

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