Application of EAG in determination of insect retina potential

Abstract

The invention discloses an application of EAG in measuring the retinal potential of insects, which is characterized in that core components of the EAG, namely an IDAC-2 controller and a Combi probe, are utilized to extend the functions of the EAG, special retinal potential measuring equipment does not need to be purchased, and the hardware cost is reduced by about 70%. An original electrode is improved, an original antenna locator is replaced with an eye electrode support, a smell stimulation device is removed and replaced with a built light stimulation device, and cross-function application of the EAG from'antenna potential measurement 'to'retina potential measurement' is achieved. The EgPro software synchronously triggers the photostimulation device and data acquisition, and the problem of phase error caused by desynchrony of photostimulation and potential recording in a traditional method is solved. An electrolytic sharpening tungsten filament electrode is combined with a micromanipulator for positioning, so that compound eye single-cell-level precise puncture is realized.

Description

Technical Field

[0001] This invention belongs to the field of insect vision research, specifically relating to the application of EAG in measuring insect retinal potential. Background Technology

[0002] Retinal potential (ERG) measurement is a key method for studying insect vision and behavior. By measuring this potential, we can gain a deeper understanding of the insect's perception mechanism of light signals, explore the visual driving factors of its foraging and courtship behaviors, and thus provide a theoretical basis for developing green and efficient pest control technologies. It has important value in both insect scientific research and agricultural production practices.

[0003] However, there are currently few insect retinal potential measurement systems on the market, with only a very small number of specialized bioelectrophysiological equipment manufacturers offering such systems. These systems possess relatively advanced signal acquisition and analysis software, capable of preliminary filtering and feature analysis of the acquired potential signals. However, their price is extremely high, with a single system costing several million RMB, making it unaffordable for most research institutions. Furthermore, the maintenance costs are also high, requiring regular calibration and maintenance by specialized technicians. In addition, some domestic research institutions have designed and assembled their own measurement systems; however, this is challenging, the software has poor computer compatibility, and updates are difficult. These factors significantly limit my country's research in retinal potential measurement. Summary of the Invention

[0004] To address the shortcomings described in the prior art, this invention provides an application of EAG in measuring insect retinal potentials. By using an antennal potential measurement system (EAG) and replacing the odor stimulation device with a light stimulation device, the retinal potential of insects can be measured. This method is low-cost, easy to operate, and can meet the research needs of measuring insect retinal potentials.

[0005] The technical solution adopted in this invention is: an application of EAG in measuring insect retinal potential, using the EAG's IDAC-2 dual-channel USB interface signal acquisition controller and Combi probe, in conjunction with a photostimulation device to measure insect retinal potential; The IDAC-2 dual-channel USB interface signal acquisition controller is connected to a computer via a USB communication interface and a USB cable. The computer has EggPro software built in. The output of the Combi probe is securely connected to the IDAC-2 signal acquisition controller to ensure stable transmission of the acquired signal, facilitating its transmission to the IDAC-2 signal acquisition controller. The recording electrode of the Combi probe is connected to a glass electrode, which is then inserted into the compound eye of the insect. A hole is drilled in the compound eye according to the measurement requirements. The glass electrode is inserted gently and precisely to avoid damaging the retinal structure. The reference electrode of the Combi probe is inserted into the pronotum of the insect; The original odor stimulation device used in EAG was removed and replaced with a self-built light stimulation device. The light stimulation device stimulates the compound eyes of insects with light. The changes in retinal potential caused by the light stimulation collected by the Combi probe are transmitted to the IDAC-2 dual-channel USB interface signal acquisition controller, which then uploads the data to the EggPro software. EggPro software processes the acquired retinal potential data. Its data processing function first removes noise using a noise filtering algorithm, and then uses waveform analysis tools to perform feature analysis on the potential waveform, such as measuring peak potentials, calculating latency, and analyzing waveform variation patterns. Based on the analysis results, the retinal potential response characteristics of different parts of the insect compound eye to different light stimuli are summarized.

[0006] As a preferred embodiment of the present invention, the photostimulation device includes a light source, a narrowband interference filter, a neutral density filter, a circular variable filter, and a transmission optical fiber; the light source is a 300 W halogen lamp, and the wavelength range of the light filtered by the narrowband interference filter is 340-700 nm; the neutral density filter and the circular variable filter are mainly used to adjust the light intensity so that the light intensity between different wavelengths remains consistent.

[0007] A narrowband interference filter, a neutral density filter, a circular variable filter, and a transmission fiber are sequentially arranged in the propagation optical path of the light source; the beam output end of the transmission fiber is 1 cm away from the insect's eye. A 6.3 mm diameter quartz fiber is preferably used for the transmission fiber.

[0008] As a preferred embodiment of the present invention, the distance between the light source and the narrowband interference filter is 10cm; the light source is a halogen lamp.

[0009] As a preferred embodiment of the present invention, the EggPro software synchronously triggers the photostimulation device and the IDAC-2 dual-channel USB interface signal acquisition controller. This enables the IDAC-2 dual-channel USB interface signal acquisition controller to synchronously acquire changes in retinal potential signals in real time, and to closely observe the potential waveforms displayed on the EggPro software interface during the acquisition process, promptly detecting and handling abnormal situations to ensure the integrity and accuracy of data acquisition.

[0010] As a preferred embodiment of the present invention, the reference electrode is a platinum-plated tungsten wire electrode.

[0011] As a preferred embodiment of the present invention, the insect is a compound-eyed insect, and holes are drilled in different areas of the compound eyes of the insect, and the drilled holes are the insertion points for the recording electrodes.

[0012] As a preferred embodiment of the present invention, the compound eye perforation area is the central area, the back edge area, and the intermediate area between the central area and the back edge area.

[0013] As a preferred embodiment of the present invention, a noise reduction device is also provided, which includes a Faraday cage and a light-blocking cloth.

[0014] As a preferred embodiment of the present invention, the measurement steps are as follows: Insect sample processing and fixation: Select healthy and active adult insects, lightly anesthetize them, cut off their appendages, fix them in a special container with paraffin, place them on the operating table, and gently adjust the insect's posture with tweezers to fully expose the eyes. After fixation, in a dark room environment, the eye is located with the help of an anatomical microscope, and holes are drilled in the compound eye according to the set position; Following the electrophysiological experimental procedures, the recording electrodes were precisely inserted into the opening area, and the reference electrodes were inserted into the anterior thoracic dorsal plate. Set up a light stimulation device: Select the required narrowband interference filter, neutral density filter, and circular variable filter; and place the beam output end of the light stimulation device 1 cm away from the insect's eye; Initialization settings: In the EggPro software, select the IDAC-2 dual-channel USB interface signal acquisition controller, set the data acquisition parameters, and set the parameters of the photostimulation device; Data collection: The insect compound eye was stimulated according to the set light stimulation sequence; the EggPro software collected and recorded the changes in retinal potential signals through the IDAC-2 dual-channel USB interface signal acquisition controller; during the acquisition process, the potential waveform displayed on the EggPro software interface was closely observed to ensure that the data acquisition was normal. Data processing and analysis: The data processing function in the EggPro software processes the collected retinal potential data, and can ultimately obtain retinal potential information of different parts of the compound eye in response to different light stimuli.

[0015] Beneficial effects of this invention: Low-cost reuse and functional expansion of equipment: By utilizing the core components of the electroacoustic gaiter (EAG)—the IDAC-2 controller and the Combi probe—its functionality can be extended, eliminating the need to purchase dedicated retinal potential measurement equipment and reducing hardware costs by approximately 70%. Furthermore, the original electrodes have been improved; the original antennal locator has been replaced with an ocular electrode holder, and the odor stimulation device has been removed and replaced with a built-in light stimulation device, enabling the EAG to achieve cross-functional applications from "electroacoustic potential measurement" to "retinal potential measurement."

[0016] Precise synergy between light stimulation and electrophysiological signals: EggPro software synchronously triggers the photostimulation device and data acquisition (accuracy ±10 ms), solving the phase error problem caused by the asynchrony between photostimulation and potential recording in traditional methods.

[0017] High-sensitivity electrode: By using electrolytically sharpened tungsten wire electrodes in conjunction with a micromanipulator for positioning, precise single-cell-level insertion into the compound eye is achieved (error <50μm), which improves positioning accuracy by 80% compared to traditional handheld electrodes.

[0018] Noise suppression: The triple noise reduction design of Faraday cage + light-blocking cloth + software filtering (5-500 Hz) reduces background noise from ±0.1 mV to ±0.02 mV, and can clearly capture weak retinal potentials. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the present invention.

[0021] Figure 2 The following are schematic diagrams showing the drilling positions of different parts of the compound eye of the copper-green beetle of the present invention. Figure 21 shows the drilling position in the central area of ​​the compound eye, Figure 22 shows the drilling position in the edge area of ​​the back of the compound eye, and Figure 23 shows the drilling position in the middle area between the central area and the edge area of ​​the back.

[0022] Figure 3 The diagram shows the retinal potential response of the compound eye of the copper-green beetle to left-handed circularly polarized light according to the present invention. In the diagram, A represents 400 nm; B represents 500 nm; C represents 520 nm; and D represents white light.

[0023] Figure 4 This is a schematic diagram of the optical path of the photostimulation device of the present invention. Detailed Implementation

[0024] 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.

[0025] An application of EAG in measuring insect retinal potential, such as Figure 1 As shown, the insect retinal potential was measured using EAG's IDAC-2 dual-channel USB interface signal acquisition controller and Combi probe, along with a photostimulation device. The IDAC-2 dual-channel USB interface signal acquisition controller is connected to a computer via a USB communication interface and a USB cable. The computer has EggPro software built in. The output of the Combi probe is securely connected to the IDAC-2 signal acquisition controller to ensure stable transmission of the acquired signal, facilitating its transmission to the IDAC-2 signal acquisition controller. The recording electrode of the Combi probe is connected to a glass electrode, which is then inserted into the compound eye of the insect. A hole is drilled in the compound eye according to the measurement requirements. The glass electrode is inserted gently and precisely to avoid damaging the retinal structure. Insects are compound-eyed insects. Drilling holes in different areas of the insect's compound eyes creates the insertion points for recording electrodes. Typically, the selected areas for drilling holes in the compound eyes are the central region, the edge region of the back, and the intermediate region between the central region and the edge region of the back.

[0026] The reference electrode of the Combi probe is inserted into the pronotum of the insect, and the reference electrode is a platinum-plated tungsten wire electrode.

[0027] The original odor stimulation device used in EAG was removed and replaced with a built-up light stimulation device. like Figure 4 As shown, the optical stimulation device includes a light source, a narrowband interference filter, a neutral density filter, a circular variable filter, and a conductive optical fiber. The light source is a 300 W halogen lamp, and the wavelength range of the light filtered by the narrowband interference filter is 340-700 nm. The distance between the light source and the narrowband interference filter is 10 cm. The neutral density filter and the circular variable filter are mainly used to adjust the light intensity, ensuring that the light intensity remains consistent across different wavelengths. The conductive optical fiber is preferably a 6.3 mm diameter silica optical fiber.

[0028] A narrowband interference filter, a neutral density filter, a circular variable filter, and a transmission fiber are sequentially arranged in the propagation optical path of the light source; the beam output end of the transmission fiber is 1 cm away from the insect's eye.

[0029] The EggPro software synchronously triggers the photostimulation device and the IDAC-2 dual-channel USB interface signal acquisition controller. The photostimulation device applies light to the insect's eyes; the retinal potential changes caused by this light stimulation, collected by the Combi probe, are transmitted to the IDAC-2 dual-channel USB interface signal acquisition controller, which then uploads the data to the EggPro software. During the acquisition process, the potential waveforms displayed on the EggPro software interface are closely monitored to promptly identify and handle any anomalies, ensuring the integrity and accuracy of the data acquisition. Furthermore, a Faraday cage and a light-blocking cloth are used in the operating environment to physically reduce noise.

[0030] EggPro software processes the acquired retinal potential data. Its data processing function first removes noise using a noise filtering algorithm, and then uses waveform analysis tools to perform feature analysis on the potential waveform, such as measuring peak potentials, calculating latency, and analyzing waveform variation patterns. Based on the analysis results, the retinal potential response characteristics of different parts of the insect compound eye to different light stimuli are summarized.

[0031] The measurement procedure for retinal potential is as follows: Insect sample processing and fixation: Select healthy and active adult insects, lightly anesthetize them, cut off their appendages, fix them in a special container with paraffin, place them on the operating table, and gently adjust the insect's posture with tweezers to fully expose the eyes. After fixation, in a dark room environment, the eye is located with the help of an anatomical microscope, and holes are drilled in the compound eye according to the set position; Following the electrophysiological experimental procedures, the recording electrodes were precisely inserted into the opening area, and the reference electrodes were inserted into the anterior thoracic dorsal plate. Set up a light stimulation device: Select the required narrowband interference filter, neutral density filter, and circular variable filter; and place the beam output end of the light stimulation device 1 cm away from the insect's eye; Initialization settings: In the EggPro software, select the IDAC-2 dual-channel USB interface signal acquisition controller, set the data acquisition parameters, and set the parameters of the photostimulation device; Data collection: The insect compound eye was stimulated according to the set light stimulation sequence; the EggPro software collected and recorded the changes in retinal potential signals through the IDAC-2 dual-channel USB interface signal acquisition controller; during the acquisition process, the potential waveform displayed on the EggPro software interface was closely observed to ensure that the data acquisition was normal. Data processing and analysis: EggPro software's data processing function processes the acquired retinal potential data. First, it uses a noise filtering algorithm to remove noise. Then, it employs waveform analysis tools to perform feature analysis on the potential waveform, such as measuring peak potentials, calculating latency, and analyzing waveform variation patterns. Based on the analysis results, it summarizes the retinal potential response characteristics of different parts of the insect compound eye to different light stimuli.

[0032] This invention demonstrates the sensitivity of different parts of the compound eye of the African violet beetle to polarized light by taking it as an example: (1) Sample processing and fixation: Select robust and active adult *Ceratophyllum demersum* beetles. After slight anesthesia, cut off the appendages, fix them in a special container with paraffin, place them on the operating table, and gently adjust the beetle's posture with tweezers to fully expose the eyes. After fixation, locate the eyes in a dark room using a dissecting microscope, and drill holes in the compound eyes according to the set positions. In this embodiment, the retinal potential response of different regions of the compound eye to left-handed circularly polarized light stimulation was tested. Holes were drilled in the central region C, the dorsal edge region D, and the region M between the two. Figure 2 As shown in Figure 21, the location of the perforation in the central region of the compound eye is shown; Figure 22, the location of the perforation in the dorsal edge region of the compound eye is shown; and Figure 23, the location of the perforation in the middle region between the central region and the dorsal edge region is shown. Glass electrodes were inserted at each of the perforated locations, and 400 nm blue light, 500 and 520 nm green light, and full-spectrum white light, which are more sensitive to the copper beetle, were selected and combined with a left-handed circular polarizer to obtain left-handed circularly polarized light. After the copper beetle had been dark-adapted for 3 hours, the perforated areas of its compound eye were stimulated with left-handed circularly polarized light, and the retinal grid response values ​​of different areas of the compound eye were recorded.

[0033] Following the electrophysiological experimental protocol, the recording electrode was precisely inserted into the opening area, and the reference electrode was inserted into the anterior chest back plate to prepare for potential retinal aging (EAG) acquisition. The insertion process should be gentle and precise to avoid damaging the retinal structure.

[0034] (2) Start the equipment and software: Turn on the IDAC-2 controller, the photostimulation device, and the eggpro software on the computer. Perform system initialization settings in the eggpro software, including selecting the IDAC-2 device and setting data acquisition parameters. (3) Setting of light stimulation parameters: Further optimize the light stimulation parameters according to the experimental design. For example, set the stimulation time to 1 second each time and the interval time to 60 seconds. (4) Data Acquisition: Turn on the light stimulation device and stimulate the retina of the copper beetle according to the set light stimulation sequence. At the same time, the eggpro software collects and records the changes in retinal potential signals in real time through the IDAC-2 controller. During the acquisition process, closely observe the potential waveform displayed on the software interface to ensure that the data acquisition is normal. (5) Data Processing and Analysis: After data acquisition, the collected retinal potential data were processed using the data processing function in the Eggpro software. First, noise filtering algorithms were used to remove noise, and then waveform analysis tools were used to perform feature analysis on the potential waveform, such as measuring peak potential, calculating latency, and analyzing waveform change patterns. Based on the analysis results, the retinal potential response characteristics of different parts of the compound eye of the African violet beetle to different polarized light stimuli were summarized.

[0035] Test results are as follows Figure 3 As shown in the figure, after 3 hours of dark adaptation, the C, D and M regions of the compound eyes of both male and female adults can be significantly stimulated with retinal potential responses under left-handed circularly polarized light.

[0036] Under stimulation with left-handed circularly polarized light at 400 nm and 520 nm, there was no significant difference in retinal potential responses in the three regions of the compound eye between male and female adults (400 nm, male: df =2; F =0.084, P =0.92, female insect: df =2; F =0.215, P =0.809; 520nm, male insect: df =2; F =0.319, P =0.733, Female insect: df =2; F =1.030, P =0.387).

[0037] The female insect stimulated by 500 nm left-handed circularly polarized light ( df =2; F =4.883, P =0.022) and male insects under white light stimulation ( df =2; F =12.094, P =0.002) Significant differences were found in the retinal electrical grid responses in the C, D, and M regions of the compound eye.

[0038] Under stimulation with 500 nm left-handed circularly polarized light, the retinal potential response value of female insects in region M was significantly higher than that in regions C and D, while there was no significant difference between regions C and D. Under stimulation with white light, the retinal potential value of male insects in region D was significantly higher than that in regions C and M, the retinal potential value in region C was significantly higher than that in region M, and the retinal grid value in region M was the smallest.

[0039] Male insects stimulated by 500 nm left-handed circularly polarized light ( df =2; F =2.167, P =0.154) and female insects under white light stimulation ( df =2; F =0.690, P =0.524) There was no significant difference in retinal potential responses in the C, D and M regions of the compound eye.

[0040] In this specification, the terms "an embodiment," "example," "specific example," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0041] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An application of EAG in determining insect retinal potential, characterized in that: The insect retinal potential was measured using EAG's IDAC-2 dual-channel USB interface signal acquisition controller and Combi probe, along with a photostimulation device. The IDAC-2 dual-channel USB interface signal acquisition controller is connected to a computer via a USB communication interface and a USB cable. The computer has EggPro software built in. The output of the Combi probe is connected to the IDAC-2 signal acquisition controller; The recording electrode of the Combi probe is connected to a glass electrode, which is then inserted into the compound eye of the insect. The reference electrode of the Combi probe is inserted into the pronotum of the insect; The light stimulation device stimulates the insect's eyes with light. The changes in retinal potential caused by the light stimulation collected by the Combi probe are transmitted to the IDAC-2 dual-channel USB interface signal acquisition controller, which then uploads the data to the EggPro software. EggPro software processes the acquired retinal potential data.

2. The application of EAG in determining insect retinal potential according to claim 1, characterized in that: The optical stimulation device includes a light source, a narrowband interference filter, a neutral density filter, a circular variable filter, and a transmission optical fiber; A narrowband interference filter, a neutral density filter, a circular variable filter, and a transmission fiber are sequentially arranged in the propagation optical path of the light source; the beam output end of the transmission fiber is 1 cm away from the insect's eye.

3. The application of EAG in determining insect retinal potential according to claim 2, characterized in that: The distance between the light source and the narrowband interference filter is 10 cm; the light source is a halogen lamp.

4. The application of EAG according to claim 2 or 3 in determining insect retinal potential, characterized in that: EggPro software synchronously triggers the photostimulation device and the IDAC-2 dual-channel USB interface signal acquisition controller.

5. The application of EAG according to claim 4 in determining insect retinal potential, characterized in that: The reference electrode is a platinum-plated tungsten wire electrode.

6. The application of EAG according to claim 4 in determining insect retinal potential, characterized in that: The insects in question are compound-eyed insects. Holes are drilled in different areas of the insect's compound eyes, and these holes are the insertion points for the recording electrodes.

7. The application of EAG according to claim 6 in determining insect retinal potential, characterized in that: The compound eye perforation area is the central area, the back edge area, and the intermediate area between the central area and the back edge area.

8. The application of EAG according to claim 7 in determining insect retinal potential, characterized in that: It is also equipped with noise reduction equipment, which includes a Faraday cage and a light-blocking cloth.

9. The application of EAG according to claim 7 in determining insect retinal potential, characterized in that, The measurement steps are as follows: Insect sample processing and fixation: Select healthy, active adult insects, lightly anesthetize them, cut off their appendages, fix them in a special container with paraffin, place them on the operating table, and gently adjust the insect's posture with tweezers to fully expose the eyes. After fixation, in a dark room, the eye is located using a dissecting microscope, and holes are drilled in the compound eye according to the set positions; Following the electrophysiological experimental procedures, the recording electrodes were precisely inserted into the opening area, and the reference electrodes were inserted into the anterior thoracic dorsal plate. Set up a light stimulation device: Select the required narrowband interference filter, neutral density filter, and circular variable filter; and place the beam output of the light stimulation device 1 cm away from the insect's eye; Initialization settings: In the EggPro software, select the IDAC-2 dual-channel USB interface signal acquisition controller, set the data acquisition parameters, and set the parameters of the photostimulation device; Data collection: The insect compound eye was stimulated according to the set light stimulation sequence; the EggPro software collected and recorded the changes in retinal potential signals through the IDAC-2 dual-channel USB interface signal acquisition controller; during the acquisition process, the potential waveform displayed on the EggPro software interface was closely observed to ensure that the data acquisition was normal. Data processing and analysis: The data processing function in the EggPro software processes the collected retinal potential data, and can ultimately obtain retinal potential information of different parts of the compound eye in response to different light stimuli.

Images