Use of citrus volatiles in repelling Pardosa pseudoannulata

Abstract

The present invention relates to the use of citrus volatiles to repel the Pardosa pseudoannulata, and belongs to the technical field of spider repellents. The present invention provides the use of citrus volatiles to repel the Pardosa pseudoannulata. Based on this use, the present invention also provides the use of citrus volatiles to prepare a Pardosa pseudoannulata repellent, the use of the citrus volatile α-terpineol to repel the Pardosa pseudoannulata, and the use of the citrus volatile α-terpineol to prepare a Pardosa pseudoannulata repellent. The present invention utilizes α-terpineol, which has a repellent effect on the Pardosa pseudoannulata, to develop a spider repellent, which has great development potential and is of great significance.

Description

Technical Field

[0001] The invention belongs to the technical field of spider repellent, and particularly relates to use of citrus volatiles in repelling Pardosa pseudoannulata. Background Art

[0002] Pardosa pseudoannulata ( & Strand, 1906), a member of the family Lycosauridae and order Araneae. It is found in Guizhou, Henan, Shaanxi, Hebei, Beijing, Shanghai, Gansu, Jilin, Liaoning, Jiangxi, and Yunnan.

[0003] The Pardosa pseudoannulata (Pardosa pseudoannulata) is a ferocious spider with a terrifying appearance. Although it is a predatory natural enemy, people still want to keep it at a distance. Currently, common methods of repelling spiders include physical repelling or spraying with chemical agents. The former is ineffective and passive, while the latter is prone to pollution and toxicity.

[0004] At present, the development of a repellent for the Pardosa pseudoannulata has not been reported, and there is no report in the field on the use of citrus volatiles to repel the Pardosa pseudoannulata. Summary of the Invention

[0005] In order to solve the technical problem of filling the above-mentioned gap in the prior art in this field, the present invention provides the use of citrus volatiles to repel Pardosa pseudoannulata.

[0006] The technical solutions of the present invention are as follows:

[0007] Use of citrus volatiles to repel Pardosa pseudoannulata.

[0008] The citrus volatiles are: α-terpineol.

[0009] Use of citrus volatiles in preparing a repellent for the Pardosa pseudoannulata.

[0010] The Pardosa pseudoannulata repellent contains citrus volatiles as active ingredients;

[0011] Preferably, the citrus volatiles are α-terpineol.

[0012] Use of citrus volatile α-terpineol in repelling Pardosa pseudoannulata.

[0013] The effective dosage of citrus volatile α-terpineol to repel Pardosa pseudoannulata is 10-100 μL.

[0014] Use of citrus volatile α-terpineol in preparing a repellent for the Pardosa pseudoannulata.

[0015] The annular Pardosa spider repellent contains citrus volatile α-terpineol as an active ingredient;

[0016] Preferably, the Pardosa annularis repellent further comprises an auxiliary material.

[0017] The beneficial effects of the present invention are as follows:

[0018] The present invention unexpectedly discovered that α-terpineol, a citrus volatile, has a repellent effect on the spider Pardosa pseudoannulata. The researchers tested the spider's preference for α-terpineol through behavioral experiments, and verified its repellent effect by measuring the electrophysiological response of the spider's sensilla to α-terpineol using EAG (insect antennal potential). The behavioral test results showed that α-terpineol had a highly significant (P < 0.001) repellent effect on the spider.

[0019] The present invention also further tests and obtains the repellent effect of the concentration gradient of citrus extract α-terpineol and the volatile mixture, screens out the optimal repellent concentration, and is mainly used for indoor repellent of Pardosa pseudoannulata. Its significance is that: there are widespread patients with arachnophobia worldwide. They feel a sense of fear when seeing spiders or when they are exposed to spiders. They are even afraid of being in a closed environment where they think there may be spiders (such as the presence of spider webs, etc., which remind people of spiders). After the fear of spiders is stimulated, some people's symptoms will last for several days. During this period, they will constantly suspect that there are spiders in their surroundings, causing trouble for their normal life. Utilizing the repellent effect of α-terpineol on spiders, spider repellents can be made. On the one hand, it can be used to drive spiders away from specific environments, and on the other hand, it can alleviate the anxiety of patients with arachnophobia. Therefore, spider repellents have greater development potential and are of great significance. The present invention reflects the behavioral activity of Pardosa pseudoannulata to the citrus volatile: α-terpineol through indoor behavioral selection experiments and electroantennographic potential experiments, proving that α-terpineol has a repellent effect on Pardosa pseudoannulata. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram of the physical device of the two-way selection box system used in Section 1.2.1 of Experimental Example 1 of the present invention.

[0021] Figure 2 This is a diagram of the actual device of the glass electrode used in Section 1.2.2 of Experimental Example 1 of the present invention.

[0022] Figure 3 This is a bar graph showing the selectivity of Pardosa pseudoannulata to citrus volatiles in Section 2.1 of Experimental Example 1 of the present invention.

[0023] Figure 4 This is a bar graph of the potential values ​​of the antennal potential response of the Pardosa pseudoannulata to citrus volatiles in Section 2.2 of Experimental Example 1 of the present invention.

[0024] Figure 5This is a bar chart showing the number of webs built by the Pardosa pseudoannulata in the field in the treatment and control groups in Section 2.3 of Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to specific embodiments and experimental examples, but the scope of protection of the present invention is not limited thereto.

[0026] The first group of examples: Use of citrus volatiles to repel Pardosa pseudoannulata

[0027] This set of embodiments provides the use of citrus volatiles to repel the Pardosa pseudoannulata.

[0028] In a specific embodiment, the citrus volatile is α-terpineol.

[0029] Example 2: Use of citrus volatiles in preparing a repellent for Pardosa pseudoannulata

[0030] This group of examples provides the use of citrus volatiles in preparing a repellent for the Pardosa pseudoannulata.

[0031] In a specific embodiment, the Pardosa pseudoannulata repellent contains citrus volatiles as active ingredients;

[0032] Preferably, the citrus volatiles are α-terpineol.

[0033] The third group of examples: Use of citrus volatile α-terpineol to repel Pardosa pseudoannulata

[0034] This group of embodiments provides the use of citrus volatile α-terpineol to repel Pardosa pseudoannulata.

[0035] In a specific embodiment, the effective amount of the citrus volatile α-terpineol for repelling the Pardosa pseudoannulata is 10-100 μL.

[0036] Example 4: Use of citrus volatile α-terpineol in the preparation of Pardosa pseudoannulata repellent

[0037] This group of examples provides the use of citrus volatile α-terpineol in preparing a repellent for the Pardosa pseudoannulata.

[0038] In a specific embodiment, the Pardosa annularis repellent contains citrus volatile α-terpineol as an active ingredient;

[0039] Preferably, the Pardosa annularis repellent further comprises an auxiliary material.

[0040] Experimental Example 1

[0041] 1 Materials and Methods

[0042] 1.1 Experimental materials and treatments

[0043] Spider Collection and Rearing: Pardosa pseudoannulata (Lycodidae, Pardosa genus) were collected from lawns throughout the Jinggangshan University campus and housed individually in transparent plastic boxes (L×W×H = 16.5 cm × 11.5 cm × 5.8 cm). Moisturized sterile cotton balls were placed inside the boxes to provide moisture. The laboratory temperature was set at 25 ± 2°C, with a photoperiod of 14 L:10 D (i.e., 14 h daylight, 10 h nightlight). Advanced-aged spiderlings or adults were selected for the experiments and were starved for two days prior to the experiment.

[0044] Citrus Volatile Extraction: Citrus volatiles were extracted using organic solvents for testing. Five freshly harvested citrus leaves were picked with alcohol-wiped tweezers and placed in a clean 10 mL glass vial. Methylene chloride, an organic solvent, was added and the leaves were removed after 5 minutes of extraction. The solution in the vial was then concentrated using a nitrogen blowdown apparatus until approximately 1 mL of concentrate remained for use in EAG and behavioral testing.

[0045] Reagents and instruments: α-Terpineol (purity ≥ 95%), Tokyo Chemical Industry Development Co., Ltd. (Shanghai), Japan.

[0046] The electroantennography (EAG) instrument consisted of a stimulus airflow controller (CS-55, Syntech, the Netherlands) and a signal recording controller (Keysight 34461A, Tangshan Dinggan Technology Co., Ltd.).

[0047] 1.2 Research Methods

[0048] 1.2.1 Electroantennographic experiments on the Pardosa pseudoannulata to the citrus volatile α-terpineol

[0049] Studies have confirmed that spiders possess odor-sensing hairs on their bodies that can sense chemicals in the environment, typically located on the tarsus and metatarsus of their legs. Xiao Yonghong et al. successfully recorded the electrical responses of female Beijing ghost spiders (Arachnoptera: A ...

[0050] During the experiment, the tarsal segment of the first step of the Pardosa pseudoannulata was selected and the claw at the end was cut off. The two ends of the walking leg were quickly connected to two homemade electrodes (glass capillaries were made into a pointed shape with a wire drawer, and conductive silver wire and physiological saline were filled into the electrodes, see Figure 2The other ends of the two glass electrodes were connected to antennal potential electrodes. The potential responses of the receptors on the legs were amplified by a DG3FA-1 high-impedance microelectrode amplifier (Tangshan Dinggan Technology Co., Ltd.) and displayed on a computer. The test volatiles were dropped onto clean filter paper (length × width = 2 cm × 1 cm, Taizhou Jinao Paper Co., Ltd.) and placed at the airflow outlet. During the experiment, stimulation was performed every 3 minutes. After each stimulation, a new filter paper was replaced for the next stimulation. Air testing was performed 6 minutes before the start of the experiment, after each group of tests, and 6 minutes before the end of the test for that leg. Each experiment lasted 33 minutes and was repeated 10 times. The continuous humidification gas flow rate was set at 250 mL / min, the stimulation gas flow rate was 200 mL / min, and the stimulation method was foot-operated pulse delivery.

[0051] 1.2.2 Experiment on the selective behavioral response of Pardosa pseudoannulata to the citrus volatile α-terpineol

[0052] The spider behavior selection test device uses a two-way choice box system ( Figure 1 ), a system previously used to test the choice preference behavior of Pireneitega luctuosa and Hololena curta

[19] The two-way choice box system consists of three transparent polypropylene plastic boxes placed side by side (top 16.5 cm × 11.5 cm, bottom 14 cm × 8.5 cm, box height 5.8 cm), with two choice boxes on the left and right, and a release box in the middle. The release box is connected to the left and right choice boxes on both sides by circular holes with a diameter of approximately 4 cm. The test spider can freely choose to enter and exit the left and right choice boxes within the release box. During the experiment, the left and right choice boxes were randomly set as the treatment box (with the test agent) and the control box (without the test agent). To facilitate the spider's climbing inside the box, the inner walls of the three boxes were scratched by hand to make them rough. The two-way choice box system was used to test the preference response of the Pardosa pseudoannulata to α-terpineol. The test environment was a closed behavioral chamber with low airflow, an ambient temperature of 24-27°C, and a relative humidity of 50-60%. α-terpineol was diluted 10 times with dichloromethane.

[0053] Before the experiment, the spiders were introduced into the release box, and the two choice box connections were sealed. They were allowed to acclimate for 20 minutes. During the experiment, 10 μl of the reagent (treatment group: α-terpineol, control group: dichloromethane) was dropped onto qualitative filter paper (length × width = 3 cm × 3 cm, Taizhou Jinao Paper Co., Ltd.). After evaporation for 2 minutes, the paper was placed into the choice box. The two connections on the choice box were opened, and the experiment began. Recordings were taken every 20 minutes for the experiment, which lasted for 2 hours. After each volatile was tested, the filter paper was replaced, and the two-way choice box was wiped with 95% anhydrous ethanol. After air drying, the next volatile was tested. Each experimental group consisted of 40 spiders.

[0054] 1.2.3 Effects of citrus volatile α-terpineol on outdoor web site selection by Pardosa pseudoannulata

[0055] The test site was the large lawn in the north area of ​​the Jinggangshan University campus, home to a large number of Pardosa pseudoannularis spiders year-round. A 50µl dose of the reagent (α-terpineol for the treatment group and dichloromethane for the control group) was dripped onto a rubber lure and placed near the roots of the lawn grass. Five replicates were used per group. The test area, within a 3-meter radius of the lure, served as the test zone. Existing Pardosa pseudoannularis webs were destroyed within this area, and the number of newly constructed webs was counted three days later.

[0056] 1.3 Data Analysis

[0057] The chi-square test was used to analyze differences between the treatment and control groups in the behavioral choice experiment, and one-way analysis of variance (ANOVA) was used to analyze differences in peak EAG responses between the various reagents. A P value < 0.05 was considered significant, and a P value < 0.001 was considered extremely significant. All statistical analyses were performed using Excel and SPSS 19.0 (IBM), and plotting was performed using Origin 2018 (OringinLab).

[0058] 2 Results and Analysis

[0059] 2.1 Electroantennographic responses of Pardosa pseudoannulata to citrus volatiles and α-terpineol

[0060] The peak EAG potential responses of Pardosa pseudoannulata to citrus volatiles and α-terpineol are shown in Table 1. The data were compared between groups using one-way analysis of variance (ANOVA) and the least significant difference (LSD) method. Figure 4 ) showed that the potential response values ​​of the receptors on the legs of Pardosa pseudoannulata to citrus volatiles and α-terpineol were higher than those in the air and dichloromethane controls (P < 0.05).

[0061] Table 1. Peak antennal potentials of Pardosa pseudoannulata in response to citrus volatiles

[0062]

[0063] 2.2 Indoor choice behavior of Pardosa pseudoannulata to the citrus volatile α-terpineol

[0064] In this study, a two-way choice box system was used to study the preference of Pardosa pseudoannulata for α-terpineol, an extract from citrus. Statistical analysis showed that the number of times Pardosa pseudoannulata entered the treatment box containing α-terpineol was significantly less than the number of times it entered the control box (χ 2 =17.065, df=1, P<0.001, Figure 3 ). This indicates that α-terpineol has a very significant repellent effect on Pardosa pseudoannulata.

[0065] 2.3 Effects of citrus volatile α-terpineol on site selection and web-building behavior of Pardosa pseudoannulata in the wild

[0066] After the web of the Pardosa pseudoannulata is destroyed, it will quickly build a new web as its main habitat. If it senses that there are factors in the environment that are not conducive to its habitat, it may abandon the original web-making location and choose to build a web in another location. In the experiment, the experimental results were as follows: Figure 5 As shown, the number of webs produced by the control group was 18.40±4.56, while the number of webs produced by the treated group was 3.80±1.64, with a highly significant difference between the two groups (t=6.734, df=8, P<0.001). This suggests that P. pseudoannularis tends not to build webs in areas containing the citrus volatile α-terpineol, indicating that α-terpineol has a repellent effect on P. pseudoannularis within a range of 3 meters.

Claims

1. Use of citrus volatile α-terpineol in repelling Pardosa pseudoannulata.

2. The use of citrus volatile α-terpineol to repel Pardosa pseudoannulata according to claim 1, characterized in that: The effective dosage of citrus volatile α-terpineol to repel Pardosa pseudoannulata is 10-100 μL.

3. Use of citrus volatile α-terpineol in the preparation of Pardosa pseudoannulata repellent.

4. Use of the citrus volatile α-terpineol in preparing a repellent for Pardosa pseudoannulata according to claim 3, characterized in that: The Pardosa annularis repellent uses citrus volatile substance α-terpineol as an active ingredient.

5. Use of the citrus volatile α-terpineol according to claim 3 or 4 in preparing a repellent for Pardosa pseudoannulata, characterized in that: The Pardosa annularis repellent further comprises auxiliary materials.

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