A component of an attractant for the sex pheromone of anophthalmus formosanus and use thereof

Abstract

The application discloses a component of an Anomala corpulenta sex pheromone attractant and application thereof. The sex attractant is R-Japonilure ((R,Z)-5-(1-decene) dihydrofuran-2(3H)-ketone). The attractant can effectively kill male A. corpulenta, inhibit the growth of the number of pests, and make the pest population gradually die out, so that the purpose of preventing and treating pests is achieved. The application has the advantages of small dosage, high sensitivity, no environmental pollution, and accurate application in pest situation forecasting, large-scale trapping, and mating interference, and has important significance for the comprehensive management of A. corpulenta.

Description

Technical Field

[0002] This invention belongs to the field of pest control technology, specifically relating to a sex pheromone attractant for the copper beetle and its application. Technical Background

[0003] The green grub (Anomala corpulenta) belongs to the order Coleoptera and the family Rutelidae. It is widely distributed in China, found in Guangxi, Jiangsu, Henan, Hebei, and other regions. Adults primarily feed on the above-ground parts of host plants such as elm and mulberry trees, including leaves; in severe cases, they can completely defoliate the plants. The larvae live hidden in the soil and mainly damage the underground parts of field crops such as potatoes, peanuts, soybeans, and corn, as well as economic crops such as flowers and fruit trees, including tender roots and stems. In severe cases, this can lead to pathogen infection and secondary damage to the plants. In recent years, the implementation of no-till and reduced-tillage systems, along with straw return to the field, has created favorable conditions for the survival and reproduction of grubs, leading to increasingly serious grub damage. In my country, grubs can cause 20%-30% losses in crops such as potatoes, peanuts, soybeans, and corn; in severe cases, losses can reach 50%.

[0004] Currently, the main method for controlling adult green scarab beetles is through chemical control. However, the long-term and frequent use of chemical agents has exacerbated the development of pesticide resistance in pests and increased the risk of pesticide residues exceeding standards in agricultural products. Furthermore, it has a strong killing effect on natural enemies and disrupts the ecological balance. Therefore, harmless and green control technologies are receiving increasing attention.

[0005] Insect sex pheromones are trace chemical substances secreted externally by specialized organs of a particular sex within the same insect species. They are detected by receptors in opposite-sex individuals of the same species, triggering behavioral responses or physiological effects (such as courtship, directional mating, and mating). Since Butenandt identified the first insect pheromone component—bombyx mori L.—in 1959 from the silkworm, insect sex pheromones have become a hot topic in chemical ecology research. Research on scarab beetle sex pheromones started relatively late, with reports of such studies appearing domestically and internationally only in the early 1970s. Sex pheromone control offers advantages such as high selectivity, non-harmful effects on natural enemies, low resistance development in pests, no environmental pollution, and low dosage, making it highly valuable for research and development. The sex pheromones of more than ten scarab beetle species, including the New Zealand winged scarab beetle, the Japanese scarab beetle, and the dark-skinned scarab beetle, have been identified, providing a scientific basis for artificial synthesis and control applications.

[0006] Insect sex pheromone-related technologies and products possess advantages such as high sensitivity, strong selectivity, harmlessness to natural enemies, and environmental protection. Since the 1960s, they have been developed into a new technology for pest control both domestically and internationally. In particular, the structures of many insect sex pheromones, mainly in Lepidoptera, have been successively identified and synthesized in the laboratory and applied in the field. Currently, hundreds of sex pheromone-related products have been applied both domestically and internationally, achieving significant economic and ecological benefits. Therefore, it is necessary to accurately identify the sex pheromone components of the green scarab beetle and, based on this identification, prepare a highly effective sex pheromone attractant for trapping the green scarab beetle, for use in pest monitoring and harmless control. Summary of the Invention

[0008] This invention provides a copper-green beetle sex pheromone attractant, wherein the active ingredient is R-Japonilure ((R,Z)-5-(1-decene)dihydrofuran-2(3H)-one).

[0009] This invention also provides an application of a sex pheromone attractant for the control of the green-necked golden beetle. Specifically, the application involves adding the aforementioned sex pheromone attractant to a carrier to prepare a green-necked golden beetle attractant lure, and then placing the lure into a trap for use.

[0010] Specifically, the carrier is one or more of the following: polyethylene pipe, polyvinyl chloride pipe, polyethylene bottle, polyethylene slow-release bag, polyvinyl chloride slow-release bag, and rubber stopper. Preferably, the carrier is a polyethylene pipe.

[0011] In use, the sex pheromone of the green-skinned beetle is made into a lure, and the trap containing the lure is suspended in the area infested by the green-skinned beetle for pest monitoring and mass trapping. Preferably, the trap is a barrel-shaped trap, suspended at a height of 1m above the ground, and / or the traps are spaced 50m apart.

[0012] The beneficial effects of this invention include:

[0013] The sex pheromone attractant provided by this invention can attract a large number of adult beetles of the green-green scarab beetle, providing a pollution-free sex pheromone control technology for the control of the green-green scarab beetle. It can be used for monitoring and harmless control of the green-green scarab beetle infestation, and has the advantages of being environmentally friendly and pollution-free, with significant ecological and economic benefits. Attached Figure Description

[0014] Figure 1 The molecular structure of R-Japonilure, a sex pheromone from the copper-green beetle.

[0015] Figure 2Behavioral response of male *Gynostemma pentaphyllum* to extracts from female beetles.

[0016] Figure 3 GC-EAD reaction of male antennae of the green scarab beetle to extracts from female beetles.

[0017] Figure 4 GC-MS identification results of extracts from female *Gynostemma pentaphyllum* beetles.

[0018] Figure 5 Chiral GC-MS identification results of extracts from female Copper-green Scarab Beetles.

[0019] Figure 6 Results of field trapping experiments of sex pheromones from the green-skinned beetle. Detailed Implementation

[0020] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] The R-Japonilure used in the following examples, CAS number: 64726-91-6 ( Figure 1 ).

[0023] Example 1 is an identification of the sex pheromone components of the copper-green beetle:

[0024] (1) Extraction of active compounds from the abdomen of female adult female *Gynostemma pentaphyllum*

[0025] Solvent extraction method: The abdomens (N=20) of adult green scarab beetles in courtship state were immersed in a glass bottle containing 2000 μL of dichloromethane. Each beetle was extracted for 5 min, and then concentrated to 20 μL using a nitrogen blower. The concentrated extract was then stored in a brown glass bottle at -20°C for later use. The treatment was repeated five times.

[0026] (2) Behavioral response of male adult beetles to extracts from female adults

[0027] Behavioral measurements were conducted between 7:00 PM and 9:00 PM. The Y-type olfactory instrument consisted of a QC-3 atmospheric sampler (Beijing Kean Occupational Safety Technology Co., Ltd.), a 300ml flask containing 200ml of double-distilled water, an activated carbon air filter, and a Y-shaped glass tube (main rod 20cm long, two arms 12cm each, angle 45°, tube diameter 3.5cm). 10µl of extract was added to a 0.5cm × 2cm filter paper strip, and after the solvent evaporated, it was placed at the end of one glass arm. A filter paper strip of the same size, containing 10µl of dichloromethane, served as the control group, and after the solvent evaporated, it was placed at the end of the other arm. The airflow (500ml / min) passed through the charcoal filter and double-distilled water before entering the Y-tube. The positions of the filter paper containing the extract and the control were exchanged every 10 insects measured. In each measurement, a healthy male green beetle was placed at the entrance of the main stem and observed for 10 minutes. If it entered 5 cm into one side tube and stayed for more than 1 minute, it was considered to have made a choice. If it did not enter either side tube for more than 10 minutes, it was considered to have made no choice.

[0028] Behavioral experiments have shown that extracts from the abdomen of female *Symplocos purpurea* have a significant attraction effect on male *Symplocos purpurea*. Figure 2 ).

[0029] (3) GC-EAD reaction of male adult beetle on female adult beetle extract.

[0030] GC-EAD measurements were performed between 19:00 and 21:00. Active male insects were selected, and intact antennae were removed using tweezers. Small incisions were made at the base and tip of the antennae, and glass electrodes filled with physiological saline were connected. An Agilent 7890A gas chromatograph was used, with an HP-5 column (30m × 0.25mm inner diameter × 0.25µm thickness) of flexible quartz capillary column. The operating conditions for gas chromatography were as follows: detector: flame ionization detector; combustion gas: hydrogen, flow rate 35 mL / min; oxidizing gas: dry air, flow rate 400 mL / min; carrier gas: nitrogen, flow rate 2.5 mL / min; injection port temperature: 220°C; detector temperature: 250°C; column temperature program: initial temperature 40°C, hold for 2 minutes, increase to 250°C at 6°C / min, hold for 20 minutes; injection method: splitless injection, injection volume 1 μl. After separation by a capillary column, the analytes were split 1:1 via a split valve, with nitrogen at a rate of 10 ml / min added as makeup carrier gas. One portion of the split analytes entered a flame ionization detector (FID), while the other portion entered an electrophoretic potentiometer (EAD). The humidified gas stream was directed at a flow rate of 500 ml / min onto the electrophoresis electrode, and the output signal was connected to a 1:100 signal amplifier. Standard compounds were determined using the same procedure.

[0031] The atlas revealed that the abdominal extract of female adult *Symplocos purpurea* contained a component that caused a significant electrophysiological response in the antennae of male adult *Symplocos purpurea*. Figure 3 ), and its retention time is consistent with that of Japonilure's standard compounds ( Figure 4 ).

[0032] (4) GC-MS identification of compound components

[0033] The extract was analyzed by GC-MS using a Shimadzu single quadrupole gas chromatograph (GCMS-QP2010 SE, SKL-0108). The column was an HP-5MS UI (Agilent, USA; 30 m long × 0.25 mm inner diameter × 0.25 μm film thickness). The temperature program was: 40 °C (2 min), ramped to 250 °C at 6 °C / min, held for 20 min, with helium as the carrier gas and a flow rate of 1 mL / min. The mass spectrometry parameters were: injection temperature 250 °C, ionization energy 70 eV, scan rate 5 Hz. The injection volume was 1 μL splitless injection. The component with a retention time of 30.15 min was Japonilure. Figure 4 Chiral analysis was performed using an Agilent HP 6890 gas chromatograph equipped with a Chiraldex-GTA capillary column (20 m × 0.25 mm inner diameter, 0.125 μm film thickness; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The column oven was maintained at a constant temperature of 180°C, and the injector was operated in splitless mode at 220°C. The stereochemical structure of the natural pheromone fraction with a retention time of 30.15 min was determined by comparing the retention times of the natural compound with the enantiomer of Japonilure on a chiral column after purification. The isolated pheromone had the same retention time as R-japonilure and co-eluted when co-injected with synthetic R-japonilure or a 1:2 R / S stereoisomer mixture. Therefore, we conclude that the sex pheromone of the green beetle is R-japonilure ( Figure 5 ).

[0034] Example 2 is a field trapping experiment using the sex pheromones of the green-skinned beetle:

[0035] The field trial was conducted from July 5 to 17, 2025, in Xinxiang, Henan Province, China (35°18′13″N, 113°55′36″E). Sixteen barrel-shaped beetle traps (Biolan Biotechnology Co., Ltd., China) were randomly placed in corn and soybean fields, spaced 50 meters apart and 1 meter above ground level. Eight traps contained polyethylene-vinyl acetate particles (4-5 mm in diameter) containing 1 mg of R-Japonilure as bait, while the other eight served as control traps without bait. The sex pheromone was introduced by immersing the particles in an ether solution of R-Japonilure, followed by gentle manual rotation at room temperature to evaporate the solvent; residual solvent was removed by a nitrogen stream. The prepared particles were sealed in 2 mL glass vials and then vacuum-sealed using a FoodSaver® vacuum sealer (Sunbeam Products, Boca Raton, Florida, USA). The particles were kept sealed during transport and opened immediately before use. The traps were set at 7 p.m. and checked every 24 hours throughout the study.

[0036] Field trapping experiments showed that traps containing the sex pheromone of the green-skinned beetle captured significantly more male green-skinned beetles than the control group, which did not capture any male green-skinned beetles during the experiment; furthermore, the plastic pellet lures containing the sex pheromone of the green-skinned beetle remained effective in the field for a total of 4 days. Figure 6 ).

Claims

1. A copper-green beetle sex pheromone attractant, characterized in that, The active ingredient of the attractant, R-Japonilure, must have a purity of 95% or higher.

2. The copper-green beetle sex pheromone attractant according to claim 1, characterized in that, R-Japonilure: S-Japonilure ((S,Z)-5-(1-decene)dihydrofuran-2(3H)-one) mass ratio ≥99:

1.

3. The application of the sex pheromone attractant of the Green-green Golden Beetle as described in claim 1 or 2 in the prevention and control of the Green-green Golden Beetle.

4. The application according to claim 3, characterized in that, The application involves adding the copper-green beetle sex pheromone attractant as described in claim 1 or 2 to a carrier to obtain a copper-green beetle sex pheromone attractant core, and then placing the core into a trap for use.

5. The application according to claim 4, characterized in that, The carrier is a polyethylene bottle, rubber stopper, etc.

6. The application according to claim 4, characterized in that, The trap is suspended in the area infested by the green jay beetle.

7. The application according to claim 6, characterized in that, The trap is suspended at a height of 1m above the ground, and / or the traps are spaced 50m apart.

8. The application according to claim 4, wherein the trap is a barrel-shaped trap.

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