A sex pheromone of the large gray aphid and its application

Abstract

This invention relates to a sex pheromone of the large gray aphid fly and its application. The large gray aphid fly sex pheromone is ethyl 9,12,15-octadecyltrienoate, which can be used to attract large gray aphid flies (…). Eupeodes corollas Male adults or increase the number of large gray aphid flies ( Eupeodes corollas The mating rate between male and female adults.

Description

Technical Field

[0001] This invention relates to the field of pest control, and in particular to an attractant. Background Technology

[0002] The large gray aphid fly ( Eupeodes corollae Hedgehogs (Hypnotidae) belong to the order Diptera, family Aphididae, and are widely distributed worldwide. They are a key group of natural enemies in agronomic ecosystems, serving both predation and pollination functions. Their larvae are obligate predators, feeding on aphids and demonstrating excellent natural control efficiency in aphid biological control. Adults participate in crop pollination through flower-visiting behavior, playing a crucial role in the pollination of many flowering plants.

[0003] As a natural enemy insect, the large gray hoverfly is utilized not only through artificial breeding and release but also through chemical trapping. Sex pheromones are key chemical substances secreted by male and female insects, received by the opposite sex, and trigger courtship and mating behaviors. They are the most important means of intraspecific communication among insects, and sex pheromones developed based on them have excellent insect control effects. Therefore, developing behavioral regulators based on the large gray hoverfly's sex pheromones to precisely target aphids and other important pests can improve the control efficacy of aphids and other pests and enhance crop pollination efficiency. Summary of the Invention

[0004] One aspect of this invention provides ethyl 9,12,15-octadecyltrienoate for attracting the large gray aphid (Aphidida albicans). Eupeodes corollae Male adults or increase the number of large gray aphid flies ( Eupeodes corollae Application in the mating rate of male and female adults.

[0005] In one specific embodiment, the male adult aphids attracted by the ethyl 9,12,15-octadecyltrienoate are used for plant pollination. For example, during the flowering season when plants require pollination, the attracted male adult aphids can pollinate plants in the attracted area. The attracted area can be, for example, farmland with crops or forests with trees (e.g., orchards with fruit trees).

[0006] The second invention provides the use of a composition containing ethyl 9,12,15-octadecanotrienoate in attracting male adult Aphididae.

[0007] In one embodiment, the composition further includes a pesticide-acceptable carrier.

[0008] In one specific embodiment, during the preparation of the lure core, the pesticide-acceptable carrier can be n-hexane.

[0009] The third invention provides a lure core comprising ethyl 9,12,15-octadecyltrienoate and a sustained-release carrier.

[0010] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 0.01 micrograms or more.

[0011] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 0.1 micrograms or more.

[0012] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 1 microgram or more.

[0013] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 10 micrograms or more.

[0014] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 0.01 to 10 micrograms.

[0015] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 0.1 to 10 micrograms.

[0016] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 1 to 10 micrograms.

[0017] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is above 100 micrograms.

[0018] In one specific embodiment, the dosage of ethyl 9,12,15-octadecanotrienoic acid is above 1000 micrograms.

[0019] In one specific embodiment, the dosage of ethyl 9,12,15-octadecanotrienoic acid is above 2000 micrograms.

[0020] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is from 0.01 to 2000 micrograms.

[0021] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 0.1 to 2000 micrograms.

[0022] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 1 to 2000 micrograms.

[0023] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 10 to 2000 micrograms.

[0024] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 100 to 2000 micrograms.

[0025] In one specific embodiment, the dosage of ethyl 9,12,15-octadecadienoic acid is 1000 to 2000 micrograms.

[0026] In one specific embodiment, the sustained-release carrier is made of rubber.

[0027] In one specific embodiment, the sustained-release carrier is at least one of a rubber-tip carrier, a cotton-core carrier, and a microtubule carrier.

[0028] In one specific embodiment, the lure also includes a pesticide-acceptable carrier.

[0029] In one specific embodiment, during the preparation of the lure core, the pesticide-acceptable carrier can be n-hexane.

[0030] The fourth invention provides a lure according to any one of the third inventions for attracting male adult *Aphidius gracilis* or increasing the *Aphidius gracilis* population. Eupeodes corollae Application in the mating rate of male and female adults.

[0031] In one specific embodiment, the male adult aphids attracted by the ethyl 9,12,15-octadecyltrienoate are used for plant pollination. For example, during the flowering season when plants require pollination, the attracted male adult aphids can pollinate plants in the attracted area. The attracted area can be, for example, farmland with crops or forests with trees (e.g., orchards with fruit trees).

[0032] In one specific embodiment, the decoys are placed in the field at intervals of ≤10 meters.

[0033] The beneficial effects of this invention are as follows: This invention is the first to discover that ethyl 9,12,15-octadecanotrienoic acid is the main component of the sex pheromone secreted by female adult Aphidida albicans. It can be used to attract male adult Aphidida albicans and, as a sex attractant, to lure the flies to a designated area, thereby increasing the population density of Aphidida albicans in a local area and increasing the mating rate between male and female adult Aphidida albicans. This, in turn, can improve the utilization rate of natural enemy insects in the field. For example, it can enable Aphidida albicans to feed on aphids to improve the control of aphid pests (such as peach aphid, cotton aphid, cabbage aphid, bean aphid, potato long-tubed aphid, wheat long-tubed aphid, cereal constricted tubed aphid, apple yellow aphid, apple gall aphid, peach powdery aphid, rose long-tubed aphid, etc.), or to improve pollination efficiency by using Aphidida albicans to pollinate plants. Attached Figure Description

[0034] Figure 1 The GC-MS mass spectrometry results of extracts from female and male adult Aphididae are shown.

[0035] Figure 2 The results of EAG assays for ethyl 9,12,15-octadecanoic acid (ECA) in adult female and male Aphidida albicans at various doses are shown.

[0036] Figure 3 The results of T-type olfly assays on ethyl 9,12,15-octadecanotrienoic acid at various doses are shown in male Aphidida albicans.

[0037] Figure 4 The effect of ethyl 9,12,15-octadecadienoic acid on the mating rate of the large gray fly was shown.

[0038] Figure 5 The effect of ethyl 9,12,15-octadecadienoate on the time male Aphidius maxima search for females before mating is shown, where n is the number of mating pairs.

[0039] Figure 6 The field attraction effect of ethyl 9,12,15-octadecyltrienoate on male aphids was demonstrated. Detailed Implementation

[0040] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0041] Unless otherwise specified, the reagents and other materials used in the embodiments of this invention can be purchased commercially.

[0042] The large grey aphid was collected from a wheat field at the Langfang Base of the Chinese Academy of Agricultural Sciences (116°60′ E, 39°50′ N) and reared there. The rearing environment was maintained at a temperature of 23±1°C, a relative humidity of 50%±5%, and a photoperiod of 12 L:12 D. Pea aphids (… Acyrthosiphon pisum Rearing the larvae. After emergence, separate the female and male adults and raise them using a mixture of 10% honey water and pollen.

[0043] Example 1: Extraction and identification of reproductive gland compounds from the large gray aphid fly

[0044] 20 μL of the original concentration of 2-pentadecanone was added to 144 μL of anhydrous ethanol to obtain a 100 μg / μL 2-pentadecanone solution. This solution was then diluted with anhydrous ethanol to prepare a 0.01 μg / μL 2-pentadecanone solution.

[0045] Extraction of gonadal compounds from *Aphis maxima*: Five-day-old unmated female and male adults were selected as experimental subjects. Compounds released from the gonads of female and male *Aphis maxima* were extracted using the ethanol immersion method. The gonads of 30 female or male insects were placed in a small brown bottle containing 200 μL of anhydrous ethanol and soaked for 3 hours to obtain the extract. The extract was transferred to a new small brown bottle, and 50 μL of 0.01 μg / μL 2-pentadecanone solution was added to the new bottle to mix with the extract, obtaining a mixture. The mixture was concentrated to 10 μL using nitrogen gas to obtain a concentrate, which was placed in a liner tube and stored at -20°C for analysis. Eight biological replicates were extracted for each treatment.

[0046] Identification of gonadal compounds in *Aphidius gravidarum*: The concentrated solution was identified using gas chromatography-mass spectrometry (GC-MS). The GC program settings were as follows: helium as carrier gas, purge rate of 3.0 mL / min, injection port temperature of 260°C, and HP-5MS UI column (30 m × 0.25 mm × 0.25 μm). The temperature program was as follows: initial temperature 40°C, hold for 2 min; increase to 220°C at 30°C / min, hold for 2 min; increase to 260°C at 4°C / min, hold for 10 min. The MS program settings were as follows: EI ionization selected, ion source temperature 250°C, interface temperature 260°C, mass spectrometry scan range between 35 and 500 mass-to-charge ratio, scan frequency of 0.2 times / second, concentrated solution injection volume of 1 μL, and splitless injection. Chemical composition analysis was performed on the concentrate according to the set injection conditions, and peak chromatograms were obtained. GC-MS mass spectrometry results of the female and male adult extracts are shown below. Figure 1 The names of the compounds corresponding to each peak were determined by mass spectrometry library analysis, and further verified by standards of the corresponding compounds. The pheromones released by female and male Aphididae were compared. The results showed that females specifically released ethyl 9,12,15-octadecanoic acid, while males did not release this compound.

[0047] Example 2: Electrophysiological and behavioral responses of the large gray aphid fly to ethyl 9,12,15-octadecanoic acid.

[0048] 20 μL of the original concentration of ethyl 9,12,15-octadecadienoate was added to 156.4 μL of n-hexane to obtain an ethyl 9,12,15-octadecadienoate solution. The 100 μg / μL ethyl 9,12,15-octadecadienoate solution was diluted with n-hexane in a 10-fold decreasing concentration gradient to prepare test solutions of 10 μg / μL, 1 μg / μL, 0.1 μg / μL, 0.01 μg / μL, 0.001 μg / μL, and 0.0001 μg / μL.

[0049] Electroantennography (EAG) and a T-type olfly were used to test the electrophysiological and behavioral responses of female and male Aphidida albicans to different doses of ethyl 9,12,15-octadecanoic acid standard.

[0050] Antennae Potential Measurement System (EAG) Test: Six concentrations of test solutions (0.0001 μg / µL to 10 μg / µL) and n-hexane (control group) were each added in 10 µL to filter paper strips measuring 0.5 cm × 5 cm. These strips were then placed into standard Pasteur tubes and sealed with sealing film. The corresponding concentration parameters were marked on the tube walls. Five-day-old unmated female and male Aphidida albicans were used as test subjects. The base of the antennae was removed under a dissecting microscope using micromanipulation and fixed to a glass electrode system containing 0.1 mol / L potassium chloride solution: the reference electrode was connected to the base of the antennae, and the recording electrode was connected to the tip. Baseline stability was monitored after successful connection, and electrophysiological recording was initiated once the baseline stabilized. The stimulation delivery system connected the tip of the Pasteur tube to the gas path device via a silicone tube. Pulsed gas flow (10 mL / s, 0.2 s) was used for stimulation, with each stimulation interval at least 30 seconds to ensure sufficient time for the antennae to recover their sensitivity. The stimulation of the test solutions was performed in ascending order of concentration gradient. The n-hexane control stimulation was performed twice, once at the beginning and once at the end. The antennal potential response values ​​for each stimulation were accurately recorded using Syntech EAG Pro software. Thirty-five biological replicates were completed for both male and female adults. The response values ​​were calculated using the formula: Relative EAG response value (mV) = EAG response value of the tested compound (mV) - Average EAG response value for n-hexane stimulation (mV). The bar charts showing the EAG response of female and male Aphidida albicans to different doses of ethyl 9,12,15-octadecanoate are shown below. Figure 2 .

[0051] according to Figure 2 The results showed that male Aphididae 'Grey's fly had a significantly higher EAG response to doses of 0.01 μg, 0.1 μg, 1 μg, 10 μg, and 100 μg of ethyl 9,12,15-octadecanoic acid than females.

[0052] T-type olfly test: The effect of ethyl 9,12,15-octadecanotrienoic acid on the behavioral selection of male Aphidida albicans was determined using a T-type olfly. The experimental setup consisted of a colorless, transparent T-shaped glass tube (inner diameter: 2.5 cm, main arm length: 3 cm, side arm length: 18 cm). The T-shaped tube was placed in a behavior box (100 cm × 60 cm × 80 cm) for the experiment. The two arms of the T-type olfly were connected sequentially to a flow meter, a humidifier, an activated carbon filter, and an air sampler. The air sampler provided a constant airflow as the power system. Before the experiment, the gas flow rate of both arms was calibrated to 0.5 L / min using the flow meter. The experimental setup was connected using silicone tubing. The ambient temperature was maintained at 23 ± 1 °C, and the relative humidity at 50% ± 5%. The experiment was conducted from 8:00 to 18:00. Five-day-old unmated male adults were used as the experimental subjects, with at least 50 Aphidida albicans per treatment. In the experiment, 10 μL of the test solution at concentrations of 1 μg / μL, 0.1 μg / μL, 0.01 μg / μL, and 0.001 μg / μL were respectively added to filter paper (1.5 cm × 1.5 cm) to obtain test compound filter paper; an equal volume of n-hexane was added to another filter paper to obtain control filter paper. The test compound filter paper and control filter paper were then placed at the ends of the two arms of a T-tube, respectively. A single *Aphidius gravidae* was then placed into the main arm and observed for 10 minutes. A valid selection was recorded when the *Aphidius gravidae* moved to more than half of either arm and remained there for 30 seconds or more; otherwise, it was recorded as an invalid selection. After every 5 flies were tested, the positions of the test group and the control group were swapped to eliminate directional bias. After every 10 flies were tested, a clean T-tube was used. The used T-tube was washed with ethanol and dried in a 180°C oven for 2 hours before reuse. At least 50 replicates were performed for each dosage. The results of the determination of male Aphidius maximus at various dosages are shown in the figure. Figure 3 .

[0053] according to Figure 3 The results showed that ethyl 9,12,15-octadecanoic acid at doses of 0.1 μg, 1 μg, and 10 μg had a significant attraction effect on male aphids.

[0054] In summary, ethyl 9,12,15-octadecadienoic acid is a sex pheromone component of female Aphidida albicans.

[0055] Example 3: Effects of ethyl 9,12,15-octadecanotrienoate on mating behavior of the large gray fly.

[0056] 20 μL of the original concentration of ethyl 9,12,15-octadecanotrienoate was added to 156.4 μL of ethanol to obtain a 100 μg / μL solution of ethyl 9,12,15-octadecanotrienoate. The 100 μg / μL solution of ethyl 9,12,15-octadecanotrienoate was then diluted with ethanol in a 10-fold decreasing concentration gradient to prepare test solutions of 0.1 μg / μL, 0.01 μg / μL, and 0.001 μg / μL.

[0057] The experimental environment was maintained at a temperature of 23±1 degrees Celsius and a relative humidity of 50%±5%. Unmated female and male insects, 5 days after emergence, were selected as experimental subjects. One female and one male insect were simultaneously placed in a cylindrical mating container (5 cm in diameter and 6 cm in height) for pairing. The paired adults were divided into a control group and test groups at various concentrations. In the control group, 1 μL of ethanol was dripped onto the female insect's body, while in the test groups, 1 μL of the test solution at each concentration was dripped onto the female insect's body. Each treatment consisted of 100 to 120 pairs. After placing the paired female and male insects in the mating container, the container was placed in an insect behavior test chamber (60 cm long, 40 cm wide, and 90 cm high). The mating process of the paired female and male insects was continuously observed and recorded using a camera from 8:00 to 18:00, for a total of 10 hours. Record the following two indicators exhibited during mating: (1) Within the observation period, if the male and female insects engage in amplexus behavior, it is considered mating; if they do not engage in amplexus behavior, it is considered not mating. Calculate the mating rate: mating rate = number of mated pairs × 100% / total number of pairs. The results are shown in […]. Figure 4 (2) Record the time taken by the male insect to search for the female insect from the beginning of the pairing process to the mating. This time is the search time of the male insect before mating. The results are shown in […]. Figure 5 .

[0058] according to Figure 4 The results showed that, compared with the control group, the use of 0.01 μg and 0.1 μg of ethyl 9,12,15-octadecanoic acid significantly increased the mating rate of both female and male insects; according to Figure 5 The results showed that, compared with the control group, the use of 0.01 μg and 0.1 μg of ethyl 9,12,15-octadecanoic acid significantly shortened the search time of male insects before mating.

[0059] Example 4: Field attraction effect of ethyl 9,12,15-octadecanotrienoate on male aphids.

[0060] Application of lures prepared based on the sex pheromone ethyl 9,12,15-octadecyltrienoate from female Aphidida albicans for attracting Aphidida albicans.

[0061] Core preparation: 20 μL of the original concentration of ethyl 9,12,15-octadecanoate was added to 156.4 μL of n-hexane to prepare a 100 μg / μL ethyl 9,12,15-octadecanoate solution. 20 μL of this solution was then added to a rubber-tipped support to prepare 38 test cores. 38 control cores were prepared by adding 20 μL of n-hexane to a rubber-tipped support.

[0062] Field Trial: This experiment was conducted at the Tianjin Academy of Agricultural Sciences Research and Pilot-Scale Base, using wheat fields in the heading stage. Efforts were made to ensure that the geographical conditions, wheat varieties, and growth stages of the test and control groups were identical to minimize interference from environmental factors. Yellow sticky insect traps were used (to facilitate the counting of captured large grey aphids). The traps were fixed with fiber rods and crossbars, with the lure inserted through the trap. The test and control groups were marked on the traps. The traps were placed at 10-meter intervals within the field. For each trial replicate, sticky traps were randomly placed in the corresponding field. Every two days after placement, the number of large grey aphids on the traps was surveyed and recorded at the same time period. After each survey, the trap positions were randomly changed to reduce error. Results are shown below. Figure 6 .

[0063] according to Figure 6 The results showed that ethyl 9,12,15-octadecanotrienoate had a significant attraction effect on male Aphidida albicans compared to the control solvent n-hexane. Therefore, ethyl 9,12,15-octadecanotrienoate can be used as a sex attractant in the field to attract Aphidida albicans.

Claims

1. Ethyl 9,12,15-octadecyltrienoate in attracting the large gray aphid ( Eupeodes corollae Male adults or increase the number of large gray aphid flies ( Eupeodes corollae Application in the mating rate of male and female adults.

2. The application according to claim 1, characterized in that, The male adult giant aphids attracted by the ethyl 9,12,15-octadecyltrienoate are used for pollination of plants.

3. A lure core comprising ethyl 9,12,15-octadecadienoic acid and a sustained-release carrier.

4. The lure core according to claim 3, characterized in that, The dosage of ethyl 9,12,15-octadecyltrienoate is 0.01 micrograms or more.

5. The lure core according to claim 3, characterized in that, The dosage of ethyl 9,12,15-octadecyltrienoate is 0.1 to 2000 micrograms.

6. The lure core according to claim 3, characterized in that, The sustained-release carrier is made of rubber.

7. The lure core according to claim 3, characterized in that, The sustained-release carrier is at least one of rubber-tip carrier, cotton-core carrier, and microtubule carrier.

8. The lure according to any one of claims 3 to 7 is used to attract male adult Aphidius maxima or to increase the number of Aphidius maxima ( Eupeodes corollae Application in the mating rate of male and female adults.

9. The application according to claim 8, characterized in that, The male adult gray aphids attracted by the lure are used for pollination of plants.

10. The application according to claim 8 or 9, characterized in that, The decoys are placed in the field at intervals of ≤10 meters.

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