A host marking pheromone of Lymantria dispar and its application and identification method

Abstract

The present invention provides a host-marking pheromone for the gypsy moth egg wasp, its application, and an identification method. The host-marking pheromone for the gypsy moth egg wasp is cis-1,3-docosenol. The present invention clarifies the structure of the gypsy moth egg wasp marking pheromone. The pheromone can induce a significant electrophysiological response in the antennae of female gypsy moth egg wasps. It is used to prevent economic insect eggs from being parasitized by the gypsy moth egg wasp, and has excellent practicality. The present invention also provides a method for identifying the host-marking pheromone for the gypsy moth egg wasp. The method uses GC-MS and EAD to determine the active ingredients of the host-marking pheromone by comparing extracts of economic insect eggs that have not been parasitized by the gypsy moth egg wasp with extracts of parasitized insect eggs. This identification method has a short cycle time and highly reliable results.

Description

Technical Field

[0001] The present invention belongs to the technical field of beneficial insect protection in agriculture and forestry, and specifically relates to a host marking pheromone of the gypsy moth egg wasp, and its application and identification method. The host marking pheromone of a class of enol compounds of the gypsy moth egg wasp is used to prevent the eggs of economic insects such as tussah silkworms and ricin silkworms from being parasitized, thereby improving the hatching rate of larvae in the wild. Background Art

[0002] The gypsy moth egg wasp, Anastatus disparis, belongs to the order Hymenoptera, superfamily Apidae, family Cynomoridae, genus Anastatus. It is an important parasitic natural enemy of the egg stages of gypsy moths, tussah silkworms, and ricin silkworms. Currently, large-scale releases of this wasp are being used in the wild to control forestry pests such as gypsy moths and the ginkgo moth moth. Besides pests, this wasp also parasitizes economic insects such as the tussah silkworm Antheraea pernyi and the ricin silkworm Philosamia cynthia ricini.

[0003] The tussah silkworm, also known as the wild silkworm, belongs to the Lepidoptera order, family Bombycidae, genus Antheraea. It is a silk-spinning insect named after its diet of oak leaves. The silkworm's outer shell is composed of animal protein fibers, which can be woven into silk for clothing, decorative purposes, and industrial applications. The tussah silkworm pupae contain high levels of protein and amino acids, making them a nutritious food and also used as a medicinal herb. The castor silkworm, a subspecies of the family Bombycidae, also known as the Indian silkworm or the cassava silkworm, is a species of silkworm that originally grew wild. It feeds on castor leaves, as well as cassava, cranewood, ailanthus, pine, and cypress leaves, making it a highly adaptable and polyphagous silkworm. The parasitism of this economic insect by this wasp will inevitably affect its economic value.

[0004] Host marking pheromones (HMPs) are chemical markers associated with insect egg-laying. They are produced by insects and mark their hosts, signaling the presence of conspecifics. The primary function of insect host marking pheromones is to regulate insect egg-laying behavior, reducing competition for host resources among offspring by discouraging the insect or other conspecifics from laying eggs on previously marked hosts or by reducing egg production. Parasitic wasps often use host marking pheromones to distinguish between already parasitized and healthy hosts, avoiding overparasitism and polyparasitism, and minimizing intraspecific and interspecific competition among their offspring.

[0005] Currently, the gypsy moth egg flat-bellied wasp, a key natural enemy, is being released in the wild on a large scale to combat various lepidopteran pests. However, during the wild reproduction of economic insects such as tussah silkworms and ricin silkworms, various parasitic wasps, including the gypsy moth egg flat-bellied wasp, often use the eggs or pupae of these insects as hosts to reproduce, and the offspring they produce develop well. This has seriously affected the survival rate of these economic insects in the wild and caused considerable economic losses. Summary of the Invention

[0006] The present invention aims to provide a host marking pheromone for the gypsy moth egg wasp, which is a marking pheromone based on an enol derivative structure of the gypsy moth egg wasp, namely cis-13-docosenol (13-Docosen-1-ol), abbreviated as DOC.

[0007] Another object of the present invention is to provide an application of a host marking pheromone of the gypsy moth egg wasp for preventing economic insect eggs from being parasitized by the gypsy moth egg wasp.

[0008] The present invention also provides a method for identifying the host marking pheromone of the gypsy moth egg flat-abdomen wasp. The method adopts gas chromatography-mass spectrometry (GC-MS) and electroantennographic technique (EAD) to determine the active components of the host marking pheromone of the gypsy moth egg flat-abdomen wasp by comparing and analyzing the extracts of the eggs of economic insects that have not been parasitized by the gypsy moth egg flat-abdomen wasp and the extracts of the hosts that have been parasitized. The identification method has a short cycle and high credibility of the results.

[0009] The specific technical solutions of the present invention are as follows:

[0010] A host marking pheromone of the gypsy moth egg flat-bellied wasp is cis-13-docosenol, with the structural formula

[0011] The application of the host marking pheromone of the gypsy moth egg wasp provided by the present invention is used to prevent the eggs of economic insects from being parasitized by the gypsy moth egg wasp, thereby improving the hatching rate of larvae of economic insects bred in the wild.

[0012] The specific application method is:

[0013] A cis-13-docosenol solution is prepared using an organic solvent and sprayed on the activity area of ​​economic insects.

[0014] The concentration of the cis-13-docosenol solution is 10,000 to 50,000 mg / L;

[0015] The organic solvent is n-hexane;

[0016] The economic insects are tussah silkworms or ricin silkworms.

[0017] The present invention provides a method for identifying a host marker pheromone of a gypsy moth egg wasp, comprising the following steps:

[0018] 1) adding n-hexane solvent to fresh unparasitized and parasitized insect eggs of Lymantria dispar to obtain extracts of unparasitized and parasitized host eggs, respectively;

[0019] 2) The extract is subjected to gas chromatography-mass spectrometry (GC-MS) and antennae potential detection (EAD) to obtain corresponding spectra;

[0020] 3) Spectral analysis revealed a characteristic peak near a retention time of 22.6 min, which was identified by mass spectrometry as cis-13-docoenol. Gas chromatography comparison using a pure cis-13-docoenol solution revealed a similar retention time to the characteristic peak of an extract from insect eggs parasitized by the gypsy moth egg wasp. Furthermore, female wasp antennae exhibited a significant potentiometric response to cis-13-docoenol, and the gypsy moth egg wasp would not lay eggs on insect eggs coated with cis-13-docoenol. This confirmed that cis-13-docoenol is the host marking pheromone of the gypsy moth egg wasp.

[0021] In step 1), the number and volume of the selected unparasitized and parasitized insect eggs of the gypsy moth egg flat-abdomen wasp and the n-hexane solvent are both 20:60 μL;

[0022] In step 2), the sample airflow separated from the end of the gas chromatography capillary column is divided into two airflows at a ratio of 1:3. The gas chromatography column is HP-5msΜLtra Inert, 30m×250μmID, 0.25μm;

[0023] In step 2), the sample was injected into the gas chromatograph using splitless injection with an injection volume of 1.0 μL; the carrier gas was high-purity nitrogen with a flow rate of 2.25 mL / min; the heating program was: starting temperature 50°C → 10°C / min → 320°C (10 min);

[0024] In step 2), the mass spectrometry conditions are as follows: El ion source, electron energy 70 eV, proton scan range 35-500 amu, ion source temperature 230°C, quadrupole temperature 150°C, and transfer line temperature 250°C; a stimulated airflow generator is used to provide a stable clean airflow, and the airflow rate is controlled at 20-30 ml / min; the connector in the gas chromatograph is adjusted to 250°C to heat the sample airflow to prevent condensation of the sample airflow;

[0025] In step 2), to prevent the antenna potentiometer from being interfered with by the surrounding drifting electromagnetic field, the antenna potentiometer should be placed in a Faraday cage;

[0026] In step 2), one antennae was cut from the base of the head of a female Lymantria dispar wasp. Both ends of the antennae were fixed to a potentioantennal probe (PRG-2, Syntech) using glass capillaries and connected to a microelectrode placed on a micromanipulator (MP-15, Syntech) to test whether the antennae were in good contact with the electrodes.

[0027] In step 3), two insect eggs treated differently were presented to female bees for selection: one coated with n-hexane and the other with an n-hexane solvent containing cis-1,3-docosanol. It was found that the gypsy moth egg flattener wasp would not lay eggs on the eggs coated with cis-1,3-docosanol. All experimental procedures were recorded under camera; the female bees were 3-5 days old and the room temperature was 25-28°C.

[0028] Compared to the prior art, the present invention's enol-derivative-based marker pheromones clarify the structure of the marker pheromone of the gypsy moth egg wasp. This pheromone can elicit a significant electrophysiological response in female gypsy moth egg wasps. These wasps recognize and avoid the marker pheromones described in the present invention, and can be used to protect the eggs of economic insects such as tussah silkworms, laying the foundation for preventing parasitism of economic insect eggs by gypsy moth egg wasps in the wild. By spraying the host-marking pheromone of the gypsy moth egg wasp in breeding areas of economic insects such as tussah silkworms and ricin silkworms in the wild, the parasitism rate of gypsy moth egg wasps on economic insects such as tussah silkworms and ricin silkworms can be significantly reduced, achieving relatively significant results and demonstrating excellent practicality. The present invention also provides a method for identifying the host marking pheromone of the gypsy moth egg flat-abdomen wasp. The method adopts gas chromatography-mass spectrometry (GC-MS) and electroantenniocentesis (EAD) technology to determine the active components of the host marking pheromone of the gypsy moth egg flat-abdomen wasp by comparing the extracts of the eggs of economic insects that have not been parasitized by the gypsy moth egg flat-abdomen wasp with the extracts of the eggs of economic insects that have been parasitized. The identification method has a short cycle and high credibility of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Gas chromatogram and electroantennary response graph;

[0030] Figure 2 The following are the results of female bees' choices on tussah eggs smeared with n-hexane and DOC, as well as the results of female bees' choices on tussah eggs smeared with different concentrations of DOC; SPSS analysis showed that there were significant differences between the two (P<0.05). DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0032] Example 1

[0033] A host marking pheromone of the gypsy moth egg flat-bellied wasp is cis-13-docosenol, with the structural formula

[0034] The application of the host marking pheromone of the gypsy moth egg wasp provided by the present invention is used to prevent the eggs of economic insects from being parasitized by the gypsy moth egg wasp. The specific application method is:

[0035] Prepare a cis-13-docosahexenol solution using n-hexane as a solvent and spray it on the area where economic insects are active.

[0036] In the experiment of female bee selection, 10 mg of cis-13-docosanol was dissolved in 200 μL, 400 μL, and 1000 μL of n-hexane, and 10 μL of each solution was dropped on a fresh host. The host was then provided with a fresh host (without cis-13-docosanol solution, but with n-hexane) to a female bee of the Lymantria dispar. The results showed that the female bees all avoided laying eggs in n-hexane smeared with cis-13-docosanol, indicating that within this concentration range (10 The results showed that the egg-laying of the gypsy moth egg flat-abdomen wasp was disturbed when the organic solution with a concentration of 10,000 to 50,000 mg / L was placed in a backpack sprayer and sprayed in the wild on the living areas of economic insects such as tussah silkworms and ricin silkworms to avoid being parasitized by female gypsy moth egg flat-abdomen wasps, thereby obtaining a higher larval hatching rate.

[0037] Example 2

[0038] A method for identifying a host marking pheromone of a gypsy moth egg wasp comprises the following steps:

[0039] 1) adding 60 μL of n-hexane solvent to 20 tussah eggs that were not parasitized by the gypsy moth egg wasp and 20 that were parasitized by the host to obtain extracts of the eggs that were not parasitized and those that were parasitized by the host;

[0040] 2) The extract was subjected to gas chromatography-mass spectrometry (GC-MS) and electroantennary spectroscopy (EAD) detection, and cis-13-docosenol was analyzed separately to obtain corresponding spectra; the specific conditions were:

[0041] The sample was injected onto an HP-5msΜLtra Inert column (30 m × 250 μm ID, 0.25 μm film thickness, Agilent Technologies) with an injection volume of 1.0 μL. The carrier gas was high-purity helium at a flow rate of 2.25 mL / min. The temperature program was as follows: starting temperature 50°C → 10°C / min → 320°C (10 min). The mass spectrometry conditions included an El ion source, an electron energy of 70 eV, a proton scan range of 35–500 amu, an ion source temperature of 230°C, a quadrupole temperature of 150°C, and a transfer line temperature of 250°C. The extract components were qualitatively analyzed by comparing the mass spectra of standard spectral libraries (NIST, Willey) with those of standard compounds and based on the retention times of the standard compounds.

[0042] Potentenniometry measurements were performed as follows: an antenna was cut from the base of the head of a female Lymantria dispar wasp. Both ends of the antenna were fixed to a potentenniometry probe (PRG-2, Syntech) using glass capillaries and connected to a microelectrode mounted on a micromanipulator (MP-15, Syntech). Good contact between the antenna and the electrode was verified. The temperature of the gas chromatograph connector (EC-03, Syntech) was adjusted to 250°C to heat the gas phase stream and prevent sample condensation. The signal was amplified using a dual-channel USB data acquisition controller (IDAC-2, Syntech). The output was then input into a computer (Thinkpad T410I) for data acquisition, storage, and processing using the EAD signal recording and analysis program. The potentenniometry instrument was placed in a Faraday cage to protect it from interference from ambient electromagnetic fields.

[0043] 3) If Figure 1 As shown, compared with the spectrum of the extract of the parasitic host, there is a significant specific peak at retention time 22.607min in the spectrum of the extract of the parasitic host ( Figure 1 A and B), was identified by mass spectrometry as cis-13-docosenol, and the retention time was close to that of pure cis-13-docosenol solution (22.620min) ( Figure 1 C), and the antennae of the female wasp of the gypsy moth egg flat-abdomen showed a potentiometric response to cis-13-docosenol (22.55min, Figure 1 (D) Two insect eggs with different treatments were presented to female bees for selection: one coated with n-hexane and the other coated with an n-hexane solvent containing cis-1,3-docosenol. All experimental procedures were recorded under camera. The female bees were 3-5 days old and the room temperature was 25-28°C. The results showed that the gypsy moth egg flat-bellied wasp would not lay eggs on the insect eggs coated with cis-1,3-docosenol. Therefore, it is confirmed that the active compound of the host marking pheromone of the gypsy moth egg flat-bellied wasp is cis-1,3-docosenol.

Claims

1. An application of a host marking pheromone for Lymantria dispar, characterized in that: The invention is used for preventing economic insect eggs from being parasitized by the gypsy moth egg wasp, wherein the host marking pheromone of the gypsy moth egg wasp is cis-13-docosenol.

2. The use according to claim 1, characterized in that The application is specifically as follows: using an organic solvent as a solvent to prepare a cis-13-docosenol solution, and spraying the solution on the activity area of ​​economic insects.

3. The use according to claim 2, characterized in that The concentration of the cis-13-docosenol solution is 10,000 to 50,000 mg / L.

4. The use according to claim 2 or 3, characterized in that The organic solvent is n-hexane.

5. The use according to claim 2 or 3, characterized in that The economic insects are tussah silkworms or ricin silkworms.

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