A sex pheromone of locustas and its use

Abstract

The present application relates to the technical field of pest control, and further provides a method for extracting and identifying locust sex pheromone, which comprises the following steps: extracting locust volatile matter, and identifying the main components of the volatile matter as (Z)-2-pentene-1-ol, benzaldehyde, 2-ethyl-1-hexanol, 3-ethyl-benzaldehyde, 2-hexyl-1-dodecanol, 2-methoxyphenol, 4-ethyl-acetophenone, butyl benzoate, phenol, 2-ethyl-4,5-dimethylphenol, dimethyl phthalate, 2,4-di-tert-butylphenol, methyl hexanoate, isooctyl phthalate and dibutyl phthalate. Experiments show that dibutyl phthalate (DBP) as locust sex pheromone has a good attraction to male locusts, and can be applied to the biological control of locusts. The present application provides a new method for the biological control of locusts, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pest control technology, specifically relating to methods for extracting, identifying, and analyzing locust sex pheromones, and the applications of locust sex pheromones. In particular, it relates to the application of dibutyl phthalate as a locust sex pheromone, and to attractants and lures containing dibutyl phthalate. Technical Background

[0002] Sex pheromones are used by insects to identify and locate mates. They are an important signaling pathway for insect-mediated mating behavior and have become a crucial component of "push-pull" pest control strategies. Insect sex pheromones are trace amounts of chemical substances secreted externally by specialized organs of a particular sex within the same insect species. They can be received by the sensory organs of opposite-sex individuals of the same species, eliciting specific behavioral responses or physiological effects (such as mate search, courtship, and mating). As early as the 1960s, Butenandt extracted and identified the first sex pheromone component, Bomkol, from the gonads of silkworms, which could induce male courtship behavior. Since then, with increasingly improved analytical and identification techniques, a large number of insect sex pheromones have been identified, including those from the vast majority of agricultural pests, making research on population-specific sex pheromones a focus of attention.

[0003] Locusts are a major global agricultural pest, and locust outbreaks often cause severe economic losses. Current locust control primarily relies on the extensive, blanket spraying of traditional pesticides with relatively short-lived residues, which poses risks to the environment and human health. Therefore, it is necessary to develop better strategies for sustainable locust control. Locusts exhibit density-dependent morphological changes, namely gregarious and solitary locusts. Locusts at different densities often employ different strategies during courtship. For gregarious locusts, mating location and mate selection are not the primary concerns for males; sperm competition is more important. For solitary locusts, they may simultaneously use both long-distance and short-distance chemical cues to locate and identify mates of the same species. Although locusts can rely on olfactory signals to identify mates and perform mate location behaviors, the composition of their sex pheromones remains unknown.

[0004] Numerous studies have investigated the chemical composition of volatile substances emitted by locusts, but the pheromone components related to mating behavior remain controversial. Analysis of locust volatile components revealed that the majority are aromatic compounds with benzene rings. Among these, gregarious males release large amounts of phenylacetonitrile. Previous studies reported phenylacetonitrile as a sex pheromone, but recent research indicates it plays a warning role in locusts. The release of 4-vinylanisole in gregarious males is also significantly higher than in solitary males; it has been identified as a locust aggregation pheromone that attracts individuals of the same species. Furthermore, 2-heptanone, specifically released by male locust adults, has been found. This compound may mediate sexual dimorphism in locusts, but whether it affects mating behavior remains unclear.

[0005] Currently, there is limited research on locust sex pheromones both domestically and internationally. The application of locust sex pheromones for locust control offers advantages such as specificity, high efficiency, and environmental friendliness; therefore, its use in locust control is of great significance.

[0006] However, whether a substance can serve as a locust sex pheromone is unpredictable, and there are no reports in the art regarding the application of dibutyl phthalate as a locust sex pheromone. The applicant of this invention has discovered that dibutyl phthalate has a strong attraction effect on locusts and can be used as a locust sex pheromone, providing a new target molecule for the green control of locusts and offering good potential for locust control. Summary of the Invention

[0007] This invention provides a sex pheromone that has a strong attraction effect on locusts, which can reduce the use of chemical pesticides and is of great significance for the integrated management of locusts.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] In one aspect, the present invention provides a locust sex pheromone, wherein the locust sex pheromone is dibutyl phthalate.

[0010] Another aspect of the present invention provides a locust attractant containing a locust sex pheromone, wherein the locust sex pheromone is dibutyl phthalate.

[0011] The present invention also provides a locust lure, the lure comprising a slow-release carrier and a locust sex pheromone loaded on the slow-release carrier, wherein the locust sex pheromone is dibutyl phthalate.

[0012] This invention also discloses the application of the locust sex pheromone of this invention, as well as locust attractants and locust lures of the locust sex pheromone in the control of locusts.

[0013] Finally, the present invention also provides a method for extracting the locust sex pheromone disclosed in the present invention, comprising the following steps:

[0014] 1) A dynamic headspace collection system was used to collect volatiles from female and male adult insects;

[0015] 2) Extract the volatiles from female and male adult insects using organic solvents and store them for later use;

[0016] 3) Gas chromatography-mass spectrometry was used to compare and analyze the extracted volatiles from male and female adults to identify informational compounds related to locust mate location.

[0017] Beneficial effects

[0018] This invention, through chemical composition analysis of the natural pheromones of locusts, has for the first time identified dibutyl phthalate as a chemical component of their sex pheromone. This substance has a strong attraction to male locusts and can be applied to the biological control of locusts.

[0019] Compared to chemical control methods for locusts, using the sex pheromones of this invention for control has the following advantages:

[0020] 1. Compared to chemical control, sex pheromones leave no residue and are harmless to plants and other beneficial insects in the environment;

[0021] 2. It has a very significant effect on attracting male locusts, and can be artificially released to interfere with mating behavior, thereby indirectly controlling the damage caused by the next generation of locust larvae;

[0022] 3. The required dose of sex pheromones in the lure core is low, which can significantly reduce prevention and control costs and make the prevention and control effect highly efficient.

[0023] 4. The method for extracting locust sex pheromones provided by this invention has the advantages of being simple, easy to implement, and accurate in analysis. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0025] Figure 1 Total ion flow diagram of volatiles released by mature male and female locusts

[0026] Figure 2 The proportion of peak area of ​​15 compounds in the volatiles emitted from the body surface of mature female and male locusts to the total amount

[0027] Figure 3 Content of six highly abundant volatile substances on the female body surface at different developmental stages of female insects

[0028] Figure 4 Standard curves of 6 female high-abundance surface volatiles

[0029] Figure 5 Wind tunnel experiment diagram

[0030] Figure 6 Displacement distance of mature male migratory locusts stimulated by different doses of a mixture of six female compounds

[0031] Figure 7(A) Distance of mature male locusts under different mixture stimuli

[0032] Figure 7(B) Displacement distance of male insects stimulated by a mixture of six female-specific compounds and by DBP alone.

[0033] Figure 8 Displacement distance of mature male migratory locusts under different doses of DBP stimulation

[0034] Figure 9 Dose-response curves of the antennae of mature male and female migratory locusts to six compounds, EAG.

[0035] Figure 10 Schematic diagram of a double-selection cage experiment in the wild and the selection behavior of mature male locusts towards DBP. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specification. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This application specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] This invention provides a locust sex pheromone, namely dibutyl phthalate. The use of dibutyl phthalate as a locust sex pheromone is a first-time study and report by the applicant, and dibutyl phthalate exhibits excellent attraction effects on locusts.

[0042] This invention also provides a locust attractant containing a locust sex pheromone, wherein the locust sex pheromone is dibutyl phthalate. The attractant of this invention exhibits good attractant effects.

[0043] Preferably, the attractant may further contain one or more of the following: (Z)-2-penten-1-ol, benzaldehyde, 2-ethyl-1-hexanol, 3-ethyl-benzaldehyde, 2-hexyl-1-dodecanool, 2-methoxyphenol, 4-ethyl-acetophenone, butyl benzoate, phenol, 2-ethyl-4,5-dimethylphenol, dimethyl phthalate, 2,4-di-tert-butylphenol, methyl hexanoate, and isooctyl phthalate.

[0044] Preferably, the attractant described above can be formulated into a pesticide-acceptable formulation according to agricultural use requirements. Formulating a suitable formulation is achievable using conventional techniques in the art.

[0045] The present invention also provides a locust lure, the lure comprising a slow-release carrier and a locust sex pheromone loaded on the slow-release carrier, wherein the locust sex pheromone is dibutyl phthalate.

[0046] Preferably, the slow-release carrier of the locust lure is a rubber stopper or a rubber tube.

[0047] More preferably, the load of locust sex pheromones on a single locust lure is 10 mg.

[0048] The present invention discloses the application of dibutyl phthalate, a locust sex pheromone, and attractants and lures containing this sex pheromone in the control of locusts.

[0049] Finally, the present invention also provides a method for extracting and identifying locust sex pheromones, which involves extracting locust volatiles and analyzing and identifying the volatiles.

[0050] Specifically, the present invention provides a method for extracting and identifying sex pheromones in locusts, comprising the following steps:

[0051] 1) A dynamic headspace collection system was used to collect the volatiles of male and female adult insects;

[0052] 2) Extract the volatiles from male and female adult insects using organic solvents and store them for later use;

[0053] 3) Gas chromatography-mass spectrometry was used to compare and analyze the extracted volatiles from male and female adults to identify informational compounds related to locust mate location.

[0054] Preferably, step 1) of the above method further includes loading locusts into a container, and passing compressed air through three processing containers to purify and humidify the gas. The three processing containers are respectively filled with molecular sieve (0.5nm), activated carbon and distilled water, and the containers and processing containers are preferably glass bottles.

[0055] Furthermore, in step 1), the air passes through the treatment container and then enters the container, which is connected to the collector, into which an adsorbent (Porapak Q 80–100mesh, Supelco Bellefonte, PA, USA) is added.

[0056] Preferably, the organic solvent in step 2) of the above method is dichloromethane, and the locust extract is stored at -20°C.

[0057] Preferably, the gas chromatography-mass spectrometry (GC-MS) coupling in step 3) of the above method is performed using an Agilent gas chromatograph 6890N coupled with a 5973 mass spectrometer.

[0058] Preferably, in step 3), the capillary column used is a 30m long DB-WAX capillary column (30m×0.25mm ID, 0.25μm film, J&W Scientific, Folsom, CA, USA).

[0059] Further, in step 3), the injection port temperature is 250℃, the temperature program is 40℃ for 4 min, then increased to 180℃ at 5℃ / min, then increased to 230℃ at 10℃ / min, held at 230℃ for 3 min, and the injection volume is 2μL.

[0060] The analytical and identification method of this invention can more accurately analyze the chemical composition of locust sex pheromones.

[0061] Extraction and analysis revealed that the main volatile components were (Z)-2-penten-1-ol, benzaldehyde, 2-ethyl-1-hexanol, 3-ethyl-benzaldehyde, 2-hexyl-1-dodecanool, 2-methoxyphenol, 4-ethyl-acetophenone, butyl benzoate, phenol, 2-ethyl-4,5-dimethylphenol, dimethyl phthalate, 2,4-di-tert-butylphenol, methyl hexanoate, isooctyl phthalate, and dibutyl phthalate. Experiments showed that dibutyl phthalate (DBP) has a good attraction effect on male locusts as a sex pheromone and can be applied to the biological control of locusts.

[0062] The inventive concept of the present invention is further illustrated below with reference to the embodiments. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0063] Example

[0064] 1. Headspace collection of volatiles and chemical analysis

[0065] 1.1 Implementation Method

[0066] A dynamic headspace collection system was used to collect volatiles from male and female locusts. Thirty male and thirty female locusts were placed in separate 1000 mL round-bottom glass flasks. Compressed air was passed through three 500 mL glass bottles to purify and humidify the gas. Each bottle was filled with a molecular sieve (0.5 nm), activated carbon (Beijing Chemical Company), and distilled water, respectively. After the air was pushed into the round-bottom glass flasks, it was passed through a glass collector (a 3 mm inner diameter glass tube containing 100 mg of the adsorbent Porapak Q80–100mesh, Supelco Bellefonte, PA, USA). An atmospheric sampler was used at the outlet to evacuate the air at a rate of 400 mL / min. The evacuation time was 3 hours, typically from 11:00 AM to 2:00 PM. Simultaneously, to eliminate contamination, the empty round-bottom flasks were evacuated, and the collected compounds were used as a control. The adsorbent, which had adsorbed volatile compounds from 30 locusts, was rinsed with 1 mL of dichloromethane, and then the sample was concentrated to 60 μL (2 μL per female locust) using nitrogen. The extract was then stored at -20°C until chemical analysis was performed.

[0067] The experimental apparatus employed an Agilent 6890N gas chromatograph coupled with a 5973 mass spectrometer. A 30m long DB-WAX capillary column (30m × 0.25mm ID, 0.25μm film, J&W Scientific, Folsom, CA, USA) was used. The injection port temperature was 250℃, and the temperature program was as follows: hold at 40℃ for 4 min, increase to 180℃ at 5℃ / min, then increase to 230℃ at 10℃ / min, and hold at 230℃ for 3 min. To quantify the content of individual compounds in female insects, an external standard curve (from 0.1 ng to 10 ng) of the pheromone from a single female insect was constructed.

[0068] 1.2 Results

[0069] Gas chromatography-mass spectrometry (GC-MS) was used to compare and analyze volatile substances from the body surface of sexually mature male and female locusts to identify informational compounds involved in mate localization. Figure 1 As shown in Table 1, among the 15 compounds identified in the locust odor profile, six compounds were released at significantly higher levels in female locusts than in males. These six compounds were (Z)-2-penten-1-ol; benzaldehyde; 2-ethyl-1-hexanol; 3-ethyl-benzaldehyde; 4-ethylacetophenone; and dibutyl phthalate. DBP was the most abundant volatile component in female locusts, accounting for 20.24% of the total emissions. Figure 2 It can be seen that DBP accounts for only 2.76% of the male volatile components. Figure 3 The study showed that female insects had higher levels of dibutyl phthalate and 4-ethyl acetophenone, which increased significantly 7 to 14 days after emergence, but the levels did not change much in the 5th instar and pre-sexual maturity stages. The release of the other four odors did not show this characteristic.

[0070] Table 1. Components and retention times of compounds on the body surface of female and male locusts.

[0071]

[0072] Note: Data are expressed as mean ± sem. A t-test was used; different letters indicate statistically significant differences between groups.

[0073] To quantify the six candidate pheromone components, this invention first established a linear regression analysis of the content (x) (from 0.1 ng to 10 ng) of the six odors released most frequently by females and their integration area (y). The regression equations are shown below. Figure 4The average concentrations of the following six compounds per locust were 1.92, 0.31, 1.79, 1.62, 0.88, and 2.12 ng, respectively: (Z)-2-penten-1-ol; benzaldehyde; 2-ethyl-1-hexanol; 3-ethyl-benzaldehyde; 4-ethylacetophenone; and dibutyl phthalate.

[0074] 2. Wind tunnel behavioral experiments

[0075] 2.1 Implementation Method

[0076] Figure 5 This is a schematic diagram of a wind tunnel experiment. The experiment was conducted in a resin glass flat tunnel, measuring 220cm × 50cm × 100cm. A small wire mesh chamber (14×7×7cm) was located at the downwind end of the wind tunnel for releasing test insects. A small cage (10×10×10cm) was placed 220cm upwind to conceal the locusts placed inside. The airflow speed inside the wind tunnel was approximately 20cm / s. For uniform illumination, red lights were placed above the wind tunnel, evenly spaced according to its length. The internal temperature of the wind tunnel was maintained at 28±1℃ during the experiment. Before the experiment, sexually mature male and female locusts were placed in the environment of the wind tunnel device to acclimatize for at least 1 hour, and the wings of the females were taped to restrict their vibration and prevent any sound signals from being generated.

[0077] 2.2 Results

[0078] Figure 6 The study showed the displacement distance of male locusts under different doses of a mixture of six female compounds. Based on the odor analysis results, this invention selected six compounds with high release rates from female locusts and formulated them into mixtures according to their volatile content. First, effective doses attracting males were screened. It was found that release rates of 10-1000 female locusts as a stimulus source elicited significant attraction behavior in male locusts; that is, the displacement distance of male locusts was significantly higher than that of the control group and when release rates of 0.01-1 female locusts were used as a stimulus source.

[0079] To further investigate the chemical signals that attract male insects during mate placement, this invention conducted wind tunnel behavioral tests on solitary insects after removing one compound from a mixture containing six compounds. The results showed that when DBP was removed, male insects did not exhibit any displacement changes, showing no significant difference from the control group, but the displacement distance was significantly lower than that observed when the mixture of the six compounds was used. Furthermore, this invention found that when male insects were stimulated with mixtures containing the other five compounds, the displacement changes were not significantly different among them, nor were they different from those stimulated with the mixture of the six compounds. However, the displacement changes were significantly higher than those observed in the control group and the mixture with DBP removed. The results are detailed below. Figure 7A To verify the effect of DBP, this invention used DBP alone to stimulate male insects. See also... Figure 7B It was found that when male locusts were stimulated with DBP alone, they moved a significantly longer distance than the control group, while there was no significant difference in the distance moved compared to stimulation with a mixture containing six compounds. Therefore, DBP is both necessary and sufficient for male locusts to locate their mates.

[0080] The present invention further uses DBP alone to test the behavioral response of male insects at different concentrations. Figure 8 The results showed that when the DBP dosage for male insects reached 20 ng or more, the displacement distance was significantly higher than that of the control and the male insects stimulated with low concentrations of 0.2 ng and 2 ng.

[0081] 3. Reaction of male and female antennae to six compounds, EAG

[0082] 3.1 Implementation Plan

[0083] Cut off the root of a healthy locust antenna with a blade, then cut off the tip of the antenna with the blade, about 1 mm in length; apply conductive glue to both ends of the electrode, connect the prepared antenna to both ends of the electrode, connect the circuit, and open the software to prepare for recording.

[0084] Turn on the gas pump, ensure the inlet bottle contains distilled water, and begin recording the data after the baseline stabilizes. Stimulate with a Pasteur tube gas stream containing solvent, pausing for 1 minute after stimulation; then stimulate with a Pasteur tube gas stream containing the corresponding odor, pausing for 1 minute after stimulation; then switch to a different odor stimulation, pausing for 1 minute between each stimulation. After all odor stimulations have been completed, use a new grasshopper antenna for stimulation and recording. The order of odor stimulation on the antennae is random. For statistical analysis, this invention subtracts the voltage value of the solvent stimulation from the voltage value of the odor stimulation, and then performs statistical analysis.

[0085] 3.2 Results

[0086] This invention utilizes EAG (electroecogenomic angle) technology to detect the dose-response of six compounds to EAG in the antennae of sexually mature male and female locusts. The results showed that DBP induced an EAG response in both males and females, with males exhibiting a stronger antennal potential response at 20 ng compared to females. The other five compounds also induced EAG responses in both male and female locusts, but there was no significant difference in EAG response between the antennae of males and females. (See attached results). Figure 9 .

[0087] 4. Application Example (Field Cage Experiment)

[0088] 4.1 Implementation Plan

[0089] Decoy preparation: DBP was prepared to a concentration of 100 μg / μL using dichloromethane as a solvent. 100 μL was added to the decoy slow-release rubber stopper. After the solvent was allowed to evaporate completely in a fume hood, it was used as the decoy.

[0090] Two-way selection experiment: In the two-way selection experiment, this invention was conducted in a 2m×2m×2m cage. Sticky boards containing DBP and dichloromethane were alternately placed on both sides of the cage. Each sticky board contained either one DBP lure or one control lure containing the solvent. Fifteen male locusts were released into the cage simultaneously, and the number of locusts stuck to each board was recorded after 9 hours.

[0091] Attraction efficiency statistics and analysis: Data from field attraction experiments were analyzed using arcsin(X). 1 / 2 The formula was converted, and the difference in attraction index between the lure and the solvent was compared using a two-tailed unpaired t-test. The attraction index (%) was calculated as follows: O / (TN) × 100, where O is the number of locusts attracted by the lure or solvent, N is the number of locusts that did not make a choice, and T is the total number of locusts tested.

[0092] 4.2 Results

[0093] Two adhesive plates containing DBP and solvent lures are placed in a large cage. The positions of the adhesive plates with different lures are alternated during each experiment. Figure 10 When male locusts were released into cages, sticky traps containing DBP lures captured a significant number of male locusts. Multiple field experiments showed that an average of over 75% of the locusts were male. This result confirms that DBP has an attractive effect on male locusts in the field, further demonstrating that this compound is a sex pheromone for locusts.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a locust attractant in locust control, characterized in that, The attractant contains dibutyl phthalate, a locust sex pheromone.

2. The application according to claim 1, characterized in that, The attractant also comprises one or more of (Z)-2-penten-1-ol, benzaldehyde, 2-ethyl-1-hexanol, 3-ethyl-benzaldehyde, 2-hexyl-1-dodecanool, 2-methoxyphenol, 4-ethyl-acetophenone, butyl benzoate, phenol, 2-ethyl-4,5-dimethylphenol, dimethyl phthalate, 2,4-di-tert-butylphenol, methyl hexanoate, and isooctyl phthalate.

3. The application according to claim 2, characterized in that, The attractant is prepared into a pesticide-acceptable formulation.

4. The application of a locust attractant in locust control, characterized in that, The locust lure includes a slow-release carrier and a locust sex pheromone loaded on the slow-release carrier, wherein the locust sex pheromone is dibutyl phthalate.

5. The application according to claim 4, characterized in that, The sustained-release carrier is a rubber stopper or a rubber tube.

6. The application according to claim 4 or 5, characterized in that, The loading of locust sex pheromones on a single locust lure is 10 mg.

7. The application according to claim 2, characterized in that, The method for extraction and analysis of dibutyl phthalate, a locust sex pheromone, includes the following steps: 1) A dynamic headspace collection system was used to collect volatiles from female and male adult insects; 2) Extract the volatiles from female and male adults using organic solvents and store them for later use; 3) Gas chromatography-mass spectrometry was used to compare and analyze the extracted volatiles from male and female adults in order to identify informational compounds related to locust mate location.

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