Type II pheromones of the cotton bollworm and compounds that enhance their effects

Abstract

This invention relates to the bollworm type II pheromone and compounds that enhance its effect. The bollworm type II pheromone is cis-3,6,9-docosatriene, which can be used to attract bollworms.

Description

Technical Field

[0001] This invention relates to the field of biological control, and in particular to the type II pheromone of the cotton bollworm and compounds that enhance its efficacy. Background Technology

[0002] The external environment in which insects live is complex and ever-changing. Various chemical substances in the environment, as well as important environmental signals such as temperature and humidity, can transmit important information such as the location of food, individuals of the same or different species, alarm signals, and egg-laying sites, which are crucial for the normal reproduction and survival of insects.

[0003] cotton bollworm Helicoverpa armigera Belonging to the family Noctuidae in the order Lepidoptera, this is a major global agricultural pest. Its larvae have a diverse and extensive diet, damaging over 100 crops including wheat, corn, cotton, soybeans, and tomatoes, causing severe economic losses and hindering the sustainable and healthy development of agriculture. The adults are highly migratory, and the females have a high oviposition rate; reports indicate that a single female can lay up to 1000 eggs. Summary of the Invention

[0004] One invention provides the use of cis-3,6,9-docosatriene in attracting cotton bollworms.

[0005] In one specific embodiment, the cotton bollworm is a male adult.

[0006] The second invention provides the application of 5-hydroxy-2-decenoic acid-δ-lactone in increasing the attraction of cis-3,6,9-docosatriene to cotton bollworms.

[0007] In one specific embodiment, the cotton bollworm is a male adult.

[0008] The third invention provides a composition comprising cis-3,6,9-docosatriene and 5-hydroxy-2-decenoic acid-δ-lactone.

[0009] In one specific embodiment, the mass ratio of the cis-3,6,9-docosatriene to the 5-hydroxy-2-decenoic acid-δ-lactone is 1:0.5 to 1:2.

[0010] In one specific embodiment, the mass ratio of the cis-3,6,9-docosatriene to the 5-hydroxy-2-decenoic acid-δ-lactone is 1:1.

[0011] In one embodiment, the composition further includes a pesticide-acceptable carrier.

[0012] In one specific embodiment, the pesticide-acceptable carrier includes a solvent.

[0013] The fourth invention provides a lure core comprising a composition and a sustained-release carrier as described in any one of the third inventions.

[0014] In one specific embodiment, the sustained-release carrier is made of rubber.

[0015] 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.

[0016] The fifth invention provides the use of the composition described in any one of the fourth inventions for attracting cotton bollworms.

[0017] In one specific embodiment, the cotton bollworm is a male adult.

[0018] The beneficial effects of this invention are:

[0019] This invention discovers that the type II sex pheromone 3Z,6Z,9Z-21:H in male cotton bollworms plays an important role in their mating behavior. Applying this compound to male cotton bollworms lacking the type II sex pheromone 3Z,6Z,9Z-21:H increases the receptivity of females to that male, indicating its attractiveness to female cotton bollworms. Therefore, this compound can be used to make lures to increase the receptivity of female cotton bollworms to males of different ages, allowing for early and concentrated trapping and reducing the cotton bollworm population in the area, thereby achieving pest control. Furthermore, this invention also discovers that 5-hydroxy-2-decenoic acid-δ-lactone can increase the attractiveness of cis-3,6,9-docosatriene to females. Attached Figure Description

[0020] Figure 1 The results of GC-MS analysis of the abdominal epidermis and gonads of male cotton bollworms are shown.

[0021] Figure 2 The standard curves of cis-3,6,9-docosatriene (3Z,6Z,9Z-21:H) standard concentration versus peak area are shown.

[0022] Figure 3 The content of cis-3,6,9-docosatriene in the abdominal epidermis of male insects of different ages is shown.

[0023] Figure 4 The diagram shows a behavioral test and video experiment.

[0024] Figure 5The selectivity of male cotton bollworms for the test compounds in Examples 2 through 4 is shown. The significance of each example was analyzed using a t-test, where ns indicates no significant difference from the comparison (P > 0.05); * indicates a significant difference from the comparison (P < 0.05); ** indicates a significant difference from the comparison (P < 0.01); and *** indicates a highly significant difference from the comparison (P < 0.001).

[0025] Figure 6 The selectivity of male cotton bollworms for the test compounds tested in Examples 5 through 7 is shown. The significance of each example was analyzed using a t-test, where ns indicates no significant difference from the comparison (P > 0.05); * indicates a significant difference from the comparison (P < 0.05); ** indicates a significant difference from the comparison (P < 0.01); and *** indicates a highly significant difference from the comparison (P < 0.001). Detailed Implementation

[0026] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0027] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0028] cis-3,6,9-cuicocarbatriene (3Z,6Z,9Z-21:H) was purchased from Pherobank BV (Wijk bijDuurstede, Netherlands).

[0029] 5-Hydroxy-2-decenoic acid-δ-lactone was purchased from Simemo Top Biotechnology Co., Ltd.

[0030] n-Hexane was purchased from Sigma (USA).

[0031] The cotton bollworms used in the experiment were all from wild-type populations reared indoors at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The specific rearing process is as follows:

[0032] After the cotton bollworm eggs hatch, each larva is reared individually in a 24-well plate containing artificial feed. When the larvae reach approximately the fifth instar, they are transferred from the plate to finger-shaped tubes for pupation. Each finger-shaped tube contains sufficient artificial feed for the larvae until pupation. After emergence, the adults are transferred to clean finger-shaped tubes and fed with cotton balls containing 10% sucrose solution. A certain number of male and female adults are retained for reproduction. The rearing conditions are: temperature 26℃±1℃, humidity 65%±5%, and photoperiod 14L:10D. Example 1

[0033] Analysis of the content of cis-3,6,9-docosatriene in male adult cotton bollworms

[0034] Using hexane as a solvent, 0.1 g (density 0.812 g / cm³) of cis-3,6,9-tetradocotriene was weighed and diluted to a total volume of 1000 μL. The mixture was thoroughly mixed to obtain a cis-3,6,9-tetradocotriene solution with a concentration of 100 μg / μL.

[0035] A 100 μg / μL solution of cis-3,6,9-docosatriene was serially diluted to prepare 10 μg / μL, 100 ng / μL, 50 ng / μL, 30 ng / μL, 20 ng / μL, and 10 ng / μL solutions of cis-3,6,9-docosatriene, which were used as standard solutions for quantitative analysis.

[0036] The compounds were collected by immersion, using n-hexane as the solvent.

[0037] Compound extraction was performed on male moths aged 1 to 5 days after emergence. Samples were collected 4 hours after the cotton bollworms entered the dark phase. The specific procedure was as follows: The abdomen of the male moth was gently squeezed, and the gonads were pulled out from the end of the abdomen using forceps. The gonads were removed from the base using dissecting scissors and placed in a brown vial. The vials were then immersed in 300 μL of n-hexane in groups of 8 moths at room temperature for approximately 30 minutes to obtain the extract. The extract was transferred to a new, clean brown vial and stored at -20°C in a sealed container. After removing the gonads, the thorax and abdomen of the female and male moths were separated at the junction. The abdominal tissue was dissected, and intra-abdominal organs and fat contents were removed (Wang et al., 2013), yielding the epidermis. Five male and five female abdominal epidermis were immersed in 1 mL of n-hexane at room temperature for approximately 30 minutes to obtain the extract. The extract was transferred to a new, clean brown vial and stored at -20°C in a sealed container. Five biological replicates were performed for both the gonads and the epidermis. Before sample injection and analysis, the extract was concentrated to approximately 20 μL using a nitrogen blower (Tianjin Hengao, HGC-16-103) and then analyzed by GC-MS (Shimadzu, QP2020 SE).

[0038] Detection was performed using a gas chromatography-mass spectrometry (GC-MS) system with an Rtx-5MS capillary column. The injection port temperature was set to 250℃, and the initial column oven temperature was set to 40℃. Splitless injection was used, with helium as the carrier gas at a flow rate of 3 mL / min. The column oven temperature program was as follows: 40℃ for 1 min, ramped up to 200℃ at 15℃ / min, ramped up to 215℃ at 3℃ / min, and finally ramped up to 260℃ at 5℃ / min, held for 5 min. The interface temperature was 260℃, the detector ion source temperature was 250℃, and the solvent delay time was 3 min. The mass scan range was 35–500 m / z. 1 μL was manually injected each time. The above program was used for both test samples and standard samples. Results for the abdominal epidermis and gonads of male insects are shown below. Figure 1 .

[0039] By comparing the 3Z, 6Z, 9Z-21:H standard, it was found that one of the compounds identified in the male gonads and abdominal epidermis had the same retention time and mass spectrum as the 3Z, 6Z, 9Z-21:H standard, indicating that the male gonads and abdominal epidermis of the cotton bollworm contain the 3Z, 6Z, 9Z-21:H compound, and it is mainly present in the abdominal epidermis.

[0040] Next, we will perform quantitative analysis of the 3Z, 6Z, and 9Z-21:H concentrations on the abdominal epidermis of male bollworms of different ages. First, we will use the external standard method to plot a standard curve between the concentration of the standard and the peak area, see [link to external standard curve]. Figure 2 The contents of 3Z, 6Z, and 9Z-21:H in the abdominal epidermis of male insects of different ages were calculated based on the obtained standard curve. The results are shown in [Figure number missing]. Figure 3 .

[0041] according to Figure 3 It can be seen that the content of 3Z,6Z,9Z-21:H in the abdominal epidermis of one-day-old and two-day-old male insects is very low and can be almost ignored. The content increases sharply to the highest level in three-day-old insects, and then decreases sharply. Example 2

[0042] Using hexane as a solvent, 0.1 g of cis-3,6,9-docosatriene concentrate (density 0.812 g / mL) was weighed and diluted to a total volume of 1000 μL. The mixture was thoroughly mixed to obtain a 100 μg / μL cis-3,6,9-docosatriene solution. The 100 μL / μL cis-3,6,9-docosatriene solution was then diluted with hexane to obtain a 10 μg / μL cis-3,6,9-docosatriene solution.

[0043] The experiment took place from 8:00 PM to 6:00 AM the following day, with an ambient temperature of 26-28℃ and a relative humidity of 65±5%. Cylindrical plastic containers with a diameter of 9 cm and a height of 10 cm were used for the behavioral video experiment. Each container contained a cotton ball soaked in 10% sugar water to ensure sufficient food. One sexually mature female bollworm (3 days old after emergence) and two male bollworms (1 day old after emergence, containing almost no cis-3,6,9-docosatriene) were placed in each container. The opening of the container was then covered with gauze. After all the test insects were placed in the containers, the video was recorded for 10 hours using night mode. Each container was recorded in 8 sets (i.e., 8 containers). Figure 4 (As shown), repeat 5 sets.

[0044] Before placing the male bollworms into the plastic container, they were first anesthetized with CO2, and the following treatment was performed:

[0045] Add 2 μL of 10 μg / μL cis-3,6,9-docosatriene solution to the abdomen of one male cotton bollworm (referred to as male 1); add 2 μL of n-hexane to the abdomen of another male cotton bollworm (referred to as male 2).

[0046] After the test insects have been anesthetized, place them in the aforementioned plastic container.

[0047] Based on video recordings, the selection rate of female insects choosing male insects was statistically analyzed. Specifically, the selection rate of male insect 1 = number of groups choosing male insect 1 / (number of groups choosing male insect 1 + number of groups choosing male insect 2); the selection rate of male insect 2 = number of groups choosing male insect 2 / (number of groups choosing male insect 1 + number of groups choosing male insect 2). The selection rate results are shown in […]. Figure 5 The significance analysis was performed using the t-test. Example 3

[0048] Using hexane as a solvent, 0.1 g of the original concentration of 5-hydroxy-2-decenoic acid-δ-lactone (density 0.980 g / mL) was taken and diluted to a total volume of 1000 μL. The mixture was thoroughly mixed to obtain a 100 μg / μL 5-hydroxy-2-decenoic acid-δ-lactone solution. The 100 μL / μL solution was then diluted with hexane to prepare a 10 μg / μL 5-hydroxy-2-decenoic acid-δ-lactone solution.

[0049] The difference from Example 2 is that:

[0050] Before placing the male bollworms into the plastic container, anesthetize them with CO2 by performing the following treatment:

[0051] Add 2 μL of 10 μg / μL 5-hydroxy-2-decenoic acid-δ-lactone solution to the abdomen of one male bollworm (referred to as male 1); add 2 μL of n-hexane to the abdomen of another male bollworm (referred to as male 2).

[0052] After the test insects have been anesthetized, place them in the aforementioned plastic container.

[0053] Based on video statistics, the selection rate of female insects choosing male insects is shown in the following results. Figure 5 . Example 4

[0054] The difference from Example 2 is that:

[0055] Mix 100 μL of 10 μg / μL cis-3,6,9-docosatriene solution and 100 μL of 10 μg / μL 5-hydroxy-2-decenoic acid-δ-lactone solution in a 1:1 ratio to obtain mixture 1#.

[0056] Add 2 μL of mixture 1# (referred to as male 1) to the abdomen of one male cotton bollworm; add 2 μL of n-hexane (referred to as male 2) to the abdomen of another male cotton bollworm.

[0057] After the test insects have been anesthetized, place them in the aforementioned plastic container.

[0058] Based on video recordings, the selection rate of female insects choosing male insects is statistically analyzed. The selection rate results are shown below. Figure 5 . Example 5

[0059] The difference from Example 4 is that:

[0060] Add 2 μL of mixture 1# (referred to as male 1) to the abdomen of one male cotton bollworm; add 2 μL of 10 μg / μL cis-3,6,9-docosatriene solution (referred to as male 2) to the abdomen of another male cotton bollworm.

[0061] After the test insects have been anesthetized, place them in the aforementioned plastic container.

[0062] Based on video statistics, the selection rate of female insects choosing male insects is shown in the following results. Figure 6 . Example 6

[0063] The difference from Example 2 is that:

[0064] Mix 100 μL of 10 μg / μL cis-3,6,9-docosatriene solution and 50 μL of 10 μg / μL 5-hydroxy-2-decenoic acid-δ-lactone solution at a ratio of 1:0.5 to obtain mixture 2#.

[0065] Add 2 μL of mixture 2# (referred to as male 1) to the abdomen of one male cotton bollworm; add 2 μL of 10 μg / μL cis-3,6,9-docosatriene solution (referred to as male 2) to the abdomen of another male cotton bollworm.

[0066] After the test insects have been anesthetized, place them in the aforementioned plastic container.

[0067] Based on video statistics, the selection rate of female insects choosing male insects is shown in the following results. Figure 6 . Example 7

[0068] The difference from Example 2 is that:

[0069] Mix 50 μL of 10 μg / μL cis-3,6,9-docosatriene solution and 100 μL of 10 μg / μL 5-hydroxy-2-decenoic acid-δ-lactone solution in a 1:2 ratio to obtain mixture 3#.

[0070] Add 2 μL of mixture 3# (referred to as male 1) to the abdomen of one male cotton bollworm; add 2 μL of 10 μg / μL cis-3,6,9-docosatriene solution (referred to as male 2) to the abdomen of another male cotton bollworm.

[0071] After the test insects have been anesthetized, place them in the aforementioned plastic container.

[0072] Based on video statistics, the selection rate of female insects choosing male insects is shown in the following results. Figure 6 .

[0073] The results of Example 2 above demonstrate that cis-3,6,9-tetradecanetriene plays an important role in the mating process of cotton bollworms, exhibiting a significant attraction to females. Based on the classification of sex pheromones, cis-3,6,9-tetradecanetriene is classified as a type II sex pheromone for cotton bollworms. The results of Example 3 demonstrate that 5-hydroxy-2-decenoic acid-δ-lactone does not attract females. The results of Example 4 demonstrate that 5-hydroxy-2-decenoic acid-δ-lactone can increase the attraction of cis-3,6,9-tetradecanetriene to females. The results of Examples 5 to 7 further demonstrate that 5-hydroxy-2-decenoic acid-δ-lactone can increase the attraction of cis-3,6,9-tetradecanetriene to females, and that a 1:1 mass ratio of 5-hydroxy-2-decenoic acid-δ-lactone to cis-3,6,9-tetradecanetriene provides the optimal attraction to females.

Claims

1. Application of cis-3,6,9-docosatriene in attracting female adult cotton bollworms.

2. Application of 5-hydroxy-2-decenoic acid-δ-lactone in increasing the attraction of cis-3,6,9-docosatriene to adult female cotton bollworms.

3. A composition comprising an active ingredient and a synergist, wherein the active ingredient is cis-3,6,9-docosatriene, and the synergist is 5-hydroxy-2-decenoic acid-δ-lactone, wherein the mass ratio of the cis-3,6,9-docosatriene to the 5-hydroxy-2-decenoic acid-δ-lactone is from 1:0.5 to 1:

2.

4. The composition according to claim 3, characterized in that, The mass ratio of the cis-3,6,9-docosatriene to the 5-hydroxy-2-decenoic acid-δ-lactone is 1:

1.

5. The composition according to any one of claims 3 and 4, characterized in that, The composition also includes a pesticide-acceptable carrier.

6. A decoy comprising the composition as described in any one of claims 3 to 5 and a sustained-release carrier.

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

8. The lure core according to claim 6, characterized in that, The sustained-release carrier is at least one of rubber-tip carrier, cotton-core carrier, and microtubule carrier.

9. The use of the composition according to any one of claims 3 to 5 or the lure according to any one of claims 6 to 8 in attracting female adult cotton bollworms.

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