Male ambrosia beetle adult attractant and use thereof
Patent Information
- Application Number
- CN202610903488.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-23
- Publication Date
- 2026-09-25
AI Technical Summary
由于该虫具有危害隐蔽性强、发育周期长且发育时间不整齐等特点，实际防效很难达到预期
[0004]本发明的目的是提供一种能稳定释放、防治效果好的雄性双条杉天牛成虫引诱剂。
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Abstract
Description
Technical Field
[0001] This invention relates to an insect attractant, specifically to an attractant for adult male two-striped longhorn beetle and its application. Background Technology
[0002] The two-striped cedar longhorn beetle (Semanotus bifasciatus) belongs to the order Coleoptera, family Cerambycidae, and subfamily Cerambycinae. It is an important stem-boring pest that mainly damages cypress trees in East Asia. In 1996, the two-striped cedar longhorn beetle was listed as a quarantine pest in my country's forest plants, and in 2013 it was listed as a dangerous pest in the national forestry system (Jiang Qi, Ren Lili, Luo Youqing, 2021. Classification of larval stages of the two-striped cedar longhorn beetle (Coleoptera: Cerambycidae) [J]. Journal of Environmental Entomology, 43(1): 25-30. Li Juan, Cui Yongsan, Song Yushuang et al., 2013. Characteristics of the new list of quarantine and dangerous pests in my country's forestry system [J]. Chinese Journal of Forest Pests and Diseases, 32(5): 42-47.). The two-striped longhorn beetle is widely distributed in many provinces and cities in my country, causing serious damage to cypress trees in Beijing, Hebei, Henan, Shanxi, Shaanxi, Shandong, Liaoning, and Baotou, Inner Mongolia (Zhang Ting, 2017. Study on insect diversity and occurrence patterns of major pests in the ancient cypress grove of the Yellow Emperor's Mausoleum [D]. Shaanxi: Northwest A&F University).
[0003] Currently, the control of the two-striped longhorn beetle mainly relies on measures such as clearing infested trees, baiting with bait, and chemical control. However, due to the beetle's highly concealed nature, long and uneven development cycle, the actual control effect is difficult to achieve the desired results. Based on the strategic need for green control of forest pests, pheromone regulation technology, which combines target specificity, environmental compatibility, and delayed resistance development, has become a key focus for the large-scale integrated management of the two-striped longhorn beetle. Summary of the Invention
[0004] The purpose of this invention is to provide an attractant for adult male two-striped longhorn beetles that can be stably released and has a good control effect.
[0005] An attractant for adult male two-striped longhorn beetle, including or excluding a solvent, wherein the active ingredient is 2,2-dimethyl-3-hexanone.
[0006] The solvent is n-hexane, and the concentration of 2,2-dimethyl-3-hexanone is labeled as C0; the concentration of 2,2-dimethyl-3-hexanone in the attractant is C0-10. -2 C0.
[0007] The male adult beetle attractant was prepared using the following method: the concentration of the stock solution of 2,2-dimethyl-3-hexanone was labeled as C0, and then the stock solution of 2,2-dimethyl-3-hexanone was diluted with n-hexane to the required concentration.
[0008] Application of 2,2-dimethyl-3-hexanone as an attractant in attracting male adult male *Cephalotaxus fortunei*.
[0009] The stock solution of 2,2-dimethyl-3-hexanone or a hexane solution thereof is used as an attractant for adult male two-striped longhorn beetles.
[0010] The stock solution concentration of 2,2-dimethyl-3-hexanone is labeled C0; the concentration of 2,2-dimethyl-3-hexanone used as the attractant is C0-10. -2 C0.
[0011] Based on olfactory screening, this invention identifies a compound, 2,2-dimethyl-3-hexanone, that attracts adult male *Diospyros spp.* longhorn beetles. This compound effectively attracts adult male *Diospyros spp.* longhorn beetles, is an industrial raw material, is safe for humans and animals, does not pollute the environment, and has broad application prospects. Attached Figure Description
[0012] Figure 1 This represents the selective response of female and male adults to male adults.
[0013] Figure 2 This represents the selective response of male and female adults to female adults.
[0014] Figure 3 This represents the selective response of male and female adults to each other.
[0015] Figure 4 The image shows the GC-EAD diagram of the volatile extract of *Cephalotaxus fortunei*.
[0016] Figure 5 TIC chromatogram of gaseous volatile extract from *Cephalotaxus fortunei*.
[0017] Figure 6 The TIC chromatogram for a mixture of C7-C40 n-alkane standards.
[0018] Figure 7 To induce activity in the active component of the gaseous volatiles,
[0019] In the figure: (A) the attraction ratio of 2,2-dimethyl-3-hexanone to male *A. 2,2-dimethyl-3-hexanone*; (B) the attraction ratio of dodecane to male *A. 2,2-dimethyl-3-hexanone*; (C) the attraction ratio of tridecane to male *A. 2,2-dimethyl-3-hexanone*; (D) the attraction effect of 2,2-dimethyl-3-hexanone to male *A. 2,2-dimethyl-3-hexanone*; (E) the attraction effect of 2,2-dimethyl-3-hexanone to female *A. 2,2-dimethyl-3-hexanone*. Detailed Implementation
[0020] The present invention will be further described in detail below with reference to the embodiments.
[0021] All reagents used below are commercially available.
[0022] The two-striped Chinese longhorn beetle was collected from the "Millennium Forest" in Xiong'an New Area, Hebei Province (116°03′E, 39°00′N). In November 2024, weakened trees exhibiting signs of damage were selected from the forest and felled. To prevent excessive moisture loss, both ends of the felled sections were immediately sealed with paraffin wax. The sections were then moved to a laboratory, placed at room temperature, and covered with a transparent nylon mesh. The following spring, after the adults emerged, they were promptly collected, and the sexes were separated and placed in insect rearing boxes for later use. The newly emerged adults reached sexual maturity and were used for subsequent experiments.
[0023] The concentration of the reagent used is:
[0024] 1) 2,2-Dimethyl-3-hexanone stock solution, concentration labeled as C0.
[0025] 2) 10 -1 A C0 solution of 2,2-dimethyl-3-hexanone requires n-hexane as the solvent for dilution.
[0026] 3) 10 -2 A C0 solution of 2,2-dimethyl-3-hexanone requires n-hexane as the solvent for dilution.
[0027] 4) 10 -3 A C0 solution of 2,2-dimethyl-3-hexanone requires n-hexane as the solvent for dilution.
[0028] 5) 10 -4 A C0 solution of 2,2-dimethyl-3-hexanone requires n-hexane as the solvent for dilution.
[0029] 6) 10 -5 A C0 solution of 2,2-dimethyl-3-hexanone requires n-hexane as the solvent for dilution.
[0030] The above solutions were prepared by sequentially diluting the 2,2-dimethyl-3-hexanone stock solution with n-hexane to the concentrations described above, thus preparing the male adult attractant for the longhorn beetle *Diplodocus spp.* of this invention. The 2,2-dimethyl-3-hexanone stock solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd.
[0031] Dodecane and tridecane were prepared using the same method to obtain solutions of the above concentrations.
[0032] Example 1: Location and Identification of Pairs of Two-striped Cedar Longhorn Beetles
[0033] 1.1 Behavioral response test of female and male adult *Cephalotaxus fortunei*
[0034] 1.1.1 Behavioral responses of male and female adults to female adults
[0035] The behavioral responses of male and female adults of the two-striped longhorn beetle to female adults were observed using a Y-shaped olfactory spectrometer. The main trunk of the device was 10 cm long, and the two side arms were 20 cm long (with an included angle of 45° and an inner diameter of 3.5 cm). A spherical trap, a drying tower, and an atmospheric sampler were connected to the side walls, respectively. All parts were connected by polytetrafluoroethylene (PTFE) tubing, and the airflow velocity was kept constant at 500 mL / min. -1 .
[0036] In the experiment, one female adult *Cephalotaxus fortunei* was placed in a spherical trap at one end of a Y-shaped olfactory instrument as the treatment arm, and the other end as the control arm. After 3 minutes of ventilation, one healthy, active, newly emerged adult (female or male) was selected each time and placed in the inlet of the Y-shaped olfactory instrument for behavioral measurement. Each insect was tested only once, and its behavioral response was observed over 10 minutes. Insects that entered more than two-thirds of the selection arm within 10 minutes and remained for more than 10 seconds were considered selected and their selection was recorded; otherwise, they were considered unselected (Ma Yan. Preliminary Study on Plant-Derived Attractants for *Cephalotaxus fortunei* [D]. Hangzhou: Zhejiang A&F University, 2019.). To avoid the influence of ambient light and the position of the tube arm on the selective behavior of *Cephalotaxus fortunei*, the positions of the treatment and control arms were switched after each test. After each set of experiments, the Y-shaped olfactory instrument and the spherical trap were thoroughly cleaned with alcohol, dried, and then the next set of experiments was conducted. Each group consisted of 10 female adults and 10 male adults, with a total of 5 groups.
[0037] 1.1.2 Behavioral responses of female and male adults to male adults
[0038] Using the same experimental setup and method as in 1.1.1, one male adult of the two-striped longhorn beetle was placed in a spherical trap at one end of a Y-shaped olfactory instrument as the treatment arm, and the other end as the blank control arm. The behavioral responses of female and male adults to male adults were measured separately. Ten female adults and ten male adults were tested in each group, for a total of five groups.
[0039] 1.1.3 Behavioral responses of male and female adults to female and male adults
[0040] To further compare the behavioral responses of female and male adults of the two-striped longhorn beetle to individuals of different sexes, the same experimental setup and method as in 1.1.1 were used. Female and male adults were placed in spherical traps on either side of a Y-shaped olfactory apparatus, and the selection of the two arms by the female and male adults was recorded. Ten female adults and ten male adults were tested in each group, for a total of 5 groups.
[0041] 1.1.4 Statistical Analysis
[0042] The test results were processed using the chi-square test, and statistical analysis was performed using IBM SPSS Statistics 26, with graphs plotted using Graphpad Pism 9.5.
[0043] The attraction rate and response rate are calculated using the following formulas:
[0044] Attraction rate (%) = Total number of insects in the odor arm / Total number of insects with selective response × 100%.
[0045] Response rate (%) = (Total number of insects in the odor arm + Total number of insects in the control arm) / Total number of insects tested × 100%.
[0046] 1.2 Extraction and Identification of Sex Pheromone from the Two-striped Longhorn Beetle
[0047] 1.2.1 Extraction of sex pheromones from the two-striped longhorn beetle
[0048] Volatile pheromones from female adult *Cephalotaxus fortunei* were collected using dynamic headspace adsorption (HLA). To avoid the generation of other volatile substances through biting among the adults, only one female adult was selected at a time and placed in a custom-made conical flask for sampling. During sampling, the airflow rate at the outlet of the atmospheric sampler was 500 mL / min. -1 An airflow, filtered through activated carbon, carried the volatile substances from the conical flask containing the *Cephalotaxus fortunei* to an adsorption tube containing the adsorbent Porapak Q. The other end of the adsorption tube was connected to the inlet of an atmospheric sampler. Based on prior behavioral observations, sample collection was scheduled for 8:00-12:00, a period of peak mating activity for the *Cephalotaxus fortunei*, for a total of 4 hours. After sampling, the adsorption tube was eluted three times with 350-400 μL of chromatographically pure hexane, and the eluent was collected in a sample vial. The volatile eluents obtained from three female adults were combined and concentrated to 200 μL using a nitrogen evaporator, stored at -20°C, and used for GC-EAD determination and GC-MS analysis.
[0049] 1.2.2 Identification of sex pheromones from the two-striped longhorn beetle
[0050] 1.2.2.1 GC-EAD Analysis
[0051] First, under a dissecting microscope, two male adult longhorn beetles were cut from their base using dissecting scissors, with a small portion of the terminal segment removed. Then, two glass capillary electrodes were aspirated with 0.9% sodium chloride saline, and the electrode wire tips were inserted into the pre-prepared glass capillaries. The treated antennae were placed horizontally between the two glass electrodes, ensuring good contact between the two ends of the antennae with the reference electrode and the recording electrode, respectively, thus completing the electrode connection. The entire procedure was performed under a stereomicroscope. After the baseline stabilized and the gas chromatography system was functioning normally, 2 μL of sample was injected, and data were recorded and analyzed using GcEad2014 v1.2.5 software. GC operating conditions: HP-5MS capillary column (30 m × 0.32 mm × 0.25 μm, Agilent Technologies), injector 250℃, splitless injection, detector 300℃. Column temperature program: 60℃ for 2 min, then increased at 10℃·min. -1 Heat to 200℃ at a rate of 20℃·min and hold for 2 min; -1 The temperature was increased to 280℃ at a certain rate and held for 8 minutes, for a total of 30 minutes.
[0052] 1.2.2.2 GC-MS Analysis Method
[0053] Volatile substances and a mixed standard of C7-C40 n-alkanes from female adult *Cephalotaxus fortunei* were analyzed using an Agilent 7890B-5977B gas chromatography-mass spectrometry (GC-MS) system. The injection volume was 2 μL, and the injection was splitless. GC operating conditions were the same as described in 1.2.2.1. MS operating conditions were: EI source, ionization energy of 70 eV, ion generator temperature of 250℃, and a mass-to-nuclear ratio range of 29–500 amu. Qualitative Navigator B.08.00 software was used to perform qualitative analysis and preliminary identification of compounds by searching the NIST 2020 spectral library, matching with the NIST mass spectrometry database, calculating retention indices from the C7-C40 n-alkanes mixed standard, and comparing with standard mass spectra.
[0054] Experimental results
[0055] 2.1 Location and identification of mates of the two-striped longhorn beetle
[0056] 2.1.1 Mating of the Two-striped Longhorn Beetle
[0057] 2.1.1.1 Olfactory behavioral responses of female and male adults to male adults
[0058] Using male adults as the treatment arm and the blank arm as the control, the selection results of male and female adults on both arms were recorded. The results showed that the selection rate of male adults on one side was 53.7%, compared to 46.3% in the control arm, with a response rate of 82%; the selection rate of female adults on one side was 43.2%, compared to 56.8% in the control arm, with a response rate of 74%. Further chi-square test revealed that male adults did not show a significant attraction effect on either male or female adults (female adults: χ²). 2 =0.676, P>0.05, n=50; Male adults: χ 2 =0.220, P>0.05, n=50) ( Figure 1 ).
[0059] 2.1.1.2 Olfactory behavioral responses of male and female adults to female adults
[0060] Female adults were used as the treatment arm, and the blank arm as the control. The selection results of female and male adults on both arms were recorded. The results showed that the selection rate of female adults on the female side was 40.5%, compared to 59.5% on the control side, with a response rate of 84%. There was no significant skewness of female adults towards female adults (χ²). 2 =1.524, P>0.05). The selection rate of male adults towards female adults was 77.3%, compared to 22.7% in the control group, with a response rate of 88%. Further chi-square test revealed a highly significant tendency of male adults towards female adults (χ²). 2 =13.091, P<0.01, n=50). The above results indicate that female adults have a significant attraction effect on male adults, but no significant attraction effect on female adults. Figure 2 ).
[0061] 2.1.1.3 Olfactory behavioral responses of male and female adults to female and male adults
[0062] To further verify the attraction effect of female adults on male adults, female and male adults were placed in the two arms of a Y-shaped tube, respectively, and their selection of the two arms was recorded. The results showed that the selection rate of female adults on the female side was 52.4%, while the selection rate on the male side was 47.6%, with a response rate of 42% (χ²). 2 =0.047, P>0.05, n=50). The selection rate of male adults to the female adult side was 64.6%, and the selection rate to the male adult side was 35.4%, with a response rate of 96%. Further chi-square test revealed a significant difference in the selection of male adults between the two arms (χ²). 2 =4.083, P<0.05, n=50), further indicating that female adults have a significant attraction effect on male adults ( Figure 3 ).
[0063] 2.2 Analysis of the active components of sex pheromones from the longhorn beetle *Cephalotaxus fortunei*
[0064] 2.2.1 Analysis of volatile pheromone active components in *Cephalotaxus fortunei*
[0065] Based on the aforementioned attraction effect of female adults on male adults, the gaseous volatiles of female adults were extracted, and the antennal electrophysiological responses of male adults were investigated. Four active components in the gaseous volatiles of female adult *Cephalotaxus fortunei* could induce electrophysiological activity in the antennae of male adults. Figure 4 Under the same conditions, GC-MS analysis was performed on the gaseous volatiles and C7-C40 n-alkanes of female adult insects to obtain their total ion chromatograms. Figure 5 TIC plot of C7-C40 n-alkanes mixed standard (see) Figure 6 The four active components were preliminarily identified as 2,2-dimethyl-3-hexanone, dodecane, 2-methyldodecane, and tridecane (Table 1).
[0066] Table 1. Preliminary analysis results of volatile active components in *Cephalotaxus fortunei*.
[0067] Comp1 2,2-Dimethyl-3-hexanone <![CDATA[C8H 16 O]]> 1077 5405-79-8 Comp2 dodecane <![CDATA[C 12 H 26 ]]> 1200 112-40-3 Comp.3 2-Methyldodecane <![CDATA[C 13 H 28 ]]> 1255 1560-97-0 Comp.4 Tridecane <![CDATA[C 13 H 28 ]]> 1300 629-50-5
[0068] Example 2: Analysis of the inducing activity of gaseous volatile active component standards
[0069] Behavioral experiment of the two-striped longhorn beetle as a standard.
[0070] A Y-type olfactometer was used to detect the attractant activity of the above-mentioned volatile active gaseous components (2,2-dimethyl-3-hexanone, dodecane, and tridecane) of female *Cephalotaxus fortunei* on unmated adult *Cephalotaxus fortunei*. The stock solutions of 2,2-dimethyl-3-hexanone, dodecane, and tridecane were sequentially diluted with n-hexane using an equal-volume, ten-fold serial dilution method. Using the same experimental apparatus and method as in 1.1.1, 10 μL of each sample was added to a filter paper strip and placed at one end of a Y-type tube, with n-hexane placed at the control end. Ten adults were tested for each dose, for a total of 5 groups. The test results were processed using the chi-square test, and statistical analysis was performed using IBM SPSS Statistics 26, with graphs plotted using Graphpad Pism 9.5.
[0071] Experimental results
[0072] The results showed that male *Cephalotaxus fortunei* exhibited a significant preference for 2,2-dimethyl-3-hexanone, with an overall selection rate significantly higher than that of the n-hexane control. Figure 7 (A). Male insects para-dodecane ( Figure 7 (B) and tridecane ( Figure 7(C) The overall tendency was weak, and the proportion of unselected individuals was relatively high, indicating that dodecane and tridecane had no attraction effect on male insects.
[0073] The results also showed that 2,2-dimethyl-3-hexanone exhibited significant sex differences in olfactory attraction activity towards female and male adults, and its attraction effect on males was clearly concentration-dependent. The olfactory selection results of males indicated ( Figure 7 (D), in the original solution, 10 −1 10 −2 At higher concentrations, the proportion of male insects selecting for 2,2-dimethyl-3-hexanone was significantly higher than that of the n-hexane control group (P < 0.05). When the concentration was reduced to 10... −3 At and below this age, male insects showed no significant difference in their selection of 2,2-dimethyl-3-hexanone versus n-hexane (P > 0.05). The olfactory selection results of female insects indicated that ( Figure 7 (E), in the original solution to 10 −4 Under all tested concentration gradients, there was no significant difference in the female insects' selection for 2,2-dimethyl-3-hexanone and n-hexane (P>0.05), indicating that 2,2-dimethyl-3-hexanone had no significant attraction effect on female *Cephalotaxus fortunei*.
[0074] In summary, 2,2-dimethyl-3-hexanone has a specific olfactory attraction effect only on male insects, and it only works at relatively high concentrations, without any significant behavioral regulatory effect on female insects.
Claims
1. An attractant for adult male two-striped longhorn beetle, including or excluding a solvent, wherein the active ingredient is 2,2-dimethyl-3-hexanone.
2. The attractant according to claim 1, wherein the solvent is n-hexane, and the concentration of 2,2-dimethyl-3-hexanone is labeled as CO; the concentration of 2,2-dimethyl-3-hexanone in the attractant is CO-10. -2 C0.
3. The attractant according to claim 1 is prepared by the following method: the concentration of the 2,2-dimethyl-3-hexanone stock solution is labeled as CO, and then the 2,2-dimethyl-3-hexanone stock solution is diluted with n-hexane to the desired concentration. 4.2 Application of 2-dimethyl-3-hexanone as an attractant in attracting male adult two-striped longhorn beetles.
5. According to claim 4, the stock solution of 2,2-dimethyl-3-hexanone or a hexane solution prepared therefrom is used as an attractant for adult male two-striped longhorn beetles.
6. In the application according to claim 5, the stock solution concentration of 2,2-dimethyl-3-hexanone is labeled as C0; the concentration of 2,2-dimethyl-3-hexanone used as the attractant is C0-10. -2 C0.