Application of methyl palmitate as attractant for lema fraxini in Qinghai grassland

Abstract

The application discloses application of methyl palmitate as an attractant for Eupithecia subnotata. The attractant comprises methyl palmitate solution configured by using n-hexane at a concentration of 0.1 ug / ml, and the attractant has the best attractant effect. The attractant is combined with a purple trapping device to attract Eupithecia subnotata, and the trapping of Eupithecia subnotata is the most efficient when the color and the reagent both have the best single-factor effect.

Description

Technical Field

[0001] This invention belongs to the field of insect attractant technology, specifically relating to the application of methyl palmitate as an attractant for caterpillars in the Qinghai grasslands. Background Technology

[0002] Qinghai grassland caterpillars ( Gynaephora qinghaiensis The red-headed black caterpillar (Gynaephora) belongs to the genus Gynaephora in the family Lymantriidae of the order Lepidoptera. Currently, 15 species of grassland caterpillars have been discovered and named worldwide. This insect undergoes holometabolous transformation, with its life cycle consisting of four developmental stages: egg, larva, pupa, and adult. The larval stage is crucial for nutrient intake and the outbreak of damage, and can be divided into seven instars based on the width of the larval head capsule. Adults exhibit significant sexual dimorphism; the thoracic legs, wings, and antennae of female adults are all in a degenerate state. Furthermore, its larvae possess venom glands on their backs; if ingested by livestock or wild animals, they are prone to developing oral ulcers and tongue-breaking disease, leading to the inability of affected animals to eat normally, ultimately resulting in emaciation and death. Currently, the control of grassland caterpillars in Qinghai Province still relies primarily on chemical control. Although control agents are constantly being iterated and optimized, the problem of pesticide residues inevitably has a negative impact on the ecological environment.

[0003] The gonads are a core component of the insect reproductive system. Through hormone secretion and germ cell development, they directly regulate insect reproductive capacity, thus affecting species continuity and playing a crucial role in insect reproduction. Gonadal metabolites are various chemical substances produced or secreted by insect gonads during metabolism, encompassing lipids, hormones, pheromones, purine metabolites, and other compounds. These substances not only provide energy support and building blocks for insect life activities but also participate in multidimensional regulation of the reproductive process as signaling molecules. For example, the sex pheromone glands of female lepidopteran moths can synthesize highly species-specific fatty acid derivatives (such as aldehydes, alcohols, and acetates), the proportions and release amounts of which directly determine mating success rates; male scorpionflies ( Panorpa When the genital sac everts, it releases a specific pheromone that can attract females within a 25-foot range, thereby mediating intraspecific mating behavior.

[0004] Sex pheromones serve as a key medium for intraspecific chemical communication in insects, and related research and applications have attracted considerable attention. Lepidoptera insects possess a unique pheromone communication system that distinguishes them from other groups, making this field of research central to insect behavior and ecology. The richness and diversity of Lepidoptera species are closely related to their pheromone-dependent communication patterns and complex chemosensory systems; a typical biological characteristic is that adult mating behavior is pheromone-mediated, typically involving female adults releasing sex pheromones to attract males of the same species at a distance, while males use their precise chemosensory abilities to locate females and complete mating.

[0005] With the development of modern chemical analysis techniques, metabolomics technology has enabled the comprehensive qualitative and quantitative analysis of all small molecule metabolites in organisms under specific physiological conditions. For example, metabolomics analysis of azoosperm components in insect semen has revealed the presence of various small molecules, including lipids, proteins, carbohydrates, inorganic salts, hormones, nucleic acids, and vitamins. Gas chromatography-mass spectrometry (GC-MS) analysis of the gonadal extract of the mulberry moth has identified five compounds with structures similar to known sex pheromones (such as Z9, Z12, Z15-18:Aci-4:Est), providing potential targets for the green control of this pest. Currently, various insect sex pheromones have been successfully identified and synthesized artificially, demonstrating significant application value in agricultural pest management. Due to their strong species specificity, non-toxicity to mammals, and good environmental compatibility, sex pheromones have become an important component of integrated pest management systems. The regulatory effect of sex pheromones on insect behavior is mainly reflected in the intervention of mating behavior; by interfering with the mating process of pests, the population reproduction rate can be effectively reduced, thereby reducing their harm to agricultural and livestock production. Currently, insect sex pheromone products on the Chinese market are mainly attractants and mating determinants, accompanied by mass trapping and mating disruption techniques. Large-scale promotion of sex pheromone trapping technology can significantly reduce pest population density, decrease the use of chemical pesticides, and thus mitigate negative impacts on the ecological environment. For example, it can be effective against the brown tea geometrid moth (…). Ectropis grisescens The sex pheromone trapping technology has achieved significant control effects in field applications. This not only confirms the practical value of sex pheromones in pest population regulation, but also lays the foundation for in-depth research on the mechanism of action and influencing factors of sex pheromones.

[0006] Currently, there are few reports on the mechanism of action of sex pheromone compounds in the behavioral regulation of Qinghai grassland caterpillars. Clarifying the mechanism of action of sex pheromone compounds in the behavioral regulation of Qinghai grassland caterpillars, conducting a comprehensive analysis of their gonadal metabolites using metabolomics technology, and screening out key functional pheromone compounds to serve as highly efficient sex pheromone attractants are urgent technical problems that need to be solved. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the existing technology and provide the application of methyl palmitate as an attractant for caterpillar caterpillars in the Qinghai grasslands.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] Application of methyl palmitate as an attractant for caterpillar caterpillars in the Qinghai grasslands.

[0010] Furthermore, the attractant contains a methyl palmitate solution with a concentration of 0.1 μg / ml prepared using n-hexane.

[0011] Furthermore, the attractant is used in conjunction with a purple trapping device to attract caterpillars in the Qinghai grasslands.

[0012] Furthermore, the trapping device is made of cardboard.

[0013] The beneficial effects of this invention are as follows: This invention analyzes the gonadal metabolites of the Qinghai grassland caterpillar and screens out the key pheromone compound methyl palmitate. Methyl palmitate exhibits the best attraction effect at a concentration of 0.1 μg / ml. The trapping efficiency of the Qinghai grassland caterpillar is highest when both color and reagent show optimal single-factor effects. Attached Figure Description

[0014] Figure 1 This is a bar chart showing the EAG response of male adult caterpillars in the Qinghai grasslands to different concentrations of sex pheromones.

[0015] Figure 2 This is a bar chart showing the EAG response of male adult caterpillars in the Qinghai grasslands to sex pheromone compounds.

[0016] Figure 3 This is a bar chart showing the olfactory behavioral response of Qinghai grassland caterpillars to sex pheromone compounds.

[0017] Figure 4 This is a bar chart showing the olfactory behavioral response of male adult caterpillars in the Qinghai grasslands to sex pheromone compounds. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings. Example

[0019] 1. Materials and Methods

[0020] 1.1 Test insects:

[0021] The larvae of the Qinghai steppe caterpillar were collected from Haiyan County, Haibei Prefecture, Qinghai Province (36°59′N, 100°52′E, altitude 3095m). The larvae were brought back to the laboratory and reared with sufficient sedge plants. Rearing conditions: temperature (26±1)℃, relative humidity 60%~80%, photoperiod L / D=16h / 8h], until adult males and females emerged. To prevent decomposition and contamination, the gonads were dissected under a microscope on ice. After removing the head with a scalpel, the 3rd and 4th abdominal segments were gently squeezed to force the gonads to protrude from the last abdominal segment. The glands were then cut off with surgical scissors, quickly frozen in liquid nitrogen, and subsequently stored in an ultra-low temperature freezer at -80℃ for later use.

[0022] 1.2 Reagents and Instruments:

[0023] The antennal potentiometer, consisting of a DGST-2 stimulation airflow controller, a DGPB-1 micro-motion manipulator, and a DG02 signal acquisition controller, was manufactured by Tangshan Dinggan Technology Co., Ltd. The four-arm olfactory sensor, model DJ-7000, was manufactured by Dianjiang (Shanghai) Technology Co., Ltd. The sex pheromone compound reagent was purchased from Gansu Ruide Biotechnology Co., Ltd.

[0024] 1.3 Gonadal metabolome analysis of Qinghai grassland caterpillars:

[0025] Total RNA was extracted from the gonads of *Catella qinghaiensis* using an RNA extraction kit (Solepro, Beijing). RNA purity in each tissue was determined using a micro-spectrophotometer (Kaiao Technology, Beijing). The purified RNA (OD260 / OD280 of 1.8–2.2) was reverse transcribed into cDNA using a reverse transcription kit (M-MLV) (Solepro, Beijing) and stored at -20°C for later use. Subsequently, Nanjing Jisi Huiyuan Biotechnology Co., Ltd. performed metabolomics analysis on six biological replicates of *Catella qinghaiensis* using gas chromatography-mass spectrometry (GC-MS). The chi-square test standard used was the commonly accepted standard, i.e., a p-value of less than 0.05 in the Student's t-test. Simultaneously, the variable importance in the projection (VIP) of the first principal component in the OPLS-DA model was greater than 1.

[0026] 1.4 Antennae potential response of male adult caterpillars in the Qinghai grasslands:

[0027] The antennal sensitivity of *Przewalski's steppe* caterpillars to different concentrations of sex pheromones was determined using an antennal potentiometry instrument. Five concentration gradients of sex pheromones (0.001, 0.01, 0.1, 1, and 10 μg / mL) were prepared using n-hexane, with n-hexane serving as a control. Each group was tested in triplicate. During testing, any antenna of a male adult *Przewalski's steppe* caterpillar was completely severed from its base using a scalpel, with 0.1 mm of the distal end removed. The two ends of the antenna were then fixed to the reference and measurement grades respectively using conductive adhesive. The electrode was inserted into the insect antennal potentiometry probe on the operating table, and the antenna was adjusted to maintain a 1 cm distance from the odorant tube opening. The electrode signal was connected to the signal collection device and integrated into Sntech EAG SVstem 2.6 software. Baseline fluctuations were observed, and the experiment began once the baseline stabilized. Each time, 10 μL of the analyte was pipetted and evenly dropped onto a 3.0 cm long and 0.5 cm wide filter paper. The filter paper was then placed into a 1 mL pipette tip, which was connected to the stimulant gas control device. The continuous gas flow rate and the stimulant gas flow rate in the odor tube were 200 mL / min and 20 mL / min, respectively. Stimulation was applied by gently pressing the pedal, with each stimulation lasting 0.2 s. Each dose was treated once, with a 60 s interval between two consecutive stimulations to ensure the antennae had recovered from the previous stimulation. The same chemical reagent was tested in ascending order of dosage to avoid dose-effects. The EAG correction value was calculated based on the data output from the antennal potentiometry meter: EAG correction value = treated EAG observation value - control EAG observation value.

[0028] 1.5 Olfactory behavioral responses of larvae and adult male caterpillars in the Qinghai grasslands:

[0029] The olfactory behavior of larvae and adult males of the Qinghai grassland caterpillar was determined using a four-arm olfactometer (DJ-7000). Its key feature is its ability to create a clear odor boundary between different odor sources. During the experiment, a vacuum pump was used to evacuate air from the center to each of the four arms. Each arm contained a sample bottle with a 1×2cm filter paper strip. Three of the filter paper strips contained 10 μL of the pheromone to be tested, while the remaining strip contained n-hexane as a control. The vacuum pump was then activated. Ten caterpillars were carefully introduced into the central area of ​​the four-arm olfactometer, ensuring the caterpillars were not damaged. The time was recorded after the caterpillars began crawling, and the observation period was 5 minutes. The distribution of the Qinghai grassland caterpillars in each arm area was also recorded. Twenty caterpillars were tested per treatment, with three replicates. After every 10 caterpillars were tested, the four-arm olfactometer was rotated 90° to eliminate bias caused by positional differences. After each insect test, replace the filter paper strip promptly and wipe the inside of the four-arm olfactometer with pure alcohol to remove any odor remaining in the instrument. The gas flow rate inside the four-arm olfactometer is 60 mL / min. The entire behavioral measurement process must be conducted indoors under parallel light, at a temperature of 22±1℃ and a humidity of 58±5% to minimize the influence of light on the experiment. The trapping rate = (number of insects in each arm / total number of insects) × 100%.

[0030] 1.6 Field trapping of adult male caterpillars in the Qinghai grasslands:

[0031] A self-designed triangular trap was used, with a frame made of 20×25 cm foam board and a base made of cardboard in four colors: blue, yellow, purple, and white, coated with insect glue to capture male adults attracted by sex pheromones. A rubber lure was pre-treated with sex pheromones and hung in the center of the trap during the experiment. Based on olfactory behavior and antennal potential responses, the sex pheromone concentration corresponding to the highest tropism of Qinghai grassland caterpillars was selected for the experiment. Each experiment was conducted in triplicate, with n-hexane as a control. The trapping location was in Haibei Prefecture, Qinghai Province, and the timing was chosen during the peak activity period of Qinghai grassland caterpillars, from 10:00 AM to 4:00 PM daily. The number of adult caterpillars trapped was observed and recorded every 2 hours, with each trapping lasting 6 hours, for a total of 12 hours.

[0032] 2 Results and Analysis

[0033] 2.1 Screening of sex pheromone compounds:

[0034] This experiment identified 346 gonadal metabolites, which were further analyzed and classified into 91 species, including 5 keto acids, 16 carboxylic acids, 6 fatty acids, 14 amino acids, 11 nucleosides and nucleotides, 15 sugars, and 24 other species. Using a typical sex pheromone component of lepidopteran insects such as tussock moths (trans-2-cis-13-octadecenol acetate—a long-chain unsaturated fatty acid derivative) as a structural control, and combining the physicochemical properties of methyl palmitoleate, with reference to three previously confirmed sex pheromone components—1-Hexadecanol, Phytol, and Octadecanol—and GC-MS identification results, the above four compounds were ultimately identified as sex pheromones of the Qinghai grassland caterpillar.

[0035] 2.2 Antennae potential response of male adult caterpillars in Qinghai grasslands to sex pheromones:

[0036] Male adult Qinghai grassland caterpillars exhibited strong electrophysiological responses to all four pheromones mentioned above. Figure 1 . Figure 1 In the bar chart, A: Methyl Palmitoleate; B: 1-Hexadecanol; C: Phytol; D: Octadecanol; 1:1-B:D; 3:7-B:D; different letters above the bars indicate significant differences between different concentrations of the same compound (P<0.05). 1-Hexadecanol (hexadecyl alcohol) had the highest EAG value. Within the concentration gradient range of 0.001-10 μg / mL, its EAG value remained stable and significantly higher than other groups, especially reaching a peak at 0.001 μg / mL, indicating that this substance may act as a core signaling molecule mediating the information recognition process of the Qinghai grassland caterpillar. When 1-Hexadecanol and Octadecanol were mixed in a 1:1 ratio, the EAG value was highest at 0.001 μg / mL and lowest at 0.01 μg / mL. When the mixing ratio was 3:7, the EAG value increased gradient with increasing concentration, only reaching its lowest value when both were at 10 μg / mL, second only to 1-Hexadecanol and Octadecanol alone. In contrast, methyl palmitoleate showed a moderate EAG value. Between phytol and Octadecanol, Octadecanol exhibited a higher but unstable EAG peak, while phytol showed a relatively lower but more stable EAG peak. These results provide important electrophysiological data to elucidate the regulatory mechanism of sex pheromone behavior in Qinghai grassland caterpillars.

[0037] Methyl palmitate is a naturally occurring fatty acid derivative found in plants and animals. In the natural environment, it can be degraded by soil microorganisms into fatty acids and methanol, posing no risk of residual pollution. This aligns with the concept of "green pest control" and avoids the damage to ecosystems caused by chemical pesticides. Its raw material (palmitoleic acid) is widely available and inexpensive, and the esterification process is simple (can be completed at room temperature and pressure), facilitating large-scale production. Furthermore, it can be combined with carrier materials such as natural rubber and polyethylene to prepare lures, achieving slow release of the attractant. Choosing methyl palmitate as a sex pheromone attractant for Qinghai grassland caterpillars not only offers the core advantages of stability and low toxicity but also significantly enhances the trapping effect by mimicking insect chemical signals.

[0038] Electrophysiological responses of Qinghai grassland caterpillars to different concentrations of methyl palmitate were determined using antennal potential technology. Results are shown in [Figure number missing]. Figure 2 , Figure 2 Different letters above the bars in the middle bar chart represent significant differences in concentrations of the same compound (P<0.05, Duncan's test). The results show that the electrophysiological (EAG) response of male adult *Przewalski's caterpillar* to the sex pheromone Methyl Palmitoleate remained generally stable, reaching its highest value at 0.01 μg / mL and its lowest value at 10 μg / mL. From the highest value, the response gradually decreased with increasing concentration. These results provide important electrophysiological data to elucidate the behavioral regulatory mechanism of the *Przewalski's caterpillar* sex pheromone Methyl Palmitoleate.

[0039] 2.3 Olfactory behavioral response of Qinghai grassland caterpillars to sex pheromones:

[0040] (1) Olfactory behavioral response of Qinghai grassland caterpillars to sex pheromones:

[0041] The olfactory behavioral responses of Qinghai grassland caterpillar larvae were analyzed by measuring the attraction rate of different concentrations (10, 1, 0.1, 0.01, 0.001 μg / ml) of the sex pheromone Methyl Palmitoleate. The results are as follows: Figure 3 As shown in the bar chart, the different letters above the bars represent significant differences between different concentrations of the same compound (P<0.05). Methyl palmitate exhibited the highest attraction rate at a concentration of 0.1 μg / ml, significantly higher than other concentrations and the control group. This indicates that methyl palmitate had the strongest attraction effect on Qinghai grassland caterpillars at this concentration, and both increasing and decreasing the concentration led to varying degrees of decrease in the attraction effect.

[0042] (2) Olfactory behavioral response of male adult caterpillars in the Qinghai grasslands to sex pheromones:

[0043] The attraction effect of methyl palmitate on male adult caterpillar caterpillars in the Qinghai grasslands was determined, and the results are as follows: Figure 4 As shown in the bar chart, different letters above the bars represent significant differences between different concentrations of the same compound (P<0.05). Compared to larvae, male adults showed increased attraction to methyl palmitate at all concentrations, with the highest attraction rate at a concentration of 0.1 μg / ml. This indicates that methyl palmitate has the best attraction effect on the Qinghai grassland caterpillar at a concentration of 0.1 μg / ml.

[0044] 2.4 Field trapping experiment of male adult caterpillars in the Qinghai grasslands:

[0045] Based on the results of indoor four-arm olfactory behavior measurements, a concentration of 0.1 μg / ml of Methyl Palmitoleate, which exhibits the highest olfactory attraction, was selected and combined with different colors (yellow, blue, white, and purple) with high olfactory attraction to conduct a field trapping experiment on Qinghai grassland caterpillars. Hexane was used as a control. The number of caterpillars trapped in the field over 12 hours was recorded. The results (see Table 1) showed that the combination of Methyl Palmitoleate with highly olfactory colors (yellow and purple cardboard, with purple cardboard showing the strongest attraction) was significantly effective in attracting and capturing male adults. However, the combination with less olfactory colors (blue and white cardboard) was ineffective, failing to attract any male adults, similar to the control group. This indicates that efficient trapping of Qinghai grassland caterpillars can only be achieved when both color and reagent exhibit optimal single-factor effects.

[0046] Table 1 Results of field trapping experiment of male adult caterpillars in Qinghai grassland

[0047] C CK yellow 9 0 blue 0 0 white 5 0 purple 16 0

[0048] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of methyl palmitate as an attractant for Qinghai grassland caterpillars, wherein the attractant contains a methyl palmitate solution with a concentration of 0.1 μg / ml or 0.001 μg / ml prepared with n-hexane; wherein the Qinghai grassland caterpillar is a larva or an adult male.

2. The application according to claim 1, characterized in that: The attractant, combined with a purple trapping device, is used to attract caterpillars in the Qinghai grasslands.

3. The application according to claim 2, characterized in that: The trapping device is made of cardboard.

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