Methods and applications of using sub-lethal concentrations of chlorfenapyr to control peach fruit borer

Abstract

This invention discloses a method and application for controlling peach fruit borer at a sublethal concentration of chlorfenapyr, belonging to the field of agricultural pest control technology. The method utilizes a sublethal concentration of chlorfenapyr (LC1: 4.154 mg / L, LC50: 4.154 mg / L). 10 12.143 mg / L or LC 30 Treatment of adult peach fruit borers with a concentration of 26.416 mg / L significantly inhibited their reproductive capacity, thereby achieving effective control over the peach fruit borer population. LC1, LC 10 and LC 30 Treatments can reduce the oviposition rate of F0 generation females and inhibit ovarian development, manifested as shortened lateral ovarian duct length, reduced number of eggs in the ovary, and ovarian morphological atrophy. This invention applies sublethal concentrations of chlorfenapyr to the population control of peach fruit borer, breaking through the traditional control approach that relies on lethal doses, and providing a novel pest control method with low dosage, environmental friendliness, and cross-generational sustained effectiveness.

Description

Technical Field

[0001] This invention belongs to the field of agricultural pest control technology, specifically relating to a method and application of controlling peach fruit borer at a sub-lethal concentration of chlorfenapyr. Background Technology

[0002] The peach fruit borer (Lepidoptera, Pyralidae family) is a highly destructive global agricultural pest with a wide host range, severely damaging fruit trees, corn, and other crops. The larvae bore into the fruit to feed, directly causing significant yield reductions and noticeably lowering fruit quality, resulting in substantial economic losses.

[0003] Currently, chemical pesticides remain the primary means of controlling the peach fruit borer. However, the long-term irrational use of chemical pesticides has led to increasingly prominent problems such as increased pesticide resistance in pests and environmental pollution. In practical field applications, pests are often exposed to sublethal doses of pesticides due to various biological and abiotic factors, thus inducing sublethal effects. Recent studies have shown that while exposure to sublethal concentrations of pesticides does not directly cause death, it affects insect reproduction, development, flight, feeding, and mating behaviors. Its cumulative effects on population dynamics may be more profound than lethal exposure. Sublethal exposure can influence population growth potential by altering insect physiological metabolic processes, endocrine regulation, and energy allocation patterns.

[0004] Chlorfenapyr is a novel pyrrole-based insecticide and acaricide that interferes with energy metabolism by uncoupling mitochondrial oxidative phosphorylation, leading to insect cessation of feeding and eventual death. Due to its unique mechanism of action and relatively good environmental safety, chlorfenapyr has been widely used for the control of lepidopteran pests. However, current research on chlorfenapyr mainly focuses on acute toxicity evaluation and field control efficacy, with relatively limited research on its sublethal effects; furthermore, no studies have been conducted on specific sublethal concentrations (LC1, LC50) of chlorfenapyr. 10 LC 30 This study explores a technical solution for controlling the population of the peach fruit borer. Therefore, investigating the sublethal effect of chlorfenapyr on the peach fruit borer is of great significance for a comprehensive understanding of its ecotoxicological effects and for the scientific control of the peach fruit borer. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method and application for controlling peach fruit borer at a sublethal concentration of chlorfenapyr, specifically by using a sublethal concentration (LC1, LC2) of chlorfenapyr. 10 or LC 30 Treatment of adult peach borers significantly inhibits their reproductive capacity and induces a transgenerational reproductive inhibition effect, thereby achieving effective and sustainable control of the peach borer population.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a method for controlling peach fruit borer using a sublethal concentration of chlorfenapyr. The method involves treating adult peach fruit borers with a sublethal concentration of chlorfenapyr, wherein the sublethal concentration is selected from LC1 and LC2. 10 and LC 30 At least one of them can be used to control the population by suppressing the reproductive capacity of the peach fruit borer.

[0007] Preferably, the sublethal concentration is LC1, with a concentration of 4.154 mg / L; or LC 10 The concentration was 12.143 mg / L; or LC. 30 The concentration was 26.416 mg / L.

[0008] Preferably, the peach borer adults are treated with a 10% sucrose aqueous solution containing acaricide for 72 hours.

[0009] This invention also discloses the application of a sublethal concentration of chlorfenapyr in the population control of peach fruit borer, wherein adult peach fruit borers are treated with a sublethal concentration of chlorfenapyr, wherein the sublethal concentration is selected from LC1 and LC2. 10 and LC 30 At least one of them is used to suppress the population growth of peach borer.

[0010] Preferably, the sublethal concentration is LC1, with a concentration of 4.154 mg / L; or LC 10 The concentration was 12.143 mg / L; or LC. 30 The concentration was 26.416 mg / L.

[0011] Preferably, the peach borer adults are treated with a 10% sucrose aqueous solution containing acaricide for 72 hours.

[0012] This invention also discloses a peach fruit borer population control agent, which contains an effective amount of chlorfenapyr and an agriculturally acceptable carrier, wherein the sublethal concentration of chlorfenapyr is selected from LC1 and LC2. 10 and LC 30 At least one of them.

[0013] Preferably, the sublethal concentration is LC1, with a concentration of 4.154 mg / L; or LC 10 The concentration was 12.143 mg / L; or LC. 30 The concentration was 26.416 mg / L.

[0014] Preferably, the carrier is a 10% sucrose aqueous solution.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention systematically reveals the mechanism of action of sublethal concentrations of chlorfenapyr on the peach fruit borer: by disrupting energy metabolism and endocrine homeostasis, it inhibits the reproduction of the peach fruit borer and produces a transgenerational effect. Specifically, treatment with sublethal concentrations of chlorfenapyr significantly reduces the oviposition rate of F0 generation females, inhibits ovarian development, and leads to a significant decrease in the levels of juvenile hormone (JH), ecdysone (20E), vitellogenin (Vg), and its receptor (VgR). At the molecular level, the expression of hormone degradation genes CYP18A1 and JHEH is upregulated, while the expression of the synthesis gene CYP314A1 and its downstream genes EcR and Met is downregulated, and the expression of Vg and VgR genes is also significantly reduced. Simultaneously, chlorfenapyr significantly disrupts glycolipid metabolism, resulting in a decrease in the levels of glycogen, trehalose, glucose, and triglycerides, a decrease in ATP levels, and an increase in the ADP / ATP and AMP / ATP ratios. The expression of related metabolic genes shows a trend of enhanced degradation and inhibited synthesis. Furthermore, the activities and gene expression of detoxification enzymes (P450, GST, CarE) were significantly upregulated in a time- and concentration-dependent manner. Transgenerational studies showed that the F1 generation maintained similar phenotypes of reproductive inhibition, ovarian developmental arrest, energy metabolism disorders, and persistent activation of detoxification enzymes.

[0016] This invention applies sublethal concentrations of chlorfenapyr to the population control of peach fruit borer, breaking through the traditional control approach that relies on lethal doses, and providing a novel pest control method with low dosage, environmental friendliness, and cross-generational sustained efficacy. Attached Figure Description

[0017] Figure 1 The figure shows the effect of sublethal concentration of chlorfenapyr on the reproductive capacity of female F0 generation peach borers; where: (A) effect of sublethal concentration of chlorfenapyr on the mating rate of peach borers; (B) effect of sublethal concentration of chlorfenapyr on the number of eggs laid by peach borers; (C) effect of sublethal concentration of chlorfenapyr on the length of the lateral ovarian tube of peach borers; (D) effect of sublethal concentration of chlorfenapyr on the number of ovarian eggs of peach borers; (E) effect of sublethal concentration of chlorfenapyr on ovarian development of peach borers.

[0018] Figure 2 The diagram shows the effects of sublethal concentrations of chlorfenapyr on hormone titers, Vg, VgR content, and related genes in peach fruit borer. (A) shows the change in JH titer after treatment with sublethal concentrations of chlorfenapyr; (B) shows the change in 20E titer after treatment with sublethal concentrations of chlorfenapyr; (C) shows the change in Vg content after treatment with sublethal concentrations of chlorfenapyr; (D) shows the change in VgR content after treatment with sublethal concentrations of chlorfenapyr; and (E) shows the expression levels of related genes after treatment with sublethal concentrations of chlorfenapyr.

[0019] Figure 3The diagram shows the effects of sublethal concentrations of chlorfenapyr on the glycolipid and energy metabolism and related genes of *Prunus persica var. chinensis*. Specifically: (A) Changes in glycogen content after treatment with sublethal concentrations of chlorfenapyr; (B) Changes in trehalose content after treatment with sublethal concentrations of chlorfenapyr; (C) Changes in glucose content after treatment with sublethal concentrations of chlorfenapyr; (D) Changes in triglyceride content after treatment with sublethal concentrations of chlorfenapyr; (E) Changes in ATP content after treatment with sublethal concentrations of chlorfenapyr; (F) Changes in ADP content after treatment with sublethal concentrations of chlorfenapyr; (G) Changes in AMP content after treatment with sublethal concentrations of chlorfenapyr; (H) Changes in the ADP / ATP ratio after treatment with sublethal concentrations of chlorfenapyr; (I) Changes in the AMP / ATP ratio after treatment with sublethal concentrations of chlorfenapyr.

[0020] Figure 4 The graph shows the changes in the activity of detoxification enzymes and the expression levels of related genes in peach fruit borer after treatment with sublethal concentrations of chlorfenapyr for different time periods; where: (A) shows the changes in cytochrome P450 enzyme activity; (B) shows the changes in glutathione S-transferase activity; (C) shows the changes in carboxylesterase activity; and (D) shows the changes in the expression levels of related genes.

[0021] Figure 5 The figure shows the effect of sublethal concentration of chlorfenapyr on the F1 generation of peach fruit borer; where: (A) is the hatching rate of eggs; (B) is the pupation rate; (C) is the emergence rate; (D) is the egg stage; (E) is the larval development stage; (F) is the pupal stage; (G) is the adult lifespan; (H) is the female-to-male ratio; (I) is the total number of eggs laid in the F1 generation; (J) is the pupal weight; (K) is the effect of different treatment groups on pupae; (L) is the number of eggs laid by a single female in the F1 generation; (M) is the length of the lateral ovarian duct; and (N) is the number of eggs in the ovary.

[0022] Figure 6 The graph shows the effects of sublethal concentrations of chlorfenapyr on hormones, glycolipids, and energy in the F1 generation of peach fruit borer; where: (A) is the change in JH titer; (B) is the change in 20E titer; (C) is the change in Vg content; (D) is the change in VgR content; (E) is the change in glycogen content; (F) is the change in trehalose content; (G) is the change in glucose content; (H) is the change in triglyceride content; (I) is the change in ATP content; (J) is the change in ADP content; (K) is the change in AMP content; (L) is the change in ADP / ATP ratio; and (M) is the change in AMP / ATP ratio.

[0023] Figure 7 The figure shows the effect of sublethal concentration of chlorfenapyr on the expression levels of genes related to reproduction and energy metabolism in the F1 generation of peach fruit borer; where: (A) represents the expression level of genes related to reproduction; (B) represents the expression level of genes related to energy metabolism.

[0024] Figure 8The figure shows the effects of sublethal concentrations of chlorfenapyr on the detoxification enzyme activity and related gene expression levels of the F1 generation of peach borer; where: (A) shows the changes in cytochrome P450 enzyme activity; (B) shows the changes in glutathione S-transferase activity; (C) shows the changes in carboxylesterase activity; and (D) shows the changes in related gene expression levels.

[0025] Figure 9 This is a graph showing the effect of sublethal concentrations of chlorfenapyr on the life table parameters of the F1 generation of the peach fruit borer; where: (A) represents the effect of sublethal concentrations of chlorfenapyr on the expected lifespan (e) of the age-stage characteristics of the F1 generation of the peach fruit borer. xj (A) Effect of sublethal concentration of chlorfenapyr on the age-stage characteristic survival rate of F1 generation of peach borer (l) x ), female adult age-characteristic fertility (f xj Population age-characteristic reproductive capacity (m) x ) and population age-characteristic net reproductive rate (l x m x (C) represents the effect of sublethal concentrations of chlorfenapyr on the age-stage characteristic survival rate (s) of the F1 generation of peach borer. xj The effect of (D) on the age-stage reproductive characteristics of the F1 generation of peach borer; xj The impact of ). Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments: Example 1 This example demonstrates the determination of the sublethal concentration of chlorfenapyr.

[0030] 1.1 Test insect source The peach fruit borer population was collected from an apple orchard in the Science and Education Park of Henan Agricultural University, Zhengzhou City, Henan Province. It was continuously reared indoors under standardized conditions for over 20 generations without any contact with pesticides. Larvae were fed fresh corn kernels, and adults were fed a 10% sucrose solution. The rearing environment was maintained at a temperature of 27±1℃, a relative humidity of 70%±5%, and a photoperiod of 16L:8D. Newly emerged adults of similar instar and size were selected for subsequent experiments.

[0031] 1.2 Test reagents The chlorfenapyr (99% active ingredient) was purchased from Hubei Jiahui Xingcheng Biotechnology Co., Ltd. (Hubei, China).

[0032] 1.3 Methods for determining toxicity The technical grade chlorfenapyr was dissolved in acetone to prepare a stock solution of 10,000 mg / L. This stock solution was then diluted with 10% sucrose solution to prepare a series of test concentrations: 0, 3.75, 7.5, 15, 30, 60, and 90 mg / L, ensuring that the amount of acetone in all working solutions was the same. Newly emerged peach fruit borer adults (female to male ratio 1:1) were fed sucrose solutions containing the above different concentrations of insecticide, while the control group was fed sucrose solution containing an equal amount of acetone. Three replicates were set up for each concentration, with 16 adults treated in each replicate. The sucrose solution containing the insecticide was changed daily, and mortality was recorded after 72 hours.

[0033] 1.4 Results Analysis The toxicity regression equation of chlorfenapyr against adult peach fruit borers was determined using SPSS software probabilistic unit analysis. The results showed that after 72 hours of treatment with chlorfenapyr on newly emerged adults, the toxicity regression equation was: Y = 2.18x + 3.62 (R²). 2 =0.994), the calculated sublethal concentrations were: LC1 = 4.154 mg / L (95% confidence interval: 2.181~6.340 mg / L), LC 10 = 12.143 mg / L (95% confidence interval: 8.410~15.707 mg / L), LC 30 = 26.416 mg / L (95% confidence interval: 21.277~31.860 mg / L), as shown in Table 1.

[0034] Table 1

[0035] “N” represents the number of samples in each concentration treatment group; “CL” represents the confidence interval.

[0036] The above results indicate that chlorfenapyr has a significant toxic effect on adult peach borers, and can effectively treat adult peach borers without causing mass mortality within the sublethal concentration range (4.154~26.416 mg / L), providing a suitable concentration gradient for subsequent sublethal effect studies.

[0037] Example 2 This example illustrates the effect of sublethal concentrations of chlorfenapyr on the reproductive capacity of the F0 generation of peach borer.

[0038] 2.1 Mating rate determination Newly emerged adults (female to male ratio 1:1) were placed in rearing cages (30×30×30 cm) and fed with LC1 and LC2 as determined above. 10 and LC 30 Sucrose solution containing a concentration of chlorfenapyr was used to treat the control group, which was fed sucrose solution containing an equal amount of acetone. After 72 hours of treatment, mating status was determined by touching the females to check for the presence of spermatophores. Each treatment was repeated in triplicate, with 20 females tested in each replicate.

[0039] The result is as follows Figure 1 As shown in Figure A, it can be seen from the figure that after treatment with sublethal concentrations of chlorfenapyr, the mating rate of newly emerged peach borers did not change significantly compared with the control group (F). 3, 8 = 1.383, P = 0.316), indicating that the sublethal concentration of chlorfenapyr does not affect the mating behavior of peach fruit borer.

[0040] 2.2 Measurement of egg production Adults that survived 72 hours after treatment were transferred to large plastic cups sealed with gauze and provided with 10% sucrose water without pesticides. Five pairs of adults (female to male ratio 1:1) were placed in each plastic cup, and eight replicates were set up for each treatment group. The total number of eggs laid in the plastic cups was counted at the peak of egg laying.

[0041] The results are as follows Figure 1 As shown in Figure B, it can be seen from the figure that the number of eggs laid by female insects decreased significantly in a dose-dependent manner with increasing treatment concentration (F). 3, 28 = 16.076, P<0.001); CK (control group) approximately 60 larvae / female, LC1 group approximately 40 larvae / female, LC 10 Approximately 31 larvae per female, LC 30 Approximately 16 larvae per female in the group; compared with the control group, LC1 and LC2 were higher. 10 and LC 30 The oviposition rate in the treatment groups decreased by approximately 33.3%, 48.3%, and 73.3%, respectively, indicating that the sublethal concentration of chlorfenapyr can significantly inhibit the oviposition capacity of female peach borers.

[0042] 2.3 Observation of ovarian development Newly emerged females were treated with LC1 and LC1 as described above. 10 and LC 30 After treatment with concentrated sucrose water for 72 hours, the ovaries were dissected under a stereomicroscope. Ovaries were photographed using a Leica stereomicroscope (Leica M205A), and the length of the lateral ovarian ducts and the number of ovarian oocytes were measured and counted. Thirty ovaries were analyzed for each treatment.

[0043] Results showed that ovarian anatomy revealed that sublethal concentrations of chlorfenapyr significantly inhibited ovarian development; compared with the control group, LC... 10 Processing group and LC 30 Length of lateral ovarian ducts in the treated females ( 2 =14.887; df = 3; P = 0.002) Figure 1 C shows the number of ovarian oocytes ( 2 = 25.817; df = 3; P<0.001) Figure 1 As shown in Figure D, all were significantly reduced. Specifically, the length of the lateral ovarian duct was approximately 14.2 mm in the CK (control group) and approximately 13.3 mm in the LC1 group. 10 Group approximately 12.7 mm, LC 30 The ovarian ovum size was approximately 12.3 mm; the number of ovarian ovums was approximately 183 in the CK (control group) and approximately 160 in the LC1 group. 10 Approximately 147 tablets per group, LC 30 The group contained approximately 139 pills; compared to the control group, LC 10 In the treatment group, the length of the lateral ovarian duct was shortened by approximately 10.6%, and the number of ovarian oocytes decreased by approximately 19.7%; LC 30 The treatment group experienced a 13.4% reduction in lateral ovarian duct length and a 24.0% decrease in the number of ovarian oocytes.

[0044] Figure 1 E is a microscopic image (anatomical observation) of the ovary of a female of the F0 generation of the peach fruit borer. The image shows that the CK (control group) ovary is the largest and most structurally complete, with numerous and robust oviduct branches and densely packed eggs, exhibiting a typical mature ovary morphology. In the LC1 group, the ovary is slightly smaller, and the number and fullness of the oviducts are mildly reduced. 10 In this group, the ovaries showed significant atrophy, the fallopian tubes became thinner and branched, and the number of ovaries was sparse; LC 30 In this group, the ovaries are severely degenerated, the smallest in size, the fallopian tubes are thin and weak with a loose structure, and there are very few eggs or even some empty tubes.

[0045] The above results indicate that although sublethal concentrations of chlorfenapyr do not affect the mating behavior of peach fruit borer, they significantly reduce the number of eggs laid by females by inhibiting ovarian development (shortening of ovarian ducts and reduction of egg number), and this effect is clearly dose-dependent. This suggests that sublethal concentrations of chlorfenapyr can effectively inhibit the reproductive capacity of peach fruit borer.

[0046] Example 3 This example illustrates the effects of sublethal concentrations of chlorfenapyr on hormone levels and reproductive-related gene expression in the F0 generation of peach borer.

[0047] 3.1 Hormone content determination Four abdominal tissues were collected from F0 generation female adults treated according to the method in Example 2. Physiological saline was added to each tissue, and the tissues were ground. The supernatant was diluted 100 times for later use. The contents of the above substances in the peach fruit borer were determined using an enzyme-linked immunosorbent assay (ELISA) kit for insect juvenile hormone (JH), an ELISA kit for insect ecdysone (20E), an ELISA kit for insect vitellogenin (VTG), and an ELISA kit for insect vitellogenin receptor (VgR) (all kits were from Jiangsu Enzyme Immunosorbent Assay Co., Ltd.).

[0048] The results showed that, compared with the control group, LC 10 and LC 30 The JH titer of female worms in the treatment group decreased significantly (F 3,8 =171.169, P<0.001; see Figure 2 A), while the 20E titer decreased significantly in all treatment groups (F). 3,8 = 56.514, P<0.001; see Figure 2 B); Vg and VgR contents decreased significantly in all treatment groups (F) 3,8 = 243.269, P<0.001; F 3,8 =33.698, P<0.001; see Figure 2 CD).

[0049] 3.2 Gene Expression Analysis Abdominal tissue from female adult peach fruit borers treated according to the method in Example 2 was collected, and RNA was extracted according to the instructions of the Total RNA Extraction Kit. RNA concentration and optical density (OD) were measured using a NanoDrop 1000 spectrophotometer, and RNA integrity was verified by agarose gel electrophoresis. cDNA was synthesized via reverse transcription using the HiScript III RT SuperMix kit, serving as a template for quantitative real-time PCR (qRT-PCR). qPCR primers were designed using the NCBI Primer-BLAST online tool and synthesized by Sangon Biotech (Shanghai) Co., Ltd. qRT-PCR was performed using an Applied Biosystems 7500 fast real-time PCR instrument with ChamQ Universal SYBR qPCR Master Mix. The reaction program was: 95℃ pre-denaturation for 3 min, 40 cycles (95℃ 15 s, 50℃ 30 s, 72℃ 30 s), and a final extension at 72℃ for 10 min. Amplification specificity was analyzed by melting curve analysis. The expression level of the target gene was calculated using a dual internal control method, with ribosomal protein genes RP49 and RPL13 selected as internal controls. Each sample was configured with three biological replicates and three technical replicates.

[0050] The results are as follows Figure 2 As shown in Figure E, it can be seen from the figure that the expression level of the 20E degradation gene CYP18A1 was significantly increased in all treatment groups (F). 3,8 = 98.794, P<0.001), while the 20E synthetic gene CYP314A1 (F 3,8 = 45.452, P<0.001) and downstream gene EcR (F 3,8 = 44.125, P<0.001) The expression level of ( ) showed a decreasing trend in all treatment groups, and in LC 10 and LC 30 The JH degradation gene JHEH reached a significant level in the high-concentration treatment group (LC). 30 Significantly upregulated (F) 3,8 =46.830, P<0.001), the downstream receptor gene Met decreased significantly in all treatment groups (F 3,8 = 53.325, P<0.001); Consistent with changes in protein content, the gene expression levels of Vg and VgR were also significantly downregulated in all treatment groups (F 3,8 =129.766, P<0.001; F 3,8 = 111.916, P<0.001).

[0051] The above results indicate that chlorfenapyr may inhibit vitellogenesis at the transcriptional level by interfering with hormone signaling pathways, thereby leading to a decrease in the reproductive capacity of F0 generation females. Specific mechanisms include: reducing JH and 20E titers, inhibiting the synthesis and expression of Vg and VgR, thus affecting ovarian development and oocyte maturation.

[0052] Example 4 This example illustrates the effect of sublethal concentrations of chlorfenapyr on the F0 glycolipid metabolism and energy levels of peach borer.

[0053] 4.1 Determination of glycolipid content The glycolipid content in *Prunus persica* was detected using a glucose content assay kit (G0504W), a glycogen content assay kit (G0561W), a trehalose content assay kit (G0552W), and a triglyceride content assay kit (G0910W) (all kits were from Suzhou Greens Biotechnology Co., Ltd.).

[0054] The results show that: Figure 3 As shown in AB, the glycogen and trehalose content in female insects in each treatment group was significantly lower than that in the control group (F). 3,8 = 214.395, P<0.001; F 3,8 = 214.749, P<0.001;); Figure 3 As shown in CD, glucose and triglycerides are only present in LC. 10 and LC 30 Significant decrease in the treatment group (F) 3,8 = 9.208, P = 0.006; F 3,8 = 16.741, P = 0.001).

[0055] 4.2 Energy metabolism measurement The contents of the above substances in the peach borer were determined using an enzyme-linked immunosorbent assay (ELISA) kit for adenosine triphosphate (ATP), an ELISA kit for adenosine diphosphate (ADP), and an ELISA kit for adenosine monophosphate (AMP) (all kits were from Jiangsu Enzyme Immunosorbent Assay Co., Ltd.).

[0056] The results show that: Figure 3 As shown in E, ATP levels decreased significantly in all treatment groups (F). 3,8 = 29.056, P<0.001); as Figure 3 As shown in FG, the levels of ADP and AMP showed an increasing trend (F 3,8 = 6.764, P = 0.014; F 3,8 = 37.335, P<0.001), which led to an increase in the ADP / ATP and AMP / ATP ratios with increasing treatment concentration (F3,8 = 27.905, P<0.001; F 3,8 = 49.022, P<0.001) See Figure 3 As shown in HI.

[0057] 4.3 Analysis of the expression of genes related to energy metabolism Detecting the expression levels of energy-related genes, such as Figure 3 As shown in Figure J: the glycogen phosphorylase gene GP showed an upregulation trend in all treatment groups, while the glycogen synthase gene GS showed a downregulation trend (F). 3,8 = 64.749, P<0.001; F 3,8 = 9.286, P = 0.006); Trehalase genes Treh1 and Treh2 showed an upregulation trend in all treatment groups, but only in LC... 30 Significant differences were observed in the treatment groups (F) 3,8 = 7.086, P = 0.012; F 3,8 = 7.825, P = 0.009); the expression level of hexokinase HK, a key gene for glucose utilization, was significantly increased in all treatment groups (F = 7.825, P = 0.009). 3,8 = 24.254, P<0.001); the fatty acid synthase gene FAS1 showed a decreasing trend in all treatment groups, while the lipid mobilization-related gene triglyceride lipase ATGL showed an increasing trend (F = 24.254, P<0.001); 3,8 = 11.846, P = 0.003; F 3,8 = 7.892, P = 0.009); key genes in the energy metabolism pathway, phosphoglycerate kinase (PGK) and pyruvate kinase (PK), were upregulated in all treatment groups (F 3,8 = 20.617, P<0.001; F 3,8 =169.715, P<0.001).

[0058] The above results indicate that energy metabolism disorders may weaken the physiological functions of female insects, preventing them from providing sufficient energy for yolk formation and reproductive activities, thereby exacerbating reproductive damage caused by chlorfenapyr. Chlorfenapyr further affects the reproductive capacity of the peach fruit borer by interfering with glucose and lipid metabolism and energy metabolism. Example 5 This example illustrates the effect of sublethal concentrations of chlorfenapyr on the activity and gene expression of the detoxification enzyme in the F0 generation of peach borer.

[0059] 5.1 Detoxification enzyme activity assay F0 generation newly emerged females were treated according to the method in Example 2, and their abdominal tissues were collected at 12h, 24h, 48h, and 72h after treatment. After the samples were prepared according to the method in Example 3, the activities of key detoxification enzymes were determined using specific detection kits: Insect cytochrome P450 enzyme ELISA kit (Xiamen Lunchangshuo Biotechnology Co., Ltd.), Carboxylesterase (CarE) activity assay kit (Beijing Solarbio Science & Technology Co., Ltd.), and Glutathione S-transferase (GST) activity assay kit (Suzhou Greens Biotechnology Co., Ltd.). Three biological replicates were set up for each treatment, and three technical replicates were set up for each biological replicate.

[0060] The results show: See Figure 4 As shown in Figure A, the P450 enzyme activity showed no significant difference from the control group at 12 h and 24 h after treatment (F). 3,8 =1.309, P = 0.339; F 3,8 = 1.929, P = 0.204); while 48h after treatment, LC 30 The treatment group significantly upregulated (F) 3,8 =5.791, P = 0.021); by 72 h, all treatment groups showed significant upregulation (F 3,8 = 21.399, P<0.001); see Figure 4 As shown in B, the GST enzyme activity decreased by only LC 12h after treatment. 30 The treatment group significantly upregulated (F) 3,8 = 18.091, P = 0.001); at 24 h, all treatment groups showed significant upregulation (F 3,8 = 842.264, P<0.001); at 48h, all treatment groups recovered to the control level (F 3,8 =3.147, P = 0.087); LC at 72 h 10 and LC 30 The treatment group showed a significant increase again (F 3,8 = 33.678, P<0.001); see Figure 4 As shown in C, CarE enzyme activity was significantly upregulated in all treatment groups at 12h, 24h, and 48h after treatment (F). 3,8 = 326.895, P<0.001; F 3,8 = 41.450, P<0.001; F 3,8 = 32.789, P<0.001); while at 72h, only at LC 10 and LC 30 Significant increase in the treatment group (F) 3,8 = 68.227, P<0.001).

[0061] 5.2 Analysis of expression of detoxification enzyme-related genes The expression levels of detoxification enzyme-related genes were detected at two time points, 24h and 72h. The results showed that the expression of detoxification enzyme genes in the treatment group generally showed an upward trend.

[0062] like Figure 4 As shown in Figure D, it can be seen from the figure that at 24h and 72h after treatment, CYP6AB1 and CYP4C1 in LC 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8 = 56.522, P<0.001; F 3,8 = 55.452, P<0.001; F 3,8 = 29.414, P<0.001; F 3,8 = 112.008, P<0.001), CYP6B7 was significantly upregulated in all treatment groups (F 3,8 = 178.693, P<0.001; F 3,8 = 32.428, P<0.001); GSTO3 expression was upregulated in all treatment groups at 24h (F 3,8 = 257.991, P<0.001), while at 72h, only at LC 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8 = 97.192, P<0.001); GSTs5 at 24h only in LC 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8 = 25.433, P<0.001), and at 72h, the expression levels in all treatment groups were upregulated (F 3,8 = 662.604, P<0.001); CarE1 only occurred at LC at both time points. 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8 = 109.470, P<0.001; F 3,8 = 41.477, P<0.001), while CarE2 was significantly upregulated in all treatment groups at 72h (F 3,8 = 45.951, P<0.001; F 3,8 = 117.078, P<0.001).

[0063] The above results indicate that chlorfenapyr induces a concentration-dependent upregulation of detoxification enzyme activity and gene expression. The detoxification process requires a large amount of energy, which may further exacerbate energy metabolism disorders, thereby affecting the reproductive capacity of the peach fruit borer.

[0064] Example 6 This example illustrates the effect of sublethal concentrations of chlorfenapyr on the F1 generation of peach borer.

[0065] 6.1 Measurement of growth and development indicators of F1 generation Adult F0 insects treated for 72 hours according to the method described in Example 2 were placed on an oviposition cloth to lay eggs. F1 generation eggs were collected and reared individually. Larvae were fed fresh corn kernels, and adults were fed normal sucrose water. Their growth was observed and recorded daily, and the hatching rate and pupation rate of larvae, as well as the emergence rate and male ratio of adults, were statistically analyzed. Each treatment had four replicates, with 50 insects per replicate. The developmental period of each stage (egg, larva, pupa, and adult) was recorded, with at least 80 insects recorded per treatment. The weight of 3-day-old pupae was measured, with at least 60 pupae per treatment group.

[0066] The results are as follows Figure 5 As shown, the hatching rate, pupation rate, and emergence rate of the eggs all showed a decreasing trend in all treatment groups. Specifically: Hatching rate of eggs in LC 10 and LC 30 The treatment group was significantly lower than the control group (F 3,8 = 20.846, P<0.001) See Figure 5 As shown in A; pupation rate and eclosion rate are only observed in LC. 30 Significant decrease in the treatment group ( 2 = 12.008, df = 3, P = 0.007; 2 =12.008, df = 3, P = 0.007), see Figure 5 As shown in BC; the developmental duration of each stage also showed an overall trend of prolongation, with the egg stage being significantly prolonged in all treatment groups ( 2 = 45.757, df = 3, P<0.001), see Figure 5 As shown in D; the larval stage occurs only in LC. 10 Significantly prolonged in the treatment group ( 2 = 14.757, df = 3, P = 0.002), see Figure 5 As shown in E; the pupal stage showed no significant changes in any of the treatment groups ( 2 = 2.931, df = 3, P = 0.402), see Figure 5 As shown in F; the adult lifespan was significantly prolonged only in the LC1 treatment group ( 2= 9.016, df = 3, P = 0.029), see Figure 5 As shown in G; the proportion of females in the F1 generation is in LC. 10 and LC 30 The treatment group had significantly higher numbers than the control group, but the total number of eggs laid showed a decreasing trend (F). 3,8 = 15.901, P<0.001), see Figure 5 As shown in HI; the overall pupal weight also showed a decreasing trend, and in LC 10 and LC 30 Significantly reduced in the treatment group ( 2 = 33.855, df = 3, P<0.001), see Figure 5 As shown in JK, this indicates that the sublethal effect of chlorfenapyr can be transmitted across generations, interfering with the individual development process of the F1 generation.

[0067] 6.2 Fertility test of F1 generation After the F1 generation adults emerged, males and females were paired in a 1:1 ratio, and the total number of eggs laid was recorded until all adults died. The ovaries of three-day-old females were dissected, and the length of the lateral ovarian ducts and the number of eggs in the ovary were measured. Twenty worms were counted for each treatment.

[0068] The results showed that, compared with the control group, the number of eggs laid by a single female in the F1 generation decreased in all treatment groups, with the LC generation showing the highest number of eggs laid. 30 The treatment group reached a significant level ( 2 = 10.344, df = 3, P = 0.016) See Figure 5 As shown in L; further ovarian dissection revealed that the length of the ovarian duct and the number of ovaries decreased with increasing treatment concentration. The length of the ovarian duct at LC... 30 The treatment group had significantly shorter treatment times than the control group ( 2 = 8.724, df = 3, P = 0.033), see Figure 5 M is shown; the number of eggs in LC 10 and LC 30 Significantly reduced in the treatment group ( 2 = 21.178, df = 3, P<0.402), see Figure 5 As shown in N.

[0069] The above results indicate that the sublethal effect of chlorfenapyr can be transmitted across generations, interfering with the individual development process of the F1 generation. Consistent with the trend observed in the F0 generation, the reproductive capacity of F1 females was also significantly inhibited, manifested as slowed growth and development, reduced pupal weight, decreased egg production, and impaired ovarian development.

[0070] Example 7 This example illustrates the effect of sublethal concentrations of chlorfenapyr on the physiological and biochemical indicators of the F1 generation of peach borer.

[0071] 7.1 Measurement of hormone levels, glucose and lipid metabolism, and energy levels F1 generation peach borer was raised to obtain 3-day-old female adults according to the method described in Example 6. Abdominal tissue was taken from them, and samples were prepared according to the methods in Examples 3 and 4, and various indicators were measured.

[0072] The results showed that there was no significant difference in JH titer among the treatment groups (F 3,8 = 2.770, P = 0.111), see Figure 6 As shown in A; however, the 20E titer showed a decreasing trend in all treatment groups, and in LC... 10 and LC 30 The treatment group had significantly lower levels than the control group (F). 3,8 =10.708, P = 0.004) See Figure 6 As shown in B, the contents of Vg and VgR also showed a decreasing trend in all treatment groups, and in LC... 10 and LC 30 Significantly reduced in the treatment group (F 3,8 = 5.460, P = 0.024; F 3,8 = 51.556, P<0.001), see Figure 6 CD shown.

[0073] Regarding glucose and lipid metabolism, the contents of glycogen and trehalose were significantly lower in all treatment groups than in the control group (F). 3,8 =105.329, P<0.001; F 3,8 = 108.582, P<0.001), see Figure 6 As shown in EF; glucose is only present in LC. 30 Significantly reduced in the treatment group (F 3,8 = 15.244, P<0.001), see Figure 6 As shown in G; the triglyceride content in LC 10 and LC 30 Significant decrease in the treatment group (F) 3,8 = 7.760, P = 0.009), see Figure 6 As shown in H.

[0074] Regarding energy metabolism, ATP levels showed a decreasing trend, and in LC... 30 Significant decrease in the treatment group (F) 3,8 = 5.019, P = 0.030), see Figure 6As shown in Figure I, ADP levels showed an increasing trend across all treatment groups, but no significant differences were observed (F). 3,8 = 1.971, P = 0.197), see Figure 6 As shown in J; the AMP content increased significantly with increasing treatment concentration (F). 3,8 = 22.564, P<0.001), see Figure 6 As shown in K; correspondingly, the ADP / ATP and AMP / ATP ratios also showed a significant upward trend (F). 3,8 = 10.531, P = 0.004; F 3,8 = 65.346, P<0.001), see Figure 6 As shown in LM.

[0075] 7.2 Gene Expression Analysis Further investigation was conducted to detect the expression levels of genes related to reproduction and those related to glucose and lipid metabolism.

[0076] Regarding the expression levels of reproduction-related genes, the results are as follows: Figure 7 As shown in Figure A, the expression level of CYP18A1 increased with increasing treatment concentration, and in LC... 30 The treatment group had a significantly higher rate than the control group (F 3,8 = 10.988, P = 0.003); CYP314A1 and EcR both showed a decreasing trend, and in LC 30 There was a significant difference between the treatment groups (F) 3,8 = 7.364, P = 0.011; F 3,8 = 14.996, P = 0.001); JHEH increased significantly in all treatment groups, while Met showed a decreasing trend, and in LC 30 Significant decrease in the treatment group (F) 3,8 = 36.038, P<0.001; F 3,8 = 5.135, P = 0.029). The expression levels of Vg and VgR showed a decreasing trend in all treatment groups, with Vg showing a decreasing trend in LC. 10 and LC 30 A significant decrease was observed in the treatment group, while VgR was only observed in LC. 30 Significant decrease in the treatment group (F) 3,8 = 59.091, P<0.001; F 3,8 = 13.794, P = 0.002).

[0077] Regarding genes related to glucose and lipid metabolism, the results are as follows: Figure 7 As shown in B: GP in LC 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8= 38.915, P<0.001); GS in LC 30 Significant decrease in the treatment group (F) 3,8 = 9.680, P = 0.005); Treh1 and Treh2 are both in LC 30 Significantly upregulated (F) in the treatment group 3,8 = 8.359, P = 0.008; F 3,8 = 6.494, P = 0.015); there was no significant difference between HK and FAS1 in each treatment group (F 3,8 = 2.356, P = 0.148; F 3,8 = 0.599, P = 0.633). Compared with the control, ATGL was significantly upregulated in all treatment groups (F = 0.599, P = 0.633). 3,8 = 73.939, P<0.001). The expression levels of PGK and PK showed an increasing trend in all treatment groups, and were particularly high in LC. 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8 = 12.383, P = 0.002; F 3,8 =20.654, P<0.001).

[0078] The above results indicate that JH titer recovered in the F1 generation, but the levels of 20E, Vg, and VgR, as well as the disordered glucose and lipid energy metabolism, persisted. Gene expression changes were largely consistent with those in the F0 generation, suggesting that the sublethal effect of chlorfenapyr can be transmitted across generations and continues to affect the physiological and biochemical processes of the peach fruit borer in the F1 generation.

[0079] Example 8 This example illustrates the effect of sublethal concentrations of chlorfenapyr on the detoxification enzyme activity and gene expression of the F1 generation of peach borer.

[0080] 8.1 Detoxification enzyme activity assay The F1 generation peach borer was obtained as a 3-day-old adult according to the method described in Example 6, and its detoxification enzyme activity was determined using the same method as in Example 5.

[0081] The results showed that, compared with the control, there was no significant difference in P450 enzyme activity among the treatment groups (F... 3,8 = 1.394, P = 0.313), see Figure 8 As shown in Figure A, GST activity in LC 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8 = 82.393, P<0.001), see Figure 8 As shown in B; CarE in LC 30 Significantly upregulated (F) in the treatment group 3,8= 18.158, P = 0.001) See Figure 8 As shown in C.

[0082] 8.2 Analysis of expression of detoxification enzyme-related genes Further analysis of the expression levels of detoxification enzyme-related genes revealed an overall upward trend. Figure 8 As shown in D: CYP6AB10 in LC1 and LC 10 Significantly upregulated (F) in the treatment group 3,8 = 103.503, P<0.001), CYP6B7 was significantly upregulated in all treatment groups (F 3,8 = 215.981, P<0.001), CYP4C1 in LC 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8 =529.049, P<0.001); GST03 in LC 10 and LC 30 Significantly upregulated (F) in the treatment group 3,8 = 25.355, P<0.001), GSTs5 showed no significant difference among treatment groups (F 3,8 = 2.477, P = 0.136); CarE1 in LC 10 and LC 30 Significant increase in the treatment group (F) 3,8 = 647.127, P<0.001), CarE2 only in LC 30 Significantly upregulated (F) in the treatment group 3,8 = 18.960, P = 0.001).

[0083] The above results indicate that some detoxification enzymes (GST, CarE) and genes remain activated in the F1 generation, and this sustained response may be a potential consequence of impaired growth and development in offspring. This suggests that the sublethal effect of chlorfenapyr can be transmitted across generations and continuously activate the detoxification system in the F1 generation.

[0084] Example 9 This example illustrates the effect of sublethal concentrations of chlorfenapyr on population parameters of the F1 generation of peach borer.

[0085] 9.1 Construction of Age-Stage Life Tables for Both Sexes To construct age-stage life tables for the F1 generation, 100 eggs were selected from each treatment group and individually numbered and isolated. They were fed under identical conditions until pupation. Emerging adults continued to pair 1:1, and daily egg production and survival were recorded until all individuals died. Life table parameters were calculated using TWOSEX-MSChart software. The mean and standard error of intrinsic growth rate (r), cyclic growth rate (λ), net reproductive rate (R0), and mean generation cycle (T) were calculated using a bootstrap test with 100,000 resampling. The significance of differences between the control group and each treatment group was analyzed using a paired bootstrap test.

[0086] The effects of chlorfenapyr on the age-stage life table parameters of the F1 generation of peach fruit borer are shown in Table 2.

[0087] Table 2

[0088] As can be seen from the table, compared with the control group, the intrinsic growth rate, circumferential growth rate, and net reproductive rate all showed a decreasing trend in all treatment groups, and in the LC... 30 Significant differences were observed in the treatment groups. The mean generation cycle was in the LC... 10 and LC 30 The doubling time was significantly prolonged in the treatment groups. The corresponding population doubling time showed a trend of increasing in all treatment groups, and was particularly prolonged in the LC... 30 Significantly prolonged in the treatment group.

[0089] 9.2 Analysis of Age-Stage Characteristic Parameters Age-stage characteristic life expectancy (e xj The overall trend across all treatment groups was upward, see [link / reference]. Figure 9 As shown in Figure A; compared with the control group, l in each treatment group x The curve declines at a faster rate, indicating higher reproductive capacity (f) of females at a specific age. x ) and population-specific age-specific reproductive capacity (m x The peak value of ) also decreased significantly, see Figure 9 As shown in B.

[0090] Age-stage specific survival curves xj The results showed that, compared with the control group, the survival rate of each treatment group was reduced at different developmental stages. Figure 9 As shown in C; the corresponding age-stage reproductive value (v) xj The trend also showed a decrease across all treatment groups, see [link / reference]. Figure 9 As shown in D.

[0091] The above results indicate that these negative effects at the population level are a combined manifestation of the aforementioned developmental delays, decreased fertility, and metabolic disorders, suggesting that even sublethal concentrations of chlorfenapyr exposure can adversely affect the dynamics of the peach fruit borer population through a cumulative effect across generations. Sublethal chlorfenapyr treatment can significantly reduce the growth potential of the peach fruit borer population, providing a new strategy for the sustainable control of this pest.

[0092] The above provides a detailed description of the method and application for controlling peach fruit borer at a sub-lethal concentration of chlorfenapyr disclosed in this invention. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this invention.

Claims

1. A method for controlling peach fruit borer at a sub-lethal concentration of chlorfenapyr, characterized in that: Peach fruit borer adults were treated with a sublethal concentration of chlorfenapyr, the sublethal concentration being selected from LC1 and LC2. 10 and LC 30 At least one of them can be used to control the population by suppressing the reproductive capacity of the peach fruit borer.

2. The method according to claim 1, characterized in that, The sublethal concentration was LC1 (4.154 mg / L), LC 10 (12.143 mg / L) or LC 30 (26.416 mg / L).

3. The method according to claim 1 or 2, characterized in that: Peach borer adults were treated with a 10% sucrose aqueous solution containing chlorfenapyr for 72 hours.

4. The application of a sublethal concentration of chlorfenapyr in the population control of peach fruit borer, characterized in that: Peach fruit borer adults were treated with a sublethal concentration of chlorfenapyr, the sublethal concentration being selected from LC1 and LC2. 10 and LC 30 At least one of them is used to suppress the population growth of peach borer.

5. The application according to claim 4, characterized in that, The sublethal concentration was LC1 (4.154 mg / L), LC 10 (12.143 mg / L) or LC 30 (26.416 mg / L).

6. The application according to claim 4 or 5, characterized in that: Peach borer adults were treated with a 10% sucrose aqueous solution containing chlorfenapyr for 72 hours.

7. A population control agent for peach fruit borer, characterized in that: Contains an effective amount of chlorfenapyr and an agriculturally acceptable carrier, wherein the sublethal concentration of chlorfenapyr is selected from LC1, LC2, and LC4. 10 and LC 30 At least one of them.

8. The population control agent according to claim 7, characterized in that, The sublethal concentration was LC1 (4.154 mg / L), LC 10 (12.143 mg / L) or LC 30 (26.416 mg / L).

9. The population control agent according to claim 7 or 8, characterized in that, The carrier is a 10% sucrose aqueous solution.

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