Application of Validamycin A in the Control of Grass Moths

Abstract

This invention belongs to the field of crop pest control technology, specifically relating to the application of ValidamycinA in the control of the corn borer. ValidamycinA is used as a trehalase inhibitor in the control of the corn borer. Experiments have shown that the trehalase inhibitor ValidamycinA can increase the trehalose content in the corn borer, reduce its egg production, and shorten its lifespan. Based on the effects of ValidamycinA according to this invention, inhibitors or antagonists related to the trehalose metabolism pathway in the corn borer can be screened to limit its reproduction and lifespan, thereby controlling the corn borer. This invention also has potential application value in the development of novel, environmentally friendly insecticides for the corn borer.

Description

Technical Field

[0001] This invention belongs to the field of crop pest control technology, specifically involving the application of Validamycin A in the control of grassland moth. Background Technology

[0002] The meadow moth (Pyralidae) is a globally migratory pest belonging to the order Lepidoptera. After reaching the third instar, individual moths spin webs and their appetite increases dramatically, causing the most severe damage to plants, often leaving notched leaves or only the veins visible. At the end of the fifth instar, the larvae pupate in the soil. Adult meadow moths are highly selective in their host plants, often congregating around them to feed on nectar and supplement their nutrition.

[0003] Currently, chemical control remains the primary method for controlling the meadow moth. However, due to the long-term use of chemical agents, the moth has developed resistance to many types of pesticides. Reports indicate that the moth has developed varying degrees of resistance to the active ingredients of several insecticides, including organophosphates, pyrethroids, dimethoate, and spinosad. Therefore, finding safe and effective methods for controlling the meadow moth is of great significance.

[0004] The inventors discovered through experiments that Validamycin A (Jinggangmycin A, CAS: 37248-47-8) inhibits the activity of trehalase, thereby reducing the egg-laying rate and shortening the lifespan of the meadow moth. This is of great significance for the development of novel, pollution-free insecticides for the meadow moth. No related reports have been found to date.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide the application of Validamycin A in the control of grassland moths.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of Validamycin A as a trehalase inhibitor in the control of grass moth.

[0009] This invention also provides the application of Validamycin A in inhibiting oviposition or shortening the lifespan of adult grass moths.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] This invention, through experiments, found that the trehalase inhibitor Validamycin A can increase the trehalose content in the grass moth, reduce its egg production, and shorten its lifespan. Based on the effects of Validamycin A according to this invention, inhibitors or antagonists related to the trehalose metabolism pathway in the grass moth can be screened to limit its reproduction and lifespan, control the grass moth, and have potential application value in the development of novel, environmentally friendly insecticides for the grass moth. Attached Figure Description

[0012] Figure 1 To investigate the effects of different temperature treatments on the survival rate, pre-oviposition period, and oviposition rate of adult male and female grass moths;

[0013] Figure 2 and 3 The effects of different temperature treatments on the ovaries of the grassland moth;

[0014] Figure 4 The effect of different temperature treatments on trehalose content in fat bodies and ovaries of grassland borer;

[0015] Figure 5 The effect of different temperature treatments on the relative expression level of the TRET1 gene;

[0016] Figure 6 Effect of Validamycin A treatment on trehalose content in the ovaries of the meadow moth;

[0017] Figure 7 The effects of Validamycin A treatment on oviposition period, oviposition rate, and female lifespan in the meadow moth. Detailed Implementation

[0018] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0019] Materials and Methods

[0020] 1. Materials

[0021] Grass moth: This is a long-term artificially bred population in the Migratory Pests Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, which has been continuously bred for multiple generations. It is bred under conditions of 23±1℃, 75±10% humidity, and a photoperiod of 16:8h (L:D). Adult moths are bred and lay eggs in circular plastic cages (40cm×10cm), and are supplemented daily with a 10% (w / v) glucose solution.

[0022] 2. Effects of different temperature treatments on the grassland moth

[0023] 2.1 Effects of different temperature treatments on the survival rate, pre-oviposition period, and oviposition rate of adult female and male grass moths.

[0024] Three temperature gradients were set up: 16±1℃ (low temperature), 23±1℃ (control temperature), and 30±1℃ (high temperature). Adults within 8 hours of emergence were placed in pairs in 400mL plastic cups lined with gauze and immediately randomly transferred to artificial climate incubators with different rearing temperatures. The photoperiod was 16:8h (L:D) for all treatments, with 20 pairs per treatment. They were nutriently provided with 10% glucose solution (w / v) twice daily. The survival rate of male and female adults, pre-oviposition period, and oviposition rate were recorded daily. Results are shown below. Figure 1 .

[0025] Depend on Figure 1 It can be seen that the pre-oviposition period and oviposition duration of adult grass moths both shorten with increasing temperature. Figure 1 A and 1B); in addition, to some extent, increased temperature improved mating rate (1D); compared with 23℃, female oviposition was inhibited at both 16℃ and 30℃ ( Figure 1 E); meanwhile, mortality results showed that the mortality rates of both males (1C) and females increased with rising temperature (E); Figure 1 F).

[0026] 2.2 Effects of different temperature treatments on the ovaries of the grassland moth

[0027] Twenty pairs of adult moths treated at different temperatures were dissected at 3 and 7 days. The ovaries were placed in 1× phosphate-buffered saline (PBS, pH 7.4) and their morphology was observed under a stereomicroscope. The developmental grades (I, II, III, IV) of the adult moth ovaries were graded under a 0.8x stereomicroscope (Olympus-SZX7, Japan), and images were acquired using an Opton digital camera. The length and width of the ovarian ducts were measured, and the number of eggs in each ovarian duct was counted. The results are shown below. Figure 2 and Figure 3 .

[0028] Depend on Figure 2 It can be seen that after 3 days of treatment at different temperatures, the length of the ovarian tube of the meadow moth treated with high temperature was significantly greater than that of the ovarian tube treated with low temperature. Figure 2 A); however, after 7 days of treatment, the length of the ovarian duct treated at 23℃ was significantly greater than that treated at 16℃ and 30℃. Figure 2D); Meanwhile, after 3 days of high-temperature treatment, the number of eggs in the ovarian tubes of *Eriocheir sinensis* was significantly higher than that after low-temperature treatment (2B). However, after 7 days of treatment, the number of eggs in the ovarian tubes treated at 23℃ was significantly higher than that after treatment at 16℃ or 30℃ (D). Figure 2 E);

[0029] Ovarian grading analysis showed that ovarian development accelerated with increasing temperature, with ovaries graded as low as grade I after 3 and 7 days of hypothermia treatment. Figure 2 C and 2F, Figure 3 );

[0030] 2.3 Effects of different temperature treatments on fat bodies of grassland borer

[0031] Female moths treated at different temperatures had their fat body tissues dissected in 1×PBS and fixed overnight for 12 h with 4% paraformaldehyde (Beyotime, Beijing, China), followed by washing three times with 1×PBS for 5 min each time. Next, the samples were stained with Nile Red (19123, Sigma-Aldrich, St Louis, MO, USA) at a final concentration of 1 μg / mL for 90 min at room temperature, followed by washing three times with 1×PBS. The washed samples were transferred to glass slides and covered with coverslips. Images were taken using a laser scanning confocal microscope (LSM T-PMT, Carl Zeiss, Germany). Finally, the diameter of lipid droplets (LDs) in the fat body was measured using ImageJ software (National Institutes of Health, USA). The results are shown in [Figure number missing]. Figure 4 A.

[0032] Depend on Figure 4 As shown in A, by staining the fat bodies of female grass moths with Nile Red after 7 days of treatment at different temperatures, it was found that the diameter (LD) of fat droplets showed a significant decreasing trend with increasing temperature.

[0033] 2.4 Effects of different temperature treatments on trehalose content in the ovaries of the grassland moth

[0034] Ovarian tissues (n=5) from female moths treated at different temperatures for 3 and 7 days were collected. The trehalose content was then determined using a trehalose assay kit (Nanjing Jiancheng Research Institute, Nanjing, China). The ovarian tissues were homogenized in the extraction solution and centrifuged at room temperature for 45 min. The protein concentration in each sample was determined using the BCA method. 60 μL of tissue sample was mixed with 240 μL of working solution and incubated at 90 °C for 10 min. The absorbance at 630 nm was then measured using a microplate reader, and the trehalose content was determined based on the absorbance values. The results are shown below. Figure 4 B.

[0035] Depend on Figure 4As shown in B, the trehalose content in the female worm ovaries showed an increasing trend after 3 and 7 days of treatment, with the increase of temperature.

[0036] 2.5 Effects of different temperature treatments on the relative expression level of the TRET1 gene

[0037] Legs, head, thorax, fat body and ovarian tissue of female insects were collected after treatment at different temperatures for 3 days and 7 days (10 insects per sample, n=3) and immediately stored in a -80℃ freezer for later use.

[0038] Based on the Tracytoplasmic Stoker Ovary Transcriptome Database (NCBI SRA accession number: PRJNA1082620), the Unigene sequence was found by searching the database annotation files using keywords and comparing it with the NCBI website to obtain the TrehaloseTransporter (TRET1) gene sequence. Primers for qRT-PCR amplification were designed based on the obtained TRET1 gene ORF sequence for validation.

[0039] The above samples were prepared according to the instructions of TRIzol Reagent (Invitrogen) and total RNA was extracted. The quantity and purity of total RNA were quality controlled by NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) and the integrity of RNA was detected by Bioanalyzer 2100 (Agilent, CA, USA). Then, 1 μg of RNA was used to synthesize first-strand cDNA according to the instructions of the TAKARA kit. Primers for the TRET1 gene and two internal reference genes (eIF4a and 18S rRNA) were designed using NCBI online tools (Table 1).

[0040] Table 1 Primers used for qRT-PCR

[0041]

[0042] The synthesized cDNA was diluted 5-fold and its amplification efficiency was detected using the corresponding primers. Next, the cDNA sample was mixed with the TAKARA SYBR Premix EX Taq kit and subjected to 40 cycles of 95℃ for 30 s, 60℃ for 30 s, and 60℃ for 1 min using a CFX 96 (BioRad) instrument. A melting curve was then added, followed by cycles of 95℃ for 30 s and 60℃ for 15 s. Finally, the CT value was used to quantify the amplification efficiency using 2... -ΔΔCT The relative expression levels of genes were calculated using the method described above, and the results are shown in [the table]. Figure 5 .

[0043] Depend on Figure 5The tissue expression profile results show that the TRET1 gene is expressed most highly in the ovary, followed by the head. Figure 5 A); In addition, the expression level of TRET1 showed an upward trend with increasing temperature. Figure 5 B) indicates that increased temperature promotes the accumulation of trehalose, which in turn induces high expression of TRET1 to transfer trehalose.

[0044] 2.6 Effects of Trehalose on the Life History of the Grass Moth

[0045] Trehalose degradation was inhibited using a trehalase inhibitor (Validamycin A, Med Chem Express, China). The specific method is as follows:

[0046] Newly emerged female moths (within 8 hours of emergence) were selected. 2 μL of 5 mM Validamycin A was injected into the abdomen of the female moths. The control group received an equal volume of sterile ddH2O injected into the abdomen. The injected females were paired with males and placed in 400 mL plastic cups, then reared in an incubator at 23°C. Seven days after inhibitor treatment, ovarian tissue samples were collected using the aforementioned method, and trehalose content and the expression levels of key genes were measured according to the relevant kit instructions. Each treatment was performed in at least three biological replicates, with each replicate including at least 20 female moths. Results are shown below. Figure 6 and Figure 7 .

[0047] Depend on Figure 6 and Figure 7 It was found that treating female meadow moths with Validamycin A and detecting the trehalose content in their ovaries 7 days after treatment showed that the trehalose content in the meadow moth ovaries increased after inhibitor treatment. Figure 6 This indicates that the inhibitor can significantly inhibit trehalase activity. Furthermore, compared to the H2O injection control, the inhibitor treatment had no effect on the pre-oviposition period of the meadow moth. Figure 7 A). However, compared with the control group injected with H2O, the oviposition period of the meadow moth was shorter after inhibitor treatment ( Figure 7 B) Egg production ( Figure 7 C) and female lifespan ( Figure 7 D) were all significantly lower than the control group.

[0048] In summary, this invention has revealed changes in trehalose metabolism in *Pterygota pratensis* under temperature stress, clarified the tissue expression patterns of trehalose metabolism genes and their relationship with fat body metabolism, and further demonstrated that trehalase inhibitors suppress the reproduction and lifespan of female *Pterygota pratensis* by inhibiting trehalose metabolism. Experiments showed that the trehalase inhibitor Validamycin A can increase trehalose content in *Pterygota pratensis*, reduce egg production, and shorten lifespan.

[0049] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. Application of ValidamycinA as a trehalase inhibitor in the control of grass moth.

2. Application of Validamycin A in inhibiting oviposition or shortening the lifespan of adult grass moths.

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