Method for interfering with the growth of multilocular hydatid cyst based on diapause pheromone ascr#18 and application thereof

Abstract

The application discloses a method for interfering with the growth of multilocular echinococcosis based on diapause pheromone Ascr#18 and application thereof, and belongs to the technical field of prevention and control of parasitic zoonosis. daf-9 With daf-12 The application discloses a method for interfering with the growth of multilocular echinococcosis based on diapause pheromone Ascr#18 and application thereof, and belongs to the technical field of prevention and control of parasitic zoonosis. The application discloses a method for interfering with the growth of multilocular echinococcosis based on diapause pheromone Ascr#18 and application thereof, and belongs to the technical field of prevention and control of parasitic zoonosis. The application provides a novel biological prevention and control strategy which is independent of traditional chemical deworming drugs, has high targeting, high safety, is environment-friendly and controllable, and is suitable for comprehensive prevention and control of alveolar echinococcosis, and can also provide a theoretical basis and application reference for prevention and control of other cestode larva parasitic diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of parasitic prevention, and in particular to a method for interfering with the growth of multilocular hydatid based on diapause pheromone Ascr#18 and application thereof. BACKGROUND

[0002] Parasites are widely distributed in the world and pose a significant threat to human and animal health. Among them, trematodes, cestodes and nematodes belong to endoparasites, which often parasitize in the digestive tract, liver, lungs and other organs of the host, causing tissue damage, inflammation, secondary infection and other problems. These parasites not only seriously affect the growth and development and production performance of animals, but also can infect a variety of animals and humans, and their prevention and control have important veterinary and public health significance.

[0003] As an important group of Platyhelminthes, the adult of cestode mainly parasitizes in the intestinal tract of vertebrates, and the period of development in the intermediate host is called metacestode. The metacestode larvae mainly parasitize in the tissues and organs of the host, causing serious pathological damage. Echinococcus is one of the most clinically significant groups, and the hydatid disease caused by its larvae is listed as one of the 17 neglected tropical diseases in the world by the World Health Organization. According to different pathogen species, hydatid disease is mainly divided into cystic echinococcosis (caused by Echinococcus granulosus) and alveolar echinococcosis (caused by Echinococcus multilocularis).

[0004] Alveolar echinococcosis (AE) is a serious zoonotic parasitic disease caused by Echinococcus multilocularis larvae. The adult parasite lives in the intestinal tract of the final host, and the eggs are excreted with feces. Humans and rodents are infected by ingesting food or water contaminated with eggs, and the eggs develop into hydatid in the liver and other tissues of the intermediate host. When the final host preys on the tissues and organs containing hydatid, the protoscolex in the hydatid develops into adult in the intestinal tract of the final host, thereby completing its life cycle. Epidemiological data shows that AE is highly prevalent in Central Asia and western China.

[0005] The pathological features of multilocular echinococcosis are the formation of infiltrative space-occupying lesions in the liver, which can cause abdominal mass, pain, jaundice, and even liver failure. This disease has the characteristics of strong invasiveness, slow growth, and hidden symptoms, making early diagnosis difficult. When the disease progresses to the late stage, it can cause multiple organ metastasis, such as lung, brain, and bone, leading to systemic symptoms and threatening life. Currently, the main treatments for AE include surgical resection and conservative treatment with benzimidazole drugs (such as albendazole). However, surgical treatment has limitations such as high risk, high recurrence rate, etc., while drug treatment faces challenges such as poor efficacy, obvious side effects (such as liver function damage), and drug resistance. Long-term use of albendazole can cause serious systemic damage, and these treatment challenges need to be broken through. Therefore, developing new and efficient treatment options has become an important research direction in the field of AE prevention and treatment.

[0006] In recent years, research on the molecular mechanisms of parasite development regulation has provided important insights for the development of novel drugs. Among them, small molecules such as ascarosides (also known as diapause pheromones) have attracted widespread attention due to their unique signal regulation functions. The function of ascarosides was first studied in the model organism *Caenorhabditis elegans*. They are recognized by G protein-coupled receptors (GPCRs) in the nematode's sensory neurons, and subsequently activate a conserved diapause signaling pathway via second messengers such as cyclic guanosine monophosphate (cGMP), inducing diapause in the nematode. Studies have shown that this signaling pathway is highly conserved in various parasitic nematodes (such as *Haemaphysalis contortus*, *Toxocara canaliculata*, and *Strongyloides stercoralis*), especially the downstream DAF-9 (cytochrome P450, CYP450)–DA–DAF-12 (nuclear hormone receptor) module, whose crucial role in the developmental transition and infection processes of parasitic nematodes has been widely verified. In this conserved pathway, DAF-9 is a nematode-specific steroid synthesis-related cytochrome P450 enzyme, primarily responsible for synthesizing the steroid hormone ligand dafachronic acid (DA). DA acts as a ligand for DAF-12, binding to it and regulating the expression of a series of genes, participating in key shifts in nematode developmental decisions, such as dauer induction and termination, sexual maturation initiation, and tissue remodeling. In the absence of DA stimulation, DAF-12 binds to its co-repressor DIN-1, inducing daueration; while in the presence of DA, DAF-12 forms a transcriptional activation complex, promoting continued nematode development. This regulatory model plays a crucial role not only in free-living nematodes but also in various parasitic nematodes, acting as a similar "developmental switch," participating in the transition of larvae to the infective stage, tissue invasion within the host, and the establishment of chronic infection. Therefore, the DAF-9–DA–DAF-12 module is one of the core pathways for parasites to sense environmental signals, regulate their own development and adapt to the host, and also provides a potential new intervention target for targeting and blocking the parasite life cycle.

[0007] Meanwhile, diapause pheromones (Ascarosides) not only regulate the behavior and life cycle of parasites but also have the potential to modulate the host's immune system. Recent studies have found that Ascr#7 can significantly alleviate type II inflammatory responses by inhibiting interleukin-33 (IL-33) production and reducing pathogenic type 2 helper T cells (Th2) and type 2 innate lymphocytes (ILC2), showing promising applications in the treatment of allergic diseases and providing a live-worm-free alternative strategy for live worm therapy. In agriculture, diapause pheromones (Ascarosides) can also induce innate immune responses in plants, enhancing their resistance to parasites and pathogens.

[0008] However, the presence and physiological functions of diapause pheromones (Ascarosides) in tapeworms have not been reported, providing a crucial opportunity for original exploration in this study. Using tandem mass spectrometry (MRM) targeting detection technology, this invention, for the first time, confirms the presence of Ascarosides#18 (Ascr#18) in the protoscolex of *Echinococcus multilocularis*, and further confirms that it may inhibit protoscolex development and infectivity by regulating the conserved DAF-9–DA–DAF-12 homologous signaling cascade. This discovery not only fills the gap in research on Ascarosides in tapeworm parasites but also provides a novel molecular basis for the development of new targeted drugs against echinococcosis.

[0009] Current methods for controlling echinococcosis have significant limitations, failing to meet the demands for high efficiency, safety, and sustainability. Chemical anthelmintics (such as praziquantel and albendazole) are the primary treatment, but long-term use easily induces parasite resistance, contradicting the "antibiotic reduction and replacement" green approach. Their efficacy diminishes over time, and they have significant side effects and toxicity. Therefore, chemical drugs have limited inhibitory effects on the development and infectivity of the protoscolex, making it difficult to effectively interrupt the parasite's life cycle. Existing small biological molecules, such as anacardic acid, nitazoxanide, small interfering RNA (siRNA), and exosomal microRNAs (miRNA), while possessing some antiparasitic potential, face challenges such as toxicity risks (e.g., skin sensitization of anacardic acid), insufficient in vivo validation (e.g., lack of animal experimental data for siRNA), technical complexity (e.g., poor stability of exosomal delivery), and unclear efficacy, limiting their clinical translation. Furthermore, there are currently no commercially available vaccines against Echinococcus multilocularis, and experimental vaccines (such as multi-epitope vaccines) have not yet been clinically applied due to issues with antigen selection and the persistence of immunity.

[0010] Currently, there is a particular lack of small biomolecules capable of targeting the development and infectivity of echinococcosis. The purpose of this invention is to provide a small molecule intervention strategy based on parasite-derived ascarosides. This strategy specifically inhibits the development and infectivity of the protoscolex by targeting and regulating the DAF-9–DA–DAF-12 signaling cascade of *Echinococcus multilocularis*, thereby blocking the parasite's life cycle. This strategy does not rely on chemical anthelmintics, avoiding the risks of drug resistance and toxicity. It features high specificity, safety, and environmental friendliness, is harmless to livestock, poultry, and humans, and aligns with green ecological principles, providing a green and innovative biotechnological solution for the prevention and control of *Echinococcus multilocularis*. Summary of the Invention

[0011] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method and application for intervening in the growth of *Echinococcus multilocularis* based on the diapause pheromone Ascr#18. Based on the identification and utilization of Ascaroside, a small chemical molecule naturally present in *Echinococcus multilocularis*, the following approach is proposed: First, mass spectrometry analysis confirms the presence of Ascr#18 within *Echinococcus multilocularis* for the first time, and molecular docking technology identifies its potential G protein-coupled receptor (GPCR), demonstrating its signal transduction potential. Then, qPCR experiments confirm that Ascr#18 treatment of the protoscolex significantly downregulates the expression of key downstream genes daf-9 and daf-12 in the diapause signaling pathway, thereby intervening in its developmental regulatory pathway. Simultaneously, protoscolex activity assays verify that Ascr#18 treatment significantly reduces its activity. Based on this, this invention utilizes Ascr#18 to act on the GPCR–DAF-9–DAF-12 signaling axis of the parasite, targeting and inhibiting the development and infection process of the prostomium of *Echinococcus multilocularis*, thereby blocking its life cycle and providing a novel biocontrol strategy that is highly specific, safe, and environmentally friendly. This approach not only responds to the advocacy of "antibiotic reduction and ban" but also provides a completely new technical pathway for the comprehensive prevention and control of *Echinococcus multilocularis* (vesicular) echinococcosis.

[0012] The objective of this invention is achieved through the following technical solution: a method for intervening in the growth of Echinococcus multilocularis based on the diapause pheromone Ascr#18, by adding exogenous Ascr#18, which can bind to the G protein-coupled receptor GPCR of Echinococcus multilocularis, downregulate the expression level of downstream daf-9 / daf-12 genes, and inhibit the development and infectivity of the prostomium of Echinococcus multilocularis.

[0013] Furthermore, the Ascr#18 natural small molecule present in the prostomium of Echinococcus multilocularis serves as an endogenous signaling molecule of the parasite, capable of binding to GPCR receptors to transmit signals and participate in the regulation of downstream developmental processes.

[0014] Furthermore, by utilizing Ascr#18 to act on the parasite's GPCR–daf-9–daf-12 signaling axis, the development and infection process of the prostomium of Echinococcus multilocularis were inhibited, thereby blocking the life cycle.

[0015] Furthermore, with increasing concentrations of exogenous Ascr#18, the inhibitory effect on the development and infectivity of Echinococcus multilocularis protoscolex was enhanced.

[0016] Furthermore, real-time PCR primers targeting the daf-9 and daf-12 genes were designed to analyze gene expression changes in Echinococcus multilocularis larvae after treatment with exogenous Ascr#18.

[0017] Furthermore, the qPCR primers for the daf-9 gene are: upstream primer: SEQ ID NO.1: 5'-AATGTGGTGAGCCCGTAAA-3'; downstream primer: SEQ ID NO.2: 5'-AGCAGTTCGTCAACCGTATC-3'.

[0018] Furthermore, the qPCR primers for the daf-12 gene are: upstream primer: SEQ ID NO.3: 5'-TGTTCGCCTTGTCGTCTTT-3'; downstream primer: SEQ ID NO.4: 5'-CTGAATCTTCTCCCTTCGTTCTC-3'.

[0019] Secondly, the present invention also provides the application of the diapause pheromone Ascr#18 in inhibiting the motility activity of the prostomium of Echinococcus multilocularis.

[0020] Thirdly, the present invention also provides the application of the diapause pheromone Ascr#18 in the preparation of a biological agent for the prevention and treatment of alveolar echinococcosis (AE).

[0021] The beneficial effects of this invention are:

[0022] This invention presents a biological technology solution that effectively blocks the development and infectivity of Echinococcus multilocularis larvae without relying on chemical anthelmintics. Based on the "signal sensing – developmental regulation – infection establishment" process of Echinococcus multilocularis larvae, this solution utilizes, for the first time, the naturally occurring small molecule pheromone Ascr#18 as a core intervention factor to target and regulate the GPCR–daf-9–daf-12 signaling axis, thereby blocking larval development and infectivity. It exhibits good targeting and biosafety.

[0023] Among them, Ascr#18 was identified as an endogenous small molecule signaling molecule in the parasite by liquid chromatography-mass spectrometry (LC-MS); molecular docking prediction showed that it can bind with high affinity to the G protein-coupled receptor GPCR and mediate signal transduction; Ascr#18 treatment significantly downregulated the expression levels of the key developmental genes daf-9 and daf-12 in the protoscolex; and the motility of the protoscolex treated under in vitro culture conditions was significantly reduced, indicating a significant weakening of infectivity. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is an Ascr#18 mass spectrometry identification image.

[0026] Figure 2 This is a schematic diagram showing the docking and binding energy between Ascr#18 and the G protein-coupled receptor GPCR molecule.

[0027] Figure 3 This is a schematic diagram showing the activity detection of protoscolex after treatment with different concentrations of Ascr#18 for 24 hours.

[0028] Figure 4 This is a schematic diagram showing the transcriptional levels of the daf-9 and daf-12 genes in the protoscolex after 24 hours of treatment with different concentrations of Ascr#18. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are merely illustrative and not intended to limit the invention.

[0030] This invention addresses the current lack of targeted small molecule technologies for controlling Echinococcus multilocularis infection by addressing the parasite's development and infectivity. It proposes an innovative intervention strategy based on the natural small molecule Ascaroside (Ascr#18). Ascaroside-like small molecules, as nematode-derived diapause pheromones, are known to regulate various developmental processes in nematodes, suggesting that Ascr#18 may possess similar signaling molecular functions within Echinococcus multilocularis.

[0031] Currently, ascarosides are widely studied in free-living nematodes (such as *Caenorhabditis elegans*), but systematic identification in *Echinococcus multilocularis* larvae is lacking. This invention utilizes liquid chromatography-mass spectrometry (LC-MS) to isolate and identify Ascr#18 from in vitro culture extracts of *Echinococcus multilocularis* larvae. The experiment employed a high-resolution mass spectrometer (Q Exactive Orbitrap), and comparison of the mass spectra with known standard spectra confirmed that the molecular structure (molecular weight, fragment ion pattern) of Ascr#18 was consistent with literature reports. The results indicate that Ascr#18 exists in the prostomium of *Echinococcus multilocularis* larvae. It is an endogenous signaling molecule in the parasite and can participate in the regulation of downstream developmental processes. This invention is the first to confirm the existence of Ascr#18 in *Echinococcus multilocularis* larvae, laying the foundation for subsequent functional studies. Figure 1 ).

[0032] Secondly, the binding affinity of Ascr#18 to the G protein-coupled receptor GPCR of *Echinococcus multilocularis* was predicted using molecular docking technology based on structural biology (software: AutoDock Vina 1.1.2). Three-dimensional prediction of the *Echinococcus multilocularis* GPCR protein was performed using the AlphaFold 3 website to simulate the interaction between Ascr#18 and the receptor binding site. Figure 2 (Principle block diagram). Docking results showed that Ascr#18 has a high affinity for a specific GPCR receptor (sequence ID: EmuJ_001171500) (binding energy -7.7 kcal / mol), indicating that it may transmit signals via GPCR. Figure 2 ).

[0033] To clarify the specific role and mechanism of Ascr#18 in Echinococcus multilocularis, protoscolex of Echinococcus multilocularis were isolated from gerbils (Meriones unguiculatus), evenly spread into 24-well plates, and cultured in DMEM medium. The plates were incubated at 37°C in a 5% (v / v) CO2 incubator. After treatment with Ascr#18 (concentrations: 100 nM, 10 μM) for 24 h, protoscolex activity was measured using a wMicroTracker ONE worm activity analyzer for 30 min, recording the movement distance per minute and the sucker contraction frequency. The results showed that the activity of protoscolex in the Ascr#18 treatment group was significantly reduced, and the inhibitory effect increased with increasing concentration, indicating that the addition of Ascr#18 reduced the activity of the protoscolex and inhibited its infectivity. Figure 3 ).

[0034] Simultaneously, qPCR primers targeting the daf-9 (cytochrome P450, CYPs) and daf-12 (nuclear hormone receptor) genes were designed using real-time quantitative PCR (daf-9: upstream primer: SEQ ID NO.1: 5'-AATGTGGTGAGCCCGTAAA-3'; downstream primer: SEQ ID NO.2: 5'-AGCAGTTCGTCAACCGTATC-3'; daf-12: upstream primer: SEQ ID NO.3: 5'-TGTTCGCCTTGTCGTCTTT-3'; downstream primer: SEQ ID NO.4: 5'-CTGAATCTTCTCTCCCTTCGTTCTC-3') to analyze gene expression changes in *Echinococcus multilocularis* larvae after Ascr#18 addition treatment. Protoscolex were collected and total RNA was extracted from the larvae. qPCR was used to detect the gene expression levels of daf-9 and daf-12 (instrument: LightCycler® 480 Instrument II). The results showed that, compared with the blank control, the expression levels of daf-9 and daf-12 in the Ascr#18 treatment group were significantly downregulated (P < 0.05), indicating at the transcriptional level that Ascr#18 inhibited the parasite development signaling pathway. Figure 4 ).

[0035] In summary, this invention identifies Ascr#18 in *Echinococcus multilocularis* larvae using mass spectrometry, verifies its binding to the GPCR receptor using protein structure modeling and molecular docking, and confirms its inhibition of protoscolex development and infectivity by downregulating daf-9 / daf-12 expression levels using activity assays and qPCR experiments. This demonstrates a highly efficient, specific, and safe biocontrol strategy. This approach overcomes the limitations of chemical drug resistance, toxicity, and difficulties in the transformation of existing small biological molecules, providing an innovative biotechnological pathway for the comprehensive control of *Echinococcus multilocularis* disease.

[0036] This invention utilizes liquid chromatography-high resolution mass spectrometry to identify the natural small molecule Ascr#18 within *Echinococcus multilocularis* larvae for the first time. Molecular docking of Ascr#18 with the *Echinococcus multilocularis* G protein-coupled receptor (GPCR) demonstrates its potential for downstream signal transduction. Based on this, qPCR primers targeting daf-9 and daf-12 were designed, and gene expression levels in protoscolex treated with Ascr#18 were detected. The results showed that the expression of both key developmental genes was significantly downregulated, confirming that Ascr#18 can inhibit *Echinococcus multilocularis* development through the GPCR–daf-9–daf-12 signaling axis. The effect of Ascr#18 on protoscolex motility was assessed by measuring protoscolex activity, revealing that it can reduce protoscolex motility and contraction frequency in a concentration-dependent manner, indicating that Ascr#18 effectively reduces the infectivity of *Echinococcus multilocularis* larvae.

[0037] This invention identifies the natural pheromone Ascr#18 in Echinococcus multilocularis larvae based on liquid chromatography-mass spectrometry (LC-MS). Molecular docking confirms that Ascr#18 can bind with high affinity to GPCRs, demonstrating its potential for signal transduction. Further qPCR and protoscolex activity detection confirm that Ascr#18 can downregulate the expression of genes in the daf-9 / daf-12 developmental pathway and significantly inhibit protoscolex motility, thus confirming that Ascr#18 plays a key role in inhibiting the development and infectivity of Echinococcus multilocularis protoscolex.

[0038] Example 1: Extraction and identification of Echinococcus multilocularis Ascr#18

[0039] (1) Isolation and purification of protoscolex

[0040] Echinococcal cysts adhering to the abdominal cavity and tissues were harvested from passaged long-clawed gerbils. The cysts were minced and placed on a 180-micron filter screen. During this process, PBS buffer containing 1× penicillin-streptomycin (PS) was continuously added, and the cysts were ground using a syringe stopper. The protoscolex suspension obtained by filtration was collected in a beaker. PBS solution was added repeatedly, and the mixture was allowed to stand. The supernatant was discarded, and the precipitate was collected. This filtration and collection process was repeated approximately five times until the protoscolex mixture precipitated.

[0041] (2) Mass spectrometry sample pretreatment

[0042] Add 500 μL of methanol and 2 steel beads to the sample tube, grind twice, 3 min each time, then centrifuge for 15 min (4) (℃, 20000 rpm), transfer the supernatant to a new EP tube, freeze-dry, reconstitute with 200 μL methanol, and centrifuge again for 15 min (4 ℃). o(C, 20000 rpm), collect the supernatant in an LC injection vial for LC-MS / MS analysis.

[0043] (3) Mass spectrometry analysis

[0044] Chromatographic conditions included column (Agilent EC-C18; 2.1 × 100 mm, 2.7 μm), column temperature (30 ℃), injection volume (20 μL), mobile phase (A: 5 mmol / L ammonium fluoride aqueous solution; B: acetonitrile), and mass spectrometry conditions in multiple reaction monitoring (MRM) and negative ion mode. The ion source was an electrospray ionization source (ESI) with an ion source temperature (TEM) of 400 ℃, a spray voltage (IS) of -4500 V, and a curtain gas (CUR) of 35 psi in negative ion mode.

[0045] Test results as follows Figure 1 As shown.

[0046] Example 2: Validation of Echinococcus multilocularis Ascr#18 as a diapause signaling molecule

[0047] Ascr#18 and GPCRs receptor molecular docking screening

[0048] The three-dimensional protein structures of *Echinococcus multilocularis* GPCRs were obtained in advance using AlphaFold3 simulation. The proteins were then preprocessed using the "Prepwiz" module of the Schrödinger software. This involved modeling calculations, bond sequence assignment, charge assignment, addition of hydrogen atoms, and creation of missing side chains or ring atoms to transform the target protein into a form capable of docking with ligands.

[0049] Download the chemical structure of Ascr#18 from PubChem. Using the "LigPrep" module of the Schrödinger software, convert the ligand file into the lowest-energy three-dimensional structure, and perform standardization processes such as hydrogenation, charge addition, and molecular conformation generation to facilitate subsequent docking.

[0050] Using the prepared GPCRs protein as the acceptor and Ascr#18 molecule as the ligand, protein-molecule docking was performed using AutoDock Vina 1.1.2 software, and the docking score table was output. PyMOL V2.5 (Schrödinger) software was used. The docking results were visualized and analyzed. The results are as follows: Figure 2 As shown.

[0051] Example 3: Effect of exogenous Ascr#18 addition on the infectivity of protoscolex

[0052] Activity determination of the protoscolex after Ascr#18 addition treatment

[0053] The protoscolex was evenly spread into 24-well plates and DMEM culture medium was added. The plates were then cultured in vitro at 37°C in an incubator containing 5% (v / v) CO2. After treatment with Ascr#18 (concentration: 100 nM, 10 μM) for 24 h, the protoscolex activity was measured using a worm activity meter (wMicroTracker ONE) for 30 min. The movement distance and sucker contraction frequency of the protoscolex per minute were recorded.

[0054] The obtained activity values ​​were entered into Microsoft Excel for relative quantitative analysis. Significance analysis was performed using t-tests and one-way ANOVA, and graphs were created using GraphPad Prism 9.5 software. The results are shown below. Figure 3 As shown.

[0055] Example 4: Effects of exogenous Ascr#18 addition on protoscolex development

[0056] (1) In vitro culture of protoscolex

[0057] The worms were evenly spread into 24-well plates and 500 μl of DMEM culture medium containing 1× penicillin-streptomycin was added. The plates were then cultured in vitro at 37°C in an incubator containing 5% CO2 (volume fraction). After treatment with Ascr#18 (concentration: 100 nM, 10 μM) for 24 h, the worms in the blank group and the experimental treatment group were collected.

[0058] (2) Total RNA extraction and reverse transcription reaction

[0059] RNA was extracted from the protostomes of each group using the RNeasy Plus Mini Kit (Qiagen). An appropriate amount of RNase-free water was added to dissolve the precipitate, and quality control was performed using a micro-nucleic acid analyzer and RNA gel electrophoresis. Subsequently, 1 microgram of RNA was used for reverse transcription using the FSQ-301 reagent.

[0060] (3) Real-time PCR

[0061] Using the Em-β-actin gene as an internal control, real-time quantitative PCR (qRT-PCR) was performed using specific primers.

[0062] The qPCR reaction system consisted of 10 μL ChamQ Universal SYBR qPCR Master Mix, 0.4 μL upstream primer, 0.4 μL downstream primer, 1 μL cDNA template, and 8.2 μL RNase-free water. The qPCR reaction conditions were: pre-denaturation at 95℃ for 30 seconds, followed by cycling (95℃ for 10 seconds, 60℃ for 10 seconds, 40 cycles); the melting curve reaction conditions were: 95℃ for 15 seconds, 60℃ for 60 seconds, and 95℃ for 15 seconds.

[0063] The obtained cyclic threshold (CT value) was calculated using Microsoft Excel in 2... -ΔΔCT Relative quantitative analysis was performed using the value comparison method, and significance analysis was conducted using the t-test and one-way ANOVA. GraphPad Prism 9.5 software was used for plotting to analyze the differences in transcriptional levels of daf-9 and daf-12 genes under different concentrations of Ascr#18 addition conditions. The results are as follows: Figure 4 As shown.

[0064] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. The application of a diapause pheromone Ascr#18 in the preparation of a biological agent for inhibiting the motility activity of the prostomium of *Echinococcus multilocularis*, characterized in that, In biological agents, exogenous Ascr#18 binds to the G protein-coupled receptor GPCR of Echinococcus multilocularis, downregulating downstream [receptors]. daf-9 / daf-12 Gene expression levels inhibit the development of the prostomium and infectivity of Echinococcus multilocularis larvae.

2. The application of the diapause pheromone Ascr#18 according to claim 1 in the preparation of a biological agent for inhibiting the motility activity of the prostomium of *Echinococcus multilocularis*, characterized in that... Ascr# was present in the prostomium of the multilocular echinococcosis. 18 natural small molecules, as endogenous signaling molecules in parasites, can bind to GPCR receptors to transmit signals and participate in the regulation of downstream developmental processes.

3. The application of the diapause pheromone Ascr#18 according to claim 1 in the preparation of a biological agent for inhibiting the motility activity of the prostomium of *Echinococcus multilocularis*, characterized in that... Using Ascr#18 to act on parasite GPCRs – daf-9 – daf-12 The signal axis targets and inhibits the development and infection process of the prostomium of Echinococcus multilocularis, thereby blocking the life cycle.

4. The application of the diapause pheromone Ascr#18 according to claim 1 in the preparation of a biological agent for inhibiting the motility activity of the prostomium of *Echinococcus multilocularis*, characterized in that... As the concentration of exogenous Ascr#18 increases, the inhibitory effect on the development of the prostomium and infectivity of Echinococcus multilocularis larvae is enhanced.

5. The application of the diapause pheromone Ascr#18 according to claim 1 in the preparation of a biological agent for inhibiting the motility activity of the prostomium of *Echinococcus multilocularis*, characterized in that... Design for daf-9 and daf-12 Real-time quantitative PCR primers were used to analyze gene expression changes in Echinococcus multilocularis larvae after treatment with exogenous Ascr#18.

6. The application of the diapause pheromone Ascr#18 according to claim 5 in the preparation of a biological agent for inhibiting the motility activity of the prostomium of *Echinococcus multilocularis*, characterized in that... daf-9 The qPCR primers for the gene are: upstream primer: SEQ ID NO.1: 5'-AATGTGGTGAGCCCGTAAA-3'; downstream primer: SEQ ID NO.2: 5'-AGCAGTTCGTCAACCGTATC-3'.

7. The application of the diapause pheromone Ascr#18 according to claim 5 in the preparation of a biological agent for inhibiting the motility activity of the prostomium of *Echinococcus multilocularis*, characterized in that... daf-12 The qPCR primers for the gene are: upstream primer: SEQ ID NO.3: 5'-TGTTCGCCTTGTCGTCTTT-3'; downstream primer: SEQ ID NO.4: 5'-CTGAATCTTCTCCCTTCGTTCTC-3'.

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