A method for screening and identifying an odor binding protein for regulating host positioning of parasitic wasps in biological control and function verification

Abstract

This invention relates to a method for screening, identifying, and validating the function of odor-binding protein genes that regulate the host localization behavior of parasitic wasps. It addresses the shortcomings in existing technologies regarding the insufficient understanding of the molecular mechanisms of host localization in parasitic wasps, particularly the lack of systematic research on the genes and functions of parasitic wasp odor-binding proteins (OBPs). The method includes S1, genome identification and characterization; S2, tissue-specific expression analysis; S3, gene function verification; and S4, electrophysiological and molecular binding mechanisms. The advantages of this invention are: by developing attractants targeting specific OBP proteins, natural enemies can be guided to precisely locate pests, reducing the use of chemical pesticides and meeting the sustainable development requirements of modern green agriculture and biological control; it can serve as a high-throughput molecular platform for screening parasitic wasp attractants or behavioral interventions, contributing to the development of novel natural enemy adjuvants and improving the field efficiency of biological control.

Description

Technical Field

[0001] This invention relates to the field of bioinformatics, specifically to a method for screening, identifying, and verifying the function of odor-binding proteins that regulate host localization in parasitic wasps during biological control. Background Technology

[0002] With the continuous expansion of global agricultural production, the economic losses caused by fly pests to fruit and vegetable crops are becoming increasingly serious. While traditional control methods, primarily relying on chemical pesticides, offer high short-term efficacy, long-term use easily leads to increased pesticide resistance in pests, environmental pollution, and damage from non-target organisms, making them unsuitable for green agriculture and sustainable development. Therefore, developing safe, efficient, and environmentally friendly biological control technologies for pests has become an important research direction in the field of integrated pest management in agriculture. Parasitic wasps, as important natural enemies of many agricultural pests, play an irreplaceable role in biological control systems. Specifically, parasitic wasps can effectively suppress fly populations by parasitizing fly larvae, making them an important biological control resource for fruit flies. The success of parasitic wasp behavior highly depends on their precise ability to locate the host and its habitat, a process primarily mediated by their highly developed olfactory system.

[0003] The molecular basis of insect olfactory perception mainly relies on a series of olfactory-related proteins in the peripheral receptors on the antennae. Among them, odorant-binding proteins (OBPs) are a class of small, soluble proteins widely present in the lymph of the antennal receptors. Their main function is to bind and transport hydrophobic volatile odor molecules in the environment, enabling them to reach and activate olfactory receptors, thereby initiating the olfactory signal transduction process. As OBPs are located at the very beginning of the olfactory perception chain, they play a crucial "molecular bridge" role in odor recognition, selectivity, and sensitivity regulation.

[0004] Existing research has shown that olfactory genes (OBPs) play important roles in the feeding, mating, oviposition, and host localization behaviors of various insects, and different OBPs often exhibit high selectivity for specific odor molecules. However, compared with model insects, research on the olfactory molecular mechanisms of parasitic wasps is relatively lagging. Especially for important biological control insects such as *Gallus circinus*, their olfactory-related gene resources have not been systematically explored, and the composition, chromosomal distribution characteristics, and evolutionary relationships of the OBP gene family remain unclear. Furthermore, in-depth research on key OBP genes involved in the recognition of host-related chemical cues and their mechanisms of action is lacking. Therefore, it is necessary to conduct a systematic study of the *Gallus circinus* OBP gene family at the genomic and molecular functional levels to clarify their role in the host localization process, providing a new theoretical foundation and technical support for the precision and molecularization of biological control technologies for pests. Summary of the Invention

[0005] The purpose of this invention is to address the insufficient analysis of the molecular mechanisms of host localization in parasitic wasps in the existing technology, especially the lack of research on the genes and functional systems of olfactory receptor-binding proteins (OBPs) in *Gallus circumbellatus*. This invention proposes a method for screening, identifying, and verifying the function of odor-binding proteins that regulate host localization in parasitic wasps during biocontrol, based on a combination of genomics, expression profiling, and molecular functional verification. The aim is to reveal the molecular basis for *Gallus circumbellatus* to recognize its host and its habitat chemical signals.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a method for screening, identifying, and functionally verifying odor-binding proteins that regulate host localization in parasitic wasps during biological control, comprising the following steps: S1. Genome identification and characterization: Eighteen OBP genes were identified in the genome of the ring-bellied gall wasp, including 17 classical OBPs and 1 Minus-C OBP. These genes are unevenly distributed on four chromosomes and form distinct gene clusters on chromosomes 1 and 3. S2. Tissue-specific expression analysis: Through transcriptome sequencing (TPM analysis) and qPCR verification, multiple genes were found to be tissue-specific. S3. Gene function verification: RNA interference technology was used to inhibit the expression of OBP4, which led to a significant reduction in the female bee's preference for yeast attractants in the host's living environment. S4. Electrophysiological and molecular binding mechanism: Electrophysiological (EAG) tests showed that after OBP4 was interfered with, female bees lost their electrophysiological response to key compounds of ethyl butyrate.

[0007] In the above-mentioned method for screening, identifying, and verifying the function of odor-binding proteins used to regulate host localization in parasitic wasps during biological control, step S1 includes the following steps: S1-1, Candidate Gene Mining: Based on chromosome-level whole genome data of *Gallus circumbellatus*, we used the known conserved OBP domain of insects (Pfam ID: PF00067) as a retrieval model, combined with homology comparison BLASTP to search for potential OBP sequences throughout the whole genome; S1-2, Structural Classification and Confirmation: The conserved domains of candidate sequences were confirmed using the CD-search online tool in the NCBI Conserved Domain Database (CDD), and the N-terminal signal peptide sequence was predicted using SignalP 5.0 to determine whether it possesses typical secretory protein characteristics. Based on amino acid sequence length, the distribution pattern of conserved cysteine ​​(Cys) sites, and the presence of the signal peptide, candidate OBP proteins were structurally classified and manually corrected. Specifically, based on the number and arrangement characteristics of conserved cysteine ​​residues, the OBP family was divided into different subtypes such as Classic, Minus-C, and Plus-C to improve the accuracy of gene annotation and functional prediction. S1-3. Chromosomal Localization and Gene Cluster Analysis: Chromosomal location information of the identified OBP genes, including chromosome number, start site, and termination site, was extracted from the genome annotation file (GFF / GTF). MapChart software was used to map all OBP genes onto a physical chromosome map, demonstrating their distribution characteristics and localization relationships on different chromosomes, and analyzing whether there is any chromosomal bias in their distribution.

[0008] In the above-mentioned method for screening, identifying, and functionally verifying odor-binding proteins for host localization in parasitic wasps during biological control, step S2 specifically includes the following steps: S2-1. Transcriptome Sequencing and Expression Abundance Calculation: Total RNA was extracted from the antennae, head (antennae removed), thorax, abdomen, and legs of adult bees. RNA integrity was tested using an Agilent 2100 Bioanalyzer to ensure RNA quality met subsequent sequencing requirements. Qualified RNA samples underwent mRNA enrichment, fragmentation, cDNA synthesis, and library construction, followed by high-throughput transcriptome sequencing on the Illumina NovaSeq platform. After obtaining the raw sequencing data, the raw reads were first assessed for quality using FastQC, and then fastp was used to remove adapter sequences, low-quality sequences, and reads with a high proportion of nitrogen (N) to obtain high-quality clean reads. The clean reads were then aligned to the *Gallus gallus domesticus* reference genome using HISAT2 software. Based on the reference genome annotation file, RSEM software was used to quantitatively analyze the expression levels of each gene, and TPM (Transcripts Per Million) was used as a standardized expression abundance index to eliminate the influence of differences in sequencing depth and gene length.

[0009] Further comparative analysis of OBP gene expression profiles across different tissues was conducted, and expression patterns were visualized by creating heatmaps. Candidate OBP genes with high expression levels and significant tissue specificity in antennal tissues were preliminarily screened, providing a basis for subsequent functional verification.

[0010] S2-2, Key Gene Screening: Based on the transcriptome expression data of male and female adult bee antennae, differential expression analysis of OBP genes was performed. First, OBP genes with high TPM values ​​(e.g., TPM > 50 or 100) in antennal tissue were screened to ensure strong expression activity of candidate genes. Then, the expression differences between male and female antennae were compared, focusing on genes significantly overexpressed in female bee antennae. Differential expression analysis was performed using DESeq2 software, with screening criteria set as |log2FoldChange| ≥ 1 and a significance level of p < 0.05.

[0011] Simultaneously, based on tissue expression specificity analysis results, OBP genes that are highly expressed in the antennae of female bees but lowly expressed or almost not expressed in the head, thorax, abdomen, and legs were prioritized for screening to improve the reliability of candidate genes related to olfactory recognition function. Finally, by comprehensively considering expression abundance, sex-biased expression characteristics, and tissue-specific expression patterns, 10 key OBP genes that may be involved in the host localization and odor recognition process of female bees were identified for subsequent qRT-PCR validation, RNAi interference, and functional analysis. S2-3. Quantitative PCR (qPCR) verification: Specific primers were designed for candidate genes, with tubin and β-actin as internal reference genes, and their spatial expression patterns in different tissues were detected by real-time quantitative PCR technology. In the above-mentioned method for screening, identifying, and functionally verifying odor-binding proteins for host localization in parasitic wasps during biological control, step S3 specifically includes the following steps: S3-1, dsRNA synthesis and microinjection: Primers were designed targeting specific regions of the OBP4 sequence to synthesize double-stranded RNA (dsRNA) through in vitro transcription; the dsRNA was then injected into the prepupal stage of *Gallus circumbellatus* under a microscope using a microinjection system to achieve efficient gene knockdown. S3-2, Behavioral Preference Evaluation (Y-tube): After the individuals in the interference group (dsOBP4) and the control group (dsGFP) emerged, they were placed in a Y-tube olfactometer to test their selection ratio for host-related odors; S3-3, Long-distance positioning assessment (wind tunnel experiment): Simulate natural flight conditions in a controlled wind tunnel environment and record the behavioral trajectory of parasitic wasps flying towards the odor source.

[0012] In the above-mentioned method for screening, identifying, and functionally verifying odor-binding proteins for host localization in parasitic wasps during biological control, step S4 specifically includes the following steps: S4-1, Electrophysiological response test (EAG): The antennae of the female bees in the interference group were detected to respond to 27 host-source volatiles using an antennae potential coupled with an antennae potential (EAG) instrument. The signal intensity was recorded and variance analysis was performed. S4-2, Protein Structure Modeling: The tertiary structure of the OBP4 protein was predicted using AlphaFold2 software, yielding multiple candidate protein structure models. Based on the pLDDT (predicted Local Distance Difference Test) score and PAE (predicted aligned error) matrix of the predicted models, the overall confidence and local structural reliability of the models were comprehensively evaluated, and the optimal model with the highest confidence was selected for subsequent analysis. Subsequently, PyMOL software was used to visualize the overall spatial structure of the protein, and the signal peptide region was appropriately processed to highlight the main structure of the mature protein and its internal hydrophobic binding cavity.

[0013] To further validate the model quality, the PROCHECK and ERRAT programs in the SAVES v6.0 online platform were used to assess the structural rationality of the model. PROCHECK was used to analyze the distribution of the protein Ramachandran plot, calculating the proportion of amino acid residues located in the most favored regions, additional allowed regions, and disallowed regions to evaluate the rationality of the protein's stereostructure. Generally, a model is considered to have high reliability when more than 90% of the residues are located within the allowed regions.

[0014] Simultaneously, the ERRAT program was used to perform statistical analysis on protein non-bonded interactions, and the stability of the model and the reliability of local structures were evaluated by calculating the overall quality factor. When the ERRAT score was higher than 90, it indicated that the overall structural quality of the model was high and suitable for subsequent molecular docking analysis. In addition, the integrity of the protein α-helix structure, the formation of hydrophobic binding cavities, and the spatial distribution of conserved sites were further combined to comprehensively evaluate the OBP4 model to ensure that it can truly reflect the odorant molecule binding characteristics of the OBP protein.

[0015] S4-3. Molecular docking and binding site identification: Molecular docking analysis of OBP4 protein and ethyl butyrate was performed using AutoDock Vina software. First, the small molecule three-dimensional structure of ethyl butyrate was obtained from the PubChem database, and OpenBabel software was used for structure format conversion and energy minimization. Subsequently, the OBP4 protein model was preprocessed, including water molecule removal, addition of polar hydrogen atoms, and charge correction, to improve docking accuracy.

[0016] The docking grid range is set according to the location of the typical hydrophobic binding cavity of the OBP protein, allowing ethyl butyrate to freely search for the optimal binding conformation within the potential active site region. AutoDock Vina calculates the binding affinity between the ligand and receptor using a semi-flexible molecular docking algorithm and outputs multiple possible binding modes. The conformation with the lowest binding energy is typically used as the most stable complex for subsequent analysis; the lower the binding energy value, the more stable the interaction between the protein and ligand and the stronger the affinity.

[0017] Further analysis of the interaction patterns between OBP4 and ethyl butyrate was conducted using PyMOL and the Protein-Ligand Interaction Profiler (PLIP) online tool, including hydrogen bonding, hydrophobic interactions, van der Waals forces, and electrostatic interactions. Key binding sites and participating amino acid residues were identified. Simultaneously, CASTp was used to predict potential binding pockets to analyze the spatial localization and binding stability of ethyl butyrate within the hydrophobic cavity, revealing the molecular mechanism by which OBP4 recognizes host-related ester volatiles at the structural level.

[0018] Compared with the prior art, the advantages of the present invention are as follows: 1. Precise Targeting: This approach precisely identified OBP4, which is highly expressed specifically in the antennae of female wasps, from among numerous OBP genes through whole-genome identification and tissue expression profiling. Experiments demonstrated that interfering with this gene significantly reduced the parasitic wasps' ability to locate their host environment, proving the high efficiency of this protein as a regulatory target.

[0019] 2. Clear mechanism: This scheme not only verified the protein function in macroscopic behavior (wind tunnel experiment, Y-tube experiment), but also revealed the specific mechanism of its recognition of ethyl ester compounds at the microscopic physiological (EAG electrophysiology) and molecular structure (molecular docking) levels, providing a solid theoretical basis for subsequent drug development.

[0020] 3. High biosafety: This approach utilizes the parasitic wasp's natural olfactory regulation mechanism. By developing attractants targeting specific OBP proteins, natural enemies can be guided to precisely locate pests, reducing the use of chemical pesticides and meeting the sustainable development requirements of modern green agriculture and biological control.

[0021] 4. It has broad application prospects: The identified OBP4 and its key binding site SER64 can serve as a molecular platform for high-throughput screening of parasitic wasp attractants or behavioral interventions, which will help develop new natural enemy adjuvants and improve the field efficiency of biological control. Attached Figure Description

[0022] Figure 1 This is a flowchart of the present invention; Figure 2 This is a distribution map of conserved cysteine ​​residues based on protein sequence alignment in this invention; Figure 3 This is a chromosome distribution diagram of the odor-binding protein in this invention in this species; Figure 4 This is a graph showing the relative expression levels in male and female tissues based on real-time quantitative PCR in this invention. Figure 5 This is a diagram showing the electrophysiological response of the parasitic wasp to ethyl butyrate after gene knockdown technology in this invention. Figure 6 This is a diagram showing the predicted three-dimensional structure of the protein and its molecular docking with ethyl butyrate in this invention. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0024] like Figure 1-6 As shown, a method for screening, identifying, and functionally verifying odor-binding proteins that regulate host localization in parasitic wasps during biological control includes the following steps: S1. Genome identification and characterization: Eighteen OBP genes were identified in the genome of the ring-bellied gall wasp, including 17 classic OBPs and 1 Minus-C OBP. These genes are not evenly distributed across the four chromosomes, and form distinct gene clusters on chromosomes 1 and 3; S2. Tissue-specific expression analysis: Through transcriptome sequencing (TPM analysis) and qPCR verification, multiple genes were found to be tissue-specific. The study focused on 10 genes that are highly expressed in the antennae, among which OBP4 showed significant expression levels in the female antennae, suggesting its association with foraging or oviposition behavior.

[0025] S3. Gene function verification: RNA interference technology was used to inhibit the expression of OBP4, which led to a significant reduction in the female bee's preference for yeast attractants in the host's living environment. Wind tunnel experiments further confirmed that although female bees with the gene knocked out had normal locomotion, their success rate in locating host habitats was significantly reduced.

[0026] S4. Electrophysiological and molecular binding mechanism: Electrophysiological (EAG) tests showed that after OBP4 was interfered with, female bees lost their electrophysiological response to key compounds of ethyl butyrate.

[0027] Molecular docking simulations show that the OBP4 protein mediates the odor recognition process by forming stable hydrogen bonds with ethyl butyrate through the SER64 residue in its hydrophobic cavity.

[0028] Step S1 includes the following steps: S1-1, Candidate Gene Mining: Based on chromosome-level whole genome data of *Gallus circumbellatus*, we used the known conserved OBP domain of insects (Pfam ID: PF00067) as a retrieval model, combined with homology comparison BLASTP to search for potential OBP sequences throughout the whole genome; S1-2, Structural Classification and Confirmation: The conserved structural domains of candidate sequences were confirmed using the CD-search online tool, and the N-terminal signal peptide sequence was predicted using SignalP 5.0; Based on the number and arrangement pattern of cysteine ​​residues, the 18 identified genes were classified into 17 classic OBPs (with 6 conserved cysteine ​​residues) and 1 Minus-C OBP. Figure 2 As shown.

[0029] S1-3. Chromosome localization and gene cluster analysis: MapChart software was used to map the identified OBPs onto a physical chromosome map and analyze their distribution patterns.

[0030] The results showed that the genes were not uniformly distributed across the four chromosomes, and significant tandem duplication was observed on chromosomes 1 (Chr1) and 3 (Chr3), forming multiple gene clusters, such as... Figure 3 As shown.

[0031] In detail, step S2 includes the following steps: S2-1. Transcriptome sequencing and expression abundance calculation: Total RNA was extracted and transcriptome sequencing was performed on the antennae, head (antennae removed), thorax, abdomen and foot tissues of adult gall midges. The transcripts per million mapped reads (TPM) value was calculated using RSEM software to preliminarily screen for genes specifically expressed in the antennae. S2-2, Key gene screening: By comparing the expression differences between male and female antennae, the focus was on 10 key OBP genes that were significantly highly expressed in the antennae of female bees; These genes are considered core candidate genes involved in female bees' perception of external chemical signals (such as host location cues).

[0032] S2-3. Quantitative PCR (qPCR) verification: Specific primers were designed for candidate genes, with tubin and β-actin as internal reference genes, and their spatial expression patterns in different tissues were detected by real-time quantitative PCR technology. like Figure 4 As shown, the expression abundance of OBP4 in the antennae of female bees was significantly higher than that in other tissues.

[0033] Furthermore, step S3 specifically includes the following steps: S3-1, dsRNA synthesis and microinjection: Primers were designed targeting specific regions of the OBP4 sequence to synthesize double-stranded RNA (dsRNA) through in vitro transcription; the dsRNA was then injected into the prepupal stage of *Gallus circumbellatus* under a microscope using a microinjection system to achieve efficient gene knockdown. S3-2, Behavioral Preference Evaluation (Y-tube): After the individuals in the interference group (dsOBP4) and the control group (dsGFP) emerged, they were placed in a Y-tube olfactometer to test their selection ratio for host-related odors (such as yeast fermentation products); Experiments have shown that the absence of OBP4 significantly reduces the chemotactic preference of female bees for yeast.

[0034] S3-3, Long-distance positioning assessment (wind tunnel experiment): Simulate natural flight conditions in a controlled wind tunnel environment and record the behavioral trajectory of parasitic wasps flying towards the odor source.

[0035] The results showed that the female wasps in the interference group had impaired directional flight ability when searching for host habitats, and their location success rate dropped significantly, proving that this gene directly mediates the host's search behavior.

[0036] Specifically, step S4 includes the following steps: S4-1, Electrophysiological response test (EAG): The physiological response of the antennae of female bees in the interference group to 27 host-derived volatiles was detected using an antennal potential coupled with electrophoresis (EAG). like Figure 5 The results showed that when OBP4 was inhibited, the female bee's electrical signal response to ethyl butyrate compounds disappeared.

[0037] S4-2, Protein structure modeling: The tertiary structure of the OBP4 protein was predicted using AlphaFold2 software, and Procheck and ERRAT were used to evaluate the model quality to ensure the accuracy of its hydrophobic cavity structure. S4-3. Molecular docking and binding site identification: Ethyl butyrate molecules were docked to the active site of OBP4 using AutoDock Vina software, and their interaction modes were analyzed by energy calculation.

[0038] like Figure 6 As shown, the results indicate that the SER64 residues within the protein's hydrophobic cavity form crucial hydrogen bond support with the ligand molecules, and are the core amino acid site mediating odor recognition.

[0039] In summary, the principle of this embodiment is as follows: A key olfactory gene regulating host localization in parasitic wasps has been discovered: This protocol identifies and demonstrates for the first time that the OBP4 gene in parasitic wasps is a core gene mediating the female wasp's detection of host habitats (such as yeast-derived volatiles). This is the first functionally validated olfactory receptor-binding protein gene in this species that is directly related to oviposition localization behavior.

[0040] This study reveals the molecular switch by which OBP4 recognizes ethyl butyrate compounds: Using molecular simulation techniques, the key amino acid residue SER64, crucial for the binding of the OBP4 protein to its ligand, was innovatively located. This scheme clearly demonstrates that this site can bind to ethyl butyrate via hydrogen bonds, elucidating the chemical nature of parasitic wasps' sensing of environmental signals.

[0041] A highly efficient evaluation system for the olfactory genes of parasitic wasps was established, integrating genomic identification, antennal-specific transcriptome analysis, RNAi gene interference, EAG electrophysiological detection, and wind tunnel / Y-tube behavioral experiments. This complete evaluation process, from sequence to function, provides a standard technical paradigm for the study of olfactory functions in other beneficial insects.

[0042] A novel strategy for synergistic pest control based on OBP protein targets was proposed: by artificially synthesizing OBP-sensitive compounds or their mimics, the search efficiency of parasitic wasps in complex farmland environments can be enhanced in a targeted manner, realizing a technological leap from gene discovery to biological control applications.

[0043] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for screening and identifying odor binding proteins regulating host location of parasitoid wasps in biological control, and for verifying the function of the odor binding proteins, characterized in that, Includes the following steps: S1. Genome identification and characterization: Eighteen OBP genes were identified in the genome of the ring-bellied gall wasp, including 17 classic OBPs and 1 Minus-C OBP. S2. Tissue-specific expression analysis: Through transcriptome sequencing results (TPM analysis) and qPCR verification, multiple genes were found to be tissue-specific. S3. Gene function verification: RNA interference technology was used to inhibit the expression of OBP4, which led to a significant reduction in the female bee's preference for yeast attractants in the host's living environment. S4. Electrophysiological and molecular binding mechanism: Electrophysiological (EAG) tests showed that after OBP4 was interfered with, female bees lost their electrophysiological response to ethyl isobutyrate.

2. The method according to claim 1, wherein the method is characterized by, Step S1 includes the following steps: S1-1, Candidate Gene Mining: Based on chromosome-level whole genome data of the ring-bellied gall wasp, the conserved domain of known insect OBP protein (Pfam ID: PF00067) was used as a search model, combined with homology comparison BLASTP to search for potential OBP sequences throughout the whole genome. S1-2, Structural Classification and Confirmation: The conserved domains of candidate sequences were confirmed using the CD-search online tool, and the N-terminal signal peptide sequence was predicted using SignalP 5.0; S1-3. Chromosome localization and gene cluster analysis: MapChart software was used to map the identified OBPs onto a physical chromosome map and analyze their distribution patterns.

3. The method according to claim 2, wherein the method is characterized by, Step S2 specifically includes the following steps: S2-1. Transcriptome sequencing and expression abundance calculation: Antennae tissues of female and male adult gall midges were collected for total RNA extraction and transcriptome sequencing; RSEM software was used to calculate the transcripts per million mapped reads (TPM) value, and genes specifically expressed in the antennae were preliminarily screened. S2-2, Key Gene Screening: By comparing the expression differences between male and female antennae, the focus was on 10 key OBP genes that were significantly highly expressed in the antennae of female bees; S2-3. Quantitative PCR (qPCR) Validation: Specific primers were designed for candidate genes, with tubin and β-actin as internal reference genes, and their spatial expression patterns in different tissues were detected by real-time quantitative PCR technology.

4. The method according to claim 3, wherein the method is characterized by, Step S3 specifically includes the following steps: S3-1, dsRNA synthesis and microinjection: Primers were designed targeting specific regions of the OBP4 sequence to synthesize double-stranded RNA (dsRNA) via in vitro transcription; the dsRNA was then injected into the prepupal stage of *Gallus circumbellatus* under a microscope using a microinjection system to achieve efficient gene knockdown. S3-2, Behavioral Preference Evaluation (Y-tube): After the individuals in the interference group (dsOBP4) and the control group (dsGFP) emerged, they were placed in a Y-tube olfactometer to test their selection ratio for host-related odors; S3-3, Long-distance positioning assessment (wind tunnel experiment): Simulate natural flight conditions in a controlled wind tunnel environment and record the behavioral trajectory of parasitic wasps flying towards the odor source.

5. The method according to claim 4, wherein the method is characterized by, Step S4 specifically includes the following steps: S4-1, Electrophysiological response test (EAG): The physiological response of the antennae of female bees in the interference group to 27 host-derived volatiles was detected using an antennal potential coupled with electrophoresis (EAG). S4-2, Protein structure modeling: The tertiary structure of the LbouOBP4 protein was predicted using AlphaFold2 software, and Procheck and ERRAT were used to evaluate the model quality to ensure the accuracy of its hydrophobic cavity structure. S4-3. Molecular docking and binding site identification: Ethyl butyrate molecules were docked to the active site of LbouOBP4 using AutoDock Vina software, and their interaction modes were analyzed by energy calculation.

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