Highly effective repellent for blood-feeding biting midges and method for screening and identifying same

Abstract

This invention provides a highly effective repellent for midges and a method for screening and identifying it, relating to the field of biotechnology. This invention identifies an odor receptor gene highly expressed in the head of female midges. FtaiOr1 Furthermore, by heterologously expressing this gene in Drosophila ab3A neurons, a transgenic Drosophila strain suitable for screening midge repellent compounds was constructed. Or22ab ‑ / ‑ − GAL4; UAS− FtaiOr1 Six midge repellent compounds (camphor, eucalyptol, (+)-anesinone, α-terpineol, (S)-cis-verbenol, and isoterpinene) were screened using this fruit fly strain. These compounds can be used alone or in combination. They are non-target organisms and environmentally friendly, and can be directly applied to the repellency and field control of midges, showing significant application value in reducing midge bites and lowering the risk of vector-borne disease transmission.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a highly effective repellent for vampire midges and its screening and identification method. Background Technology

[0002] Taiwan midge (Forcipomyia taiwana) Belonging to the order Diptera, family Midgeidae, and genus *Ceratophorus*, the Taiwan midge is tiny (1-1.5 mm) and widely distributed in Central, East, South, and Southwest my country. The Taiwan midge primarily feeds on human blood. Its bite often causes immediate wheals and intense itching; severe cases can lead to systemic allergic reactions, even anaphylactic shock, threatening life. Furthermore, during severe Japanese encephalitis (JE) outbreaks, researchers isolated the JE virus from Taiwan midges captured in infected areas and confirmed its pathogenicity through suckling mouse bite tests, demonstrating that the Taiwan midge is a member of the JE transmission chain and a significant vector besides mosquitoes. Currently, the main method for controlling the Taiwan midge is large-scale spraying of broad-spectrum chemical insecticides. However, large-scale use of insecticides not only harms the environment and ecological safety but also easily leads to pesticide resistance, making it difficult to achieve green and sustainable control of these blood-sucking midges.

[0003] The olfactory recognition system of insects is crucial for finding hosts, mates, and oviposition sites, with odorant receptor (OR) genes playing a central role in the recognition and transduction of odor signals. Analyzing the function of OR genes in the Formosan midge and screening for compounds that can specifically activate its olfactory system is the core approach for developing highly effective and environmentally friendly Formosan midge repellents.

[0004] The Drosophila empty neuron expression system is a mature heterologous expression system. By introducing the OR gene of the target insect into empty neurons of Drosophila, the functional expression of the target OR gene can be achieved in Drosophila. Odor compounds that can activate this OR gene can then be screened through electrophysiological experiments. This system has advantages such as simple operation, short experimental cycle, and low cost, and has been successfully applied to the study of OR gene function and repellent screening for various sanitary pests such as mosquitoes and tsetse flies. However, there are currently no reports on the application of this system to the verification of OR gene function in midges and the screening of highly effective repellents, and there is also a lack of highly effective and specific repellent compounds for midges.

[0005] Therefore, constructing a Drosophila empty neuron expression system for the midge OR gene and using this system to screen for highly efficient midge-repelling compounds has important theoretical and applied value for developing new midge control technologies and can effectively address the shortcomings of existing control methods.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a method for constructing a transgenic fruit fly strain for screening repellent compounds against midges, thereby addressing the aforementioned technical problems.

[0008] The second objective of this invention is to provide the application of the transgenic fruit fly strains constructed by the above-described method in the screening of repellent compounds for midges.

[0009] The third objective of this invention is to provide a method for screening repellent compounds for leeches, so as to overcome the technical defects of existing leech control methods, such as low efficiency, poor specificity, and insufficient control measures.

[0010] A fourth objective of this invention is to provide the use of the compound in the preparation of midge repellent products.

[0011] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a method for constructing a transgenic fruit fly strain for screening repellent compounds for midges, comprising the following steps: Will FtaiOr1 The gene was inserted into the genome of the fruit fly to obtain expression. FtaiOr1 Fruit fly strains based on genes UAS- FtaiOr1 ; then will express FtaiOr1 The Drosophila strains with the gene were crossed with the balanced sub-strain and the empty neuron strain, respectively, and homozygous strains were selected. Or22ab - / - GAL4;UAS FtaiOr1 Fruit fly strains, making FtaiOr1 Specifically expressed in the ab3A olfactory sensory neurons of Drosophila melanogaster; The FtaiOr1 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0012] As a further technical solution, the aforementioned FtaiOr1 The steps for inserting a gene into the genome of a fruit fly are as follows: Will FtaiOr1 The CDS sequence of the gene was ligated into the pUAST-attB vector to obtain a recombinant vector, which was then injected into Drosophila embryos to obtain expression. FtaiOr1 Fruit fly strains based on genes UAS-FtaiOr1 .

[0013] As a further technical solution, the balanced sub-strain of fruit flies includes w; Sp / Cyo; Dr / TM3,sb .

[0014] As a further technical solution, the empty neuron strain of fruit flies includes w;Or22ab - / - -GAL4 / Or22ab - / - - GAL4;Dr / TM3,sb .

[0015] As a further technical solution, the blood-sucking midge includes the Taiwan midge.

[0016] Secondly, this invention provides the application of transgenic fruit fly strains constructed by the above-described method in the screening of repellent compounds for midges.

[0017] Thirdly, the present invention provides a method for screening midge repellent compounds. A transgenic fruit fly strain is constructed using the above-mentioned construction method. Then, the SSR technology is used to test and record the response of the ab3A neurons of the transgenic fruit fly strain to different compound stimuli to determine whether the compound is a midge repellent compound.

[0018] As a further technical solution, the blood-sucking midge includes the Taiwan midge.

[0019] Fourthly, the present invention provides the use of compounds in the preparation of midge repellent products, said compounds including one or more of camphor, eucalyptol, (+)-anesinone, α-terpineol, (S)-cis-verbenol or isoterpinene.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. First-ever construction of an efficient research system for the OR gene in midge: The Drosophila empty neuron expression system was applied for the first time to the functional study of the OR gene in midge, successfully achieving the study of the OR gene in midge. FtaiOr1 The functional expression of genes provides important insights for further research on the olfactory recognition mechanism of vampire midges.

[0021] 2. Obtaining highly effective and specific repellent compounds: All six screened compounds showed strong repellent activity against Formosan midge. At a low concentration of 0.1%, camphor, eucalyptol, and (+)-anisone still exhibited strong repellent activity against Formosan midge, with repellent effects superior to DEET.

[0022] 3. Provides a low-cost, fast-cycle compound screening method: The screening method of this invention combines electrophysiological experiments, is simple to operate, has a short experimental cycle, and is low in cost. It can quickly screen highly effective midge repellent compounds from a large number of candidate compounds, providing an efficient technical path for the development of new midge control products. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 for FtaiOr1 Gel electrophoresis image of gene PCR products; Figure 2 for FtaiOr1 Functional analysis of different odors; where A is FtaiOr1 Reaction spectra of different compounds; B is FtaiOr1 The dose-response curve; Figure 3 An improved WHO standard space repellent measuring device; Figure 4 The repellency of different compounds against midges is shown; where A and B are the repellency indices of different compounds against midges at a 1% concentration; C and D are the repellency indices of different compounds against midges at a 0.1% concentration; different letters on the columns represent the repellency indices between treatment groups after multiple comparisons using Duncan's new multiple range method. P<0.01 The differences in levels are extremely significant. Detailed Implementation

[0025] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] In a first aspect, the present invention provides a method for constructing a transgenic fruit fly strain for screening repellent compounds for midges, comprising the following steps: Will FtaiOr1 The gene was inserted into the genome of the fruit fly to obtain expression. FtaiOr1 Fruit fly strains based on genes UAS- FtaiOr1 ; then will express FtaiOr1 The Drosophila strains with the gene were crossed with the balanced sub-strain and the empty neuron strain, respectively, and homozygous strains were selected. Or22ab - / - GAL4;UAS FtaiOr1 Fruit fly strains, making FtaiOr1 Specifically expressed in the ab3A olfactory sensory neurons of Drosophila melanogaster; The FtaiOr1 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0027]

[0028] This invention identified the OR gene highly expressed in the Taiwan midge (…). FtaiOr1 (genes), and make FtaiOr1 A transgenic fruit fly strain for screening midge repellent compounds was constructed by specifically expressing ab3A olfactory receptor neurons in Drosophila melanogaster. Studies have shown that this transgenic fruit fly strain has high screening efficiency and can accurately screen for midge repellent compounds.

[0029] In some alternative implementations, the term "will" FtaiOr1 The steps for inserting a gene into the genome of a fruit fly are as follows: Will FtaiOr1 The CDS sequence of the gene was ligated into the pUAST-attB vector to obtain a recombinant vector, which was then injected into Drosophila embryos to obtain expression. FtaiOr1 Fruit fly strains based on genes UAS-FtaiOr1 .

[0030] In some alternative implementations, the balanced sub-strain of fruit flies includes, but is not limited to, w; Sp / Cyo; Dr / TM3,sb .

[0031] In some alternative implementations, the empty neuron strain of Drosophila includes, but is not limited to, w;Or22ab - / - - GAL4 / Or22ab - / - -GAL4;Dr / TM3,sb .

[0032] In some alternative embodiments, the blood-sucking midge includes, but is not limited to, Formosan midge, and may also include other midges of the same genus known to those skilled in the art.

[0033] Secondly, this invention provides the application of transgenic fruit fly strains constructed by the above-described method in the screening of repellent compounds for midges.

[0034] The transgenic fruit fly strains constructed by the method of this invention are sensitive to repellent compounds and can be used for screening repellent compounds for blood-sucking midges.

[0035] In some alternative embodiments, the blood-sucking midge includes, but is not limited to, Formosan midge, and may also include other midges of the same genus known to those skilled in the art.

[0036] Thirdly, the present invention provides a method for screening midge repellent compounds. A transgenic fruit fly strain is constructed using the above-mentioned construction method. Then, the SSR technology is used to test and record the response of the ab3A neurons of the transgenic fruit fly strain to different compound stimuli to determine whether the compound is a midge repellent compound.

[0037] This screening method is simple, convenient, and efficient.

[0038] In some alternative embodiments, the blood-sucking midge includes, but is not limited to, Formosan midge, and may also include other midges of the same genus known to those skilled in the art.

[0039] Fourthly, the present invention provides the use of compounds in the preparation of midge repellent products, said compounds including one or more of camphor, eucalyptol, (+)-anesinone, α-terpineol, (S)-cis-verbenol or isoterpinene.

[0040] All six compounds mentioned above exhibit strong repellent activity against Formosan midge. At a low concentration of 0.1%, camphor, eucalyptol, and (+)-anestinone still showed strong repellent activity against Formosan midge, with repellent effects exceeding those of DEET.

[0041] In some alternative embodiments, the blood-sucking midge includes, but is not limited to, Formosan midge, and may also include other midges of the same genus known to those skilled in the art.

[0042] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0043] Example 1 (I) Construction of a Drosophila empty neuron expression system for the midge OR gene 1. Identification of OR genes highly expressed in Taiwan midges Female adult Formosan midges were collected, and their heads were dissected to extract RNA. After RNA testing, cDNA libraries were constructed by Novogene (Beijing) Co., Ltd., and sequenced using the Illumina HiSeq 2500 sequencing platform. The raw sequencing data underwent quality control, and clean reads were aligned to the Formosan midge reference genome. RSEM was used to calculate the expression levels of all genes in each sample, and FPKM (kilobases per million fragments of transcripts) was used to normalize the expression levels. Based on the transcriptome data from the female Formosan midge head, the expression levels of odorant receptor genes in the head were statistically analyzed. 58 odorant receptor genes were found to be substantially expressed in the head (fpkm > 1). The OR gene FTW2.9089 (named...) with high expression levels in the antennae was selected. FtaiOr1 (Gene), to obtain its complete coding region sequence.

[0044] 2. FtaiOr1 Construction of gene recombination expression vector The head of the female adult was dissected, and RNA was extracted. Subsequently, the first-strand cDNA was synthesized using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, Beijing, China) according to the kit instructions. FtaiOr1 Design specific primers based on gene CDS sequence ( FtaiOr1 -CDS-F: 5'- GGGAATTGGGAATTCATGCTAGGCTCGCCGCAG-3' (SEQ ID NO. 2) and FtaiOr1 -CDS-R: 5'-TCTGTTAACGAATTCTCACAGCTGCTGAAGAAGAGTAAAGT-3' (SEQ ID NO.3) was used to amplify PCR using cDNA from the head of a Formosan midge as a template (50 μL reaction system: 25 μL 2×Phanta Master Mix, 2 μL upstream / downstream primers, 2 μL cDNA, 19 μL ddH2O; reaction conditions: 95℃ for 3 min, 95℃ for 15 s, 56℃ for 15 s, 72℃ for 1 min, ×35 cycles, 72℃ for 10 min). The PCR products were detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 1 As shown, a specific band of the expected size was obtained. After recovery, the band was sequenced for verification, confirming the discovery of midges. FtaiOr1 The complete coding region sequence of the gene was obtained. After obtaining the target fragment, it was ligated into the pUAST-attB vector and transformed into E. coli Top10 competent cells. After PCR identification and sequencing verification, the recombinant expression vector pUAST- was obtained. FtaiOr1 .

[0045] 3. Preparation of transgenic fruit flies The recombinant expression vector pUAST- FtaiOr1 The samples were sent to United Huayi Company for injection, thereby constructing an expression of the Taiwan midge. FtaiOr1 UAS- gene strain of fruit fly FtaiOr1 Subsequently, the fruit fly UAS- FtaiOr1 The strains and balanced sub-strains w;Sp / Cyo;Dr / TM3,sb and empty neuron strain w;Or22ab - / - -GAL4 / Or22ab - / - -GAL4;Dr / TM3,sb Hybridization. The resulting homozygous individuals, Or22ab. - / - -GAL4;UAS- FtaiOr1 After PCR detection of sequence information, this functional strain was ultimately used for subsequent single-sensor recording.

[0046] (II) Screening of highly effective midge repellent compounds 1. Single-sensillum recording (SSR) 1) Compound Preparation: 186 compounds from the laboratory were selected as initial screening compounds. First, a 10% stock solution was prepared using paraffin oil or DMSO, then diluted to a 1% test concentration. Gradient concentrations of strongly reactive compounds were prepared at 1%, 0.1%, 0.01%, 0.001%, and 0.0001%. 0.4cm × 5cm filter paper was used as the compound carrier. Compound labels were affixed to glass Pasteur tubes used for odor storage beforehand, and strips of filter paper containing the added compounds were inserted into the tubes. Paraffin oil and DMSO were used as controls.

[0047] 2) Preparation of recording electrodes: Prepare two tungsten wire electrodes as reference and recording electrodes respectively. After connecting them to the electrode holder, grind them with a needle grinder. Grind the reference electrode to a fine point and the recording electrode to a needle tip that can be inserted into the fruit fly target sensor.

[0048] 3) Signal Recording: Fruit flies 2-7 days after emergence were selected as test insects. The fruit flies were fixed in a 10 μL pipette tip (with the tip removed), with the head and antennae extended, and secured with dental wax. Under a microscope, a reference electrode was inserted into the compound eye of the fruit fly, and a recording electrode was inserted into the base of the receptor on the antennae. Stimulation with two indicator odor compounds, 2-heptanone and ethyl hexanoate, was used to determine if it was the target receptor (ab3). After identifying the target receptor, the selected compounds were stimulated sequentially. The collected signals were processed using Autospike v3.9 (Syntech, Netherlands). Single-sensor recording results showed that... FtaiOr1 It can be strongly activated by (+)-fenchone, camphor, eucalyptol, terpinolene, α-terpineol, and (S)-cis-verbenol, and the intensity of the reaction increases with increasing dosage. Figure 2 ).

[0049] 2. Behavioral Measurement A modified WHO standard spatial repellent assay was used to determine the repellency behavior of female Formosan midges under indirect contact conditions against (+)-fenchone, camphor, eucalyptol, terpinolene, α-terpineol, and (S)-cis-verbenol. The experimental setup was made of acrylic. Figure 3The apparatus mainly consists of two outer treatment cylinders (15×10 cm) and two inner compound carrier cylinders (13×9 cm), designed in a nested configuration. It also includes auxiliary structures such as a midge release chamber and connecting sections. Before the experiment, a trimmed nylon stocking is placed over the opening of the inner compound carrier cylinder, and the test compound solution is dropped onto the surface of the stocking. The inner compound carrier cylinder is then placed into the outer treatment cylinder, completing the assembly of the reagent treatment end. A nylon stocking with a blank solvent is used as a control group. After the midges are placed in the central release chamber for 5 minutes to acclimate, the chamber door is simultaneously opened to release the midges, and the time is 10 minutes. After the experiment, the number of midges in each treatment cylinder is counted, and the apparatus is thoroughly cleaned with 75% ethanol after each experiment. Twenty midges are used in each experiment, and each treatment group is replicated at least six times. The experimental results were calculated using the formula Repulsion Index (RI) = (Nc - Nt) / (Nc + Nt) (where Nc is the number of midges in the control chamber and Nt is the number of midges in the treatment chamber). In addition, we also determined the repulsion behavior of Formosan midges against the commonly used insect repellent DEET.

[0050] The results showed that all six compounds had strong repellent effects against Formosan midge at a concentration of 1%; among them, camphor, eucalyptol, (+)-anestinone, α-terpineol, and isoterpinene had repellency indices of 88.83%, 87.67%, 83.17%, 71.83%, and 68.83%, respectively, all greater than DEET's 53.83%. Figure 4 A in Figure 4 (B in the text). At a concentration of 0.1%, camphor, eucalyptol, and (+)-anestinone still exhibit strong repellent effects against Formosan midge. Figure 4 C in Figure 4 (D in the text). In summary, all six compounds can be used as repellents for Formosan midge, with repellent effects higher than or comparable to DEET.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a transgenic Drosophila melanogaster strain for screening blood-feeding midge repellent compounds, characterized by, Includes the following steps: Will FtaiOr1 The gene was inserted into the genome of the fruit fly to obtain expression. FtaiOr1 Fruit fly strains based on genes UAS- FtaiOr1 ; then will express FtaiOr1 The gene-derived Drosophila strain was crossed with the balanced sub-strain and the empty neuron strain, respectively, and homozygous Or22ab strains were obtained through screening. - / - -GAL4;UAS- FtaiOr1 Fruit fly strains, making FtaiOr1 Specifically expressed in the ab3A olfactory sensory neurons of Drosophila melanogaster; The FtaiOr1 The CDS sequence of the gene is shown in SEQ ID NO.1; The midge repellent compound is camphor, eucalyptol, (+)-anesinone, α-terpineol, (S)-cis-verbenol, or isoterpinene; the mass concentration of camphor, eucalyptol, (+)-anesinone, α-terpineol, or isoterpinene is 0.1%-1%, and the mass concentration of (S)-cis-verbenol is 1%. The vampire midge mentioned is the Taiwan midge.

2. The application of the transgenic fruit fly strain constructed by the method described in claim 1 in the screening of midge repellent compounds. The transgenic fruit fly strain is Or22ab as described in claim 1. - / - -GAL4;UAS- FtaiOr1 Fruit fly strains; The blood-sucking midge is the Formosan midge; The repellent compound is camphor, eucalyptol, (+)-anesinone, α-terpineol, (S)-cis-verbenol, or isoterpinene; the mass concentration of camphor, eucalyptol, (+)-anesinone, α-terpineol, or isoterpinene is 0.1%-1%, and the mass concentration of (S)-cis-verbenol is 1%.

3. A method for screening compounds that repel midges, characterized in that, A transgenic fruit fly strain was constructed using the construction method described in claim 1. Then, the SSR technology was used to test and record the response of the ab3A neurons of the transgenic fruit fly strain to different compound stimuli, and to determine whether the compound was a midge repellent compound. The transgenic fruit fly strain is Or22ab as described in claim 1. - / - -GAL4;UAS- FtaiOr1 Fruit fly strains; The blood-sucking midge is the Formosan midge; The midge repellent compound is camphor, eucalyptol, (+)-anesinone, α-terpineol, (S)-cis-verbenol, or isoterpinene; the mass concentration of camphor, eucalyptol, (+)-anesinone, α-terpineol, or isoterpinene is 0.1%-1%, and the mass concentration of (S)-cis-verbenol is 1%.

4. The application of the compound in the preparation of midge repellent products, characterized in that, The compound is one or more selected from camphor, eucalyptol, (+)-anesinone, α-terpineol, (S)-cis-verbenol, or isoterpinene; the mass concentration of camphor, eucalyptol, (+)-anesinone, α-terpineol, or isoterpinene is 0.1%-1%, and the mass concentration of (S)-cis-verbenol is 1%. The vampire midge mentioned is the Taiwan midge.

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