Screening method for inhibitor of insect odor response, inhibitor of insect odor response, insect inhibition system, and insect inhibition method

Abstract

To provide an insect inhibition system using an inhibitor of insect odor response, an inhibition method, and a screening method for an inhibitor.SOLUTION: An insect inhibition system comprising a spraying unit that sprays an inhibitor of insect odor response containing at least one of a methylphenol derivative, a cinnamic acid derivative, and 2,4-diisopropylphenol is provided. A screening method for an inhibitor of insect odor response includes: preparing an odor detection structure expressing at least one of an insect olfactory receptor and a co-receptor for the olfactory receptor; giving an odorant corresponding to the olfactory receptor and a plurality of inhibitor candidate substances, respectively, to the odor detection structure; and screening the plurality of inhibitor candidate substances on the basis of the inflow of ions into the odor detection structure.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to environmental technology, a method for screening inhibitors of insect odor responses, The present invention relates to an inhibitor of the odor response of insects, an insect inhibition system, and an insect inhibition method. [Background technology]

[0002] The sense of smell is involved in many of the behaviors of insects (see, for example, Patent Document 1 and Non-Patent Document 1). Insects rely on olfaction for feeding, attraction between males and females for mating, and selection of oviposition sites. On the other hand, insecticides are also used to repel harmful insects. It has been proposed to block the insects' sense of smell or to use odors that repel insects. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2021 / 045233 [Non-patent literature]

[0004] [Non-Patent Document 1] Satoshi Hamada et al., "Giant vesicles functionally expressing membrane receptors for an insect pheromone," Chem. Commun., 2014,50, 2958 Summary of the Invention [Problem to be solved by the invention]

[0005] Highly effective inhibitors of insect odor responses and methods for screening for said inhibitors Therefore, the present invention provides a method for screening inhibitors of insect odor responses. The present invention provides an insect inhibitor system, an insect odor response inhibitor, an insect inhibition method, and an insect odor response inhibitor. One of the objectives is to: [Means for solving the problem]

[0006] The method for screening an inhibitor of an insect's odor response according to an embodiment of the present invention is The odor-detecting structure expressing at least one of the olfactory receptor and the co-receptor of the olfactory receptor is used. The present invention aims to provide an olfactory receptor-specific odorant and a plurality of candidate inhibitors, and based on the influx of ions into the odor detection structure, and screening for candidate inhibitors. The odor-sensing structure may be a cell-type receptor. The odor-sensing structure may be a cell. The odor-detecting structure may be an artificial structure. The odor-detecting structure expresses a co-receptor of the olfactory receptor. Good too.

[0007] In the above-mentioned method for screening inhibitors of insect odor responses, The receptor for 1-octen-3-ol is 1-octen-3-ol, and the odorant is 1-octen-3-ol. Good too.

[0008] In the above-mentioned method for screening an inhibitor of an insect's odor response, the insect is a cockroach. may be.

[0009] The above-mentioned method for screening inhibitors of insect odor responses is carried out by preparing insect antennae. The stimulating substance that stimulates the insect antennae and the candidate inhibitor selected by screening and detecting a response of the insect's antennae. The stimulating substance may be bombykol. The insect antennae may be moth antennae. The moth may be a silkworm moth.

[0010] In the above-mentioned method for screening inhibitors of insect odor responses, a receptor for 1-octen-3-ol, and the odorant is 1-octen-3-ol; The irritant may be bombykol and the insect antennae may be moth antennae. The method for screening inhibitors of the response to moth pheromones is The present invention may also be a method for screening for a

[0011] In the above-mentioned method for screening inhibitors of insect odor responses, the antennal responses of insects are In detecting the response, the potential of the antennae may be detected.

[0012] The screening method for inhibitors of the odor response of insects described above comprises preparing insects and The insects are given a stimulating substance and a candidate inhibitor selected in the screening. The method may further include applying a stimulus to the insect and detecting a response from the insect. The insect may be a moth. The moth may be a silkworm moth. Insect The cockroach may be a cockroach. The cockroach may be an American cockroach.

[0013] In the above-mentioned method for screening inhibitors of insect odor responses, a receptor for 1-octen-3-ol, and the odorant is 1-octen-3-ol; The stimulus may be bombykol and the insect may be a moth. A screening method for inhibitors was developed to screen for inhibitors of the response to moth pheromones. It may also be a method for performing

[0014] In the above-mentioned screening method for inhibitors of insect odor responses, the reaction of insects is examined. You can observe the behavior of the insects in releasing the insects. The insect behavior may be exploratory behavior.

[0015] Inhibitors of insect odor responses according to aspects of the present invention include inhibitors of ions into the odor-sensing structure. It is an inhibitor of insect odor responses, reducing influx to less than 37%.

[0016] The inhibitor of the insect's odor response according to an embodiment of the present invention is Inhibitors of insect odor responses obtained by the inhibitor screening method are used in odor detection. It is an inhibitor of insect odor responses, reducing ion influx into the exit structures by less than 37%. .

[0017] The inhibitors of the insect's odor response reduce the influx of ions into the odor-detecting structures by 20%. The following may also be used.

[0018] The inhibitor of the insect's odor response may be a methylphenol derivative.

[0019] The inhibitor of the odor response of the above insects was 4-isopropyl-3-methylphenol. It may also contain.

[0020] The inhibitor of the odor response of the above insects is 4-(tert-butyl)-2-methylphenyl. It may also contain alcohol.

[0021] In the inhibitors of the above insect odor responses, the solvents are ethanol, hexane, isopropyl alcohol, and ethanol. At least one solvent selected from the group consisting of propyl alcohol, acetone, DMSO, and water It may be one.

[0022] The inhibitor of the insect's odor response may be an inhibitor of the moth's odor response. The moth may be a silkworm moth. The inhibitor of the odor response of the insect may be an inhibitor of the odor response of a cockroach. The cockroach may be an American cockroach.

[0023] The insect inhibiting system according to an embodiment of the present invention is a spray system that sprays an inhibitor of the odor response of insects. The insect odor response inhibitor is the above-mentioned inhibitor of the insect odor response. may be.

[0024] The insect inhibition system according to an embodiment of the present invention is a screening method for an inhibitor of the odor response of the insect. The apparatus is provided with a spraying unit that sprays an inhibitor against the insect's odor response obtained by the leaning method.

[0025] In the above insect inhibition system, the spraying unit applies an inhibitor to the odor response of insects to the insects in the form of fine particles. It may also be made into

[0026] In the above insect inhibition system, the spraying unit applies an inhibitor to the odor response of insects to the insects in the form of fine particles. a two-fluid nozzle, an ultrasonic vibrator, a piezoelectric element, and an electrostatic spray nozzle; The device may include at least one selected from the above.

[0027] In the above insect inhibition system, the solvent of the inhibitor for the insect's odor response is ethanol. a solvent selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water; At least one of the above may be selected.

[0028] In the above insect inhibition system, the inhibitor of the insect's odor response is 4-isopropyl Contains 4-(tert-butyl)-3-methylphenol or 4-(tert-butyl)-2-methylphenol It's okay to be.

[0029] A method of inhibiting insects according to an aspect of the present invention includes spraying an insect inhibiting solution. The inhibitor of the odor response may be an inhibitor of the odor response of the insect.

[0030] The insect inhibition method according to an embodiment of the present invention comprises screening for inhibitors of the odor response of the insects. The method includes spraying the insect-inhibiting solution obtained by the rinsing method.

[0031] In the above-mentioned insect inhibition method, an inhibitor of the odor response of insects is sprayed. may be micronized.

[0032] In the above-mentioned insect inhibiting method, in the spraying, a two-fluid nozzle, an ultrasonic vibrator, At least one selected from the group consisting of a piezoelectric element and an electrostatic spray nozzle, The inhibitor of the response may be micronized.

[0033] In the above insect inhibition method, the solvent of the inhibitor for the insect's odor response is ethanol, A solvent selected from the group consisting of hexane, isopropyl alcohol, acetone, DMSO, and water. At least one of the above may be used.

[0034] In the above insect inhibition method, the inhibitor of the insect's odor response is 4-isopropyl- Contains 3-methylphenol or 4-(tert-butyl)-2-methylphenol That's fine. [Effects of the Invention]

[0035] According to the present invention, a method for screening an inhibitor of an insect's odor response, It is possible to provide inhibitors, insect inhibition systems, and insect inhibition methods for the answer. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 is a schematic diagram showing a method for expressing an olfactory receptor in an odor detection cell according to an embodiment. [Figure 2] 1A and 1B are schematic diagrams illustrating a method for homogenizing odor detection cells according to an embodiment. [Figure 3] FIG. 1 is a schematic diagram showing a method for expressing multiple olfactory receptors in odor detection cells according to an embodiment. [Figure 4] FIG. 1 is a schematic diagram showing a method for expressing multiple olfactory receptors in odor detection cells according to an embodiment. [Figure 5] 1 is a schematic diagram of an insect inhibition system according to an embodiment. [Figure 6] 1 is a schematic diagram of an insect inhibition system according to an embodiment. [Figure 7] 1 is a schematic diagram of an insect inhibition system according to an embodiment. FIG. [Figure 8] 1 is a schematic diagram of an insect inhibition system according to an embodiment. [Figure 9] 10 is a graph showing the change over time in the intensity of fluorescence emitted by odor detection cells according to an example. [Figure 10] 1 is a graph showing the relationship between the concentration of inhibitors and candidate inhibitors for the odor response of insects in an example and the normalized intensity of fluorescence emitted by odor detection cells. [Figure 11] 1 is a graph showing the relationship between the concentration of a candidate inhibitor of an insect's odor response and the normalized intensity of fluorescence emitted by odor detection cells in an example. [Figure 12] 1 is a graph showing the relationship between the concentration of inhibitors and candidate inhibitors for the odor response of insects in an example and the normalized intensity of fluorescence emitted by odor detection cells. [Figure 13]1 is a graph showing the relationship between inhibitors and candidate inhibitors of insect odor responses in an example and the normalized intensity of fluorescence emitted by odor detection cells. [Figure 14] 1 is a photograph showing an EAG measurement method according to an embodiment. [Figure 15] 10 is a graph showing the change over time in antennal potential according to an example. [Figure 16] 10 is a graph showing the change over time in antennal potential according to an example. [Figure 17] 1 is a table showing insect responses to multiple concentrations of bombykol according to an example. [Figure 18] 1 is a table showing the response of insects to multiple concentrations of bombykol after feeding them with inhibitors according to the examples. [Figure 19] 1 is a table showing insect responses to multiple concentrations of bombykol according to an example. [Figure 20] 1 is a table showing the response of insects to multiple concentrations of bombykol after feeding them with inhibitor candidate substances according to an example. [Figure 21] 1 is a table showing insect responses to multiple concentrations of bombykol according to an example. [Figure 22] 1 is a table showing the response of insects to multiple concentrations of bombykol after feeding them with inhibitor candidate substances according to an example. [Figure 23] 1 is a graph showing the relationship between the concentration of inhibitors and candidate inhibitors for the odor response of insects in an example and the normalized intensity of fluorescence emitted by odor detection cells. [Figure 24] 1 is a photograph of a container for observing the mating behavior of cockroaches according to an embodiment. [Figure 25] 1 is a graph showing the number of cockroaches accessing pieces of filter paper or aluminum pieces impregnated with pheromones according to the examples. [Figure 26] 1 is a graph showing the number of cockroaches accessing pieces of filter paper or aluminum pieces impregnated with pheromones according to the examples. DETAILED DESCRIPTION OF THE INVENTION

[0037] The method for screening an inhibitor of an insect's odor response according to the embodiment is a method for screening an inhibitor of an insect's odor response by detecting an olfactory receptor of the insect. An odor-detecting structure expressing at least one of an olfactory receptor and a co-receptor of the olfactory receptor is provided. The odorant corresponding to the olfactory receptor and each of the multiple inhibitor candidate substances are then subjected to the odor analysis. and multiple inhibitions based on the influx of ions into the odor sensing structure. and screening candidate drug substances.

[0038] The odor-sensing structure may be a cell or an artificial structure.

[0039] When the odor-detecting structure is a cell, the odor-detecting cell expresses olfactory receptors on the cell membrane. In odor-detecting cells, olfactory receptors can be naturally expressed or transgenic. The olfactory receptor may be an insect olfactory receptor.

[0040] The odor-detecting cells may be insect cells. The insect cells may be Spodoptera frugiperda. ra frugiperda) and Trichoplusia n i) and other moth-derived cells. Examples of cells derived from armyworm include Sf21 and Sf9 cells are derived from ovarian cells. Sf21 cells are cells that divide indefinitely. It is possible to establish a stable expression line that permanently expresses the introduced gene. Sf21 cells can survive over a wide temperature range from 18°C ​​to 40°C, and the pH of the culture medium can be adjusted. They also do not require carbon dioxide for regulation. Sf21 cells do not naturally have olfactory receptors. However, it is possible to express olfactory receptors by introducing the olfactory receptor gene. Sf9 cells are a clone of Sf21. Examples include High Five and Tni. Tni-derived cells are derived from ovarian cells. be.

[0041] Alternatively, the insect cells may be cells derived from Drosophila. Examples of cells derived from Drosophila include Drosophila S2 cells. Alternatively, the insect cells may be cells derived from a cockroach.

[0042] Olfactory receptors are ionotropic receptors. Olfactory receptors are G protein-coupled receptors. It may be a receptor or an ionotropic receptor. The body has sites where the ligands, which are odor molecules, interact and sites where ions flow in. When the ionotropic receptors of the odor-sensing cells bind to the ligand, Cations such as sodium ions and calcium ions flow in. In odor detection cells, Ion influx can occur within tens of milliseconds of ligand binding. The amount of ions that flow into the cell per one ligand binding is 10 7 Also called "pieces" It is being done.

[0043] Generally, a particular type of olfactory receptor has specificity for a particular odor molecule. In the exocytic cells, only one type of olfactory receptor corresponding to one type of odor molecule may be expressed. Alternatively, multiple types of olfactory receptors corresponding to multiple types of odor molecules may be expressed. The amount of olfactory receptors expressed may be adjusted. Insect olfactory receptors are expressed in flies, mosquitoes, moths, and bees. Many insect species, such as lice and cockroaches, have similar odorant receptor systems. Therefore, an inhibitor of an olfactory receptor of a specific insect will have an effect on the olfactory receptors of all insects. It can act as an inhibitor against

[0044] The odor-sensing structure may express an olfactory receptor as well as a co-receptor for the olfactory receptor. Co-receptors form heterocomplexes with olfactory receptors and function as receptors for odorants. Insect coreceptors are also similar in many insect species, including flies, mosquitoes, moths, bees, lice, and cockroaches. The olfactory receptors of certain insects have a mechanism for detecting similar odorants. Inhibitors for the co-receptors of the olfactory receptors of insects in general may function as co-receptors for the olfactory receptors of insects in general. It is possible.

[0045] An example of an olfactory receptor is the Drosophila receptor, which detects the musty odor of geosmium. DmOr56a, a receptor for olfactory or fruit odors, and Drosophila receptors DmOr82a, a receptor for the fruit odorant geranyl acetate, is a Drosophila receptor. It is a receptor for 2-methylphenol (o-cresol), which has the odor of human sweat. DmOr49b, a Drosophila receptor for the moldy odor 1-octene-3 DmOr13a, a receptor for 1-octen-3-ol, is a sex receptor in the silkworm moth. BmOR1, a receptor for the pheromone bombykol, in the silkworm moth BmO is a receptor for bombykal, a minor component of the sex pheromone of the Japanese bat, R3, DmOr85b, a general odorant receptor in Drosophila melanogaster, and the sex receptor in the diamondback moth. Examples of the pheromone receptor include, but are not limited to, PxOR1. "Sumin" is also called "geosmin."

[0046] When olfactory receptors are genetically engineered to be expressed in odor-detecting cells, for example, as shown in Figure 1, Then, a gene encoding an olfactory receptor is inserted into a vector, and the constructed vector is then transfected into host cells. The gene encoding the olfactory receptor may be transfected into the insect. mRNA can be extracted from the olfactory organ and cDNA can be synthesized and isolated. From the cDNA, a part of the gene encoding the olfactory receptor was cloned using PCR primers. It can be amplified using the R method.

[0047] A portion of the gene encoding the olfactory receptor was synthesized by inserting double-stranded cDNA into an appropriate vector. The vector is then used to transform E. coli or the like to create a cDNA library. cDNA can also be obtained by the conventional method using restriction enzymes and ligase. For example, the obtained cDNA is cleaved with a restriction enzyme to remove the restriction enzyme site of the vector DNA. The vector can be incorporated by inserting the vector into a vector and ligating it into the vector.

[0048] An odorant that corresponds to an olfactory receptor is an odorant that specifically reacts with an olfactory receptor. The odorant corresponding to DmOr56a is geosmin. The odorant corresponding to DmOr49b is geranyl acetate. The odorant corresponding to DmOr13a is 1-octene. The odorant corresponding to BmOR1 is 1-octen-3-ol. The odorant corresponding to BmOR3 is bombykol. The odorant corresponding to PxOR1 is the sex pheromone of the diamondback moth. That's it.

[0049] In odor-detecting cells, fluorescent proteins whose fluorescence intensity changes depending on ions are expressed. As described above, in odor detection cells, ionotropic olfactory receptors When odor molecules bind, ions flow into the odor-detecting cells. By introducing a gene that expresses a fluorescent protein whose fluorescence intensity changes into odor-detecting cells, Therefore, the change in fluorescence intensity or the rate of change in fluorescence intensity indicates that odor-detecting cells detect odor molecules. It is possible to confirm whether or not the fluorescent protein is present. These include GCaMP3, GCaMP6s, and aequorin, which emit fluorescence in response to ions. do.

[0050] When odorant molecules react with olfactory receptors, the concentration of ions flowing into the odor-detecting cells increases. Therefore, the intensity of the fluorescence emitted by odor-detecting cells is When the reaction between olfactory receptors and odor molecules is blocked by an inhibitor, The concentration of ions flowing into the cell becomes low or zero, and fewer fluorescent proteins emit fluorescence. As a result, the intensity of the fluorescence emitted by the odor-detecting cells becomes weaker. Therefore, based on the fluorescence intensity corresponding to the influx of ions into the odor-detecting cells, It is possible to evaluate whether the reaction between olfactory receptors and odor molecules is inhibited by an inhibitor. is.

[0051] The influx of ions into the odor detection cells may be measured electrically. A transistor having a source electrode, a drain electrode, and a gate electrode is disposed in the vicinity of the When odorant molecules react with olfactory receptors and ions flow into the odor-detecting cells, a transistor The gate potential of the gate electrode of the transistor is changed, and the drain current flowing between the source electrode and the drain electrode is When the reaction between olfactory receptors and odor molecules is inhibited by an inhibitor, the in-current is modulated. The concentration of ions flowing into the odor-detecting cells becomes low or zero, resulting in modulation of the drain current. Therefore, the signal corresponding to the influx of ions into the odor-detecting cells is low or absent. The response of olfactory receptors to odor molecules is detected by detecting modulation of the drain current of the olfactory transistor. It is possible to evaluate whether or not the activity is inhibited by an inhibitor.

[0052] Odor-detecting cells (a) select a subset of cells from a group of cells with olfactory receptors, and (b) select (a) Proliferating the selected cells and (c) confirming the responsiveness of the proliferated cells to odorants. Steps (a) to (c) are carried out multiple times, and the cells whose response to the odorant is greater than or equal to the standard value are selected. The cells selected in step (a) may be single cells. It is also possible.

[0053] Alternatively, odor-detecting cells may be (a) a subset of cells that have olfactory receptors. (b) Proliferate the selected cells, and (c) confirm the responsiveness of the proliferated cells to odorants. The steps (a) to (c) were carried out multiple times, and the strains with the highest response to odorants were grown. The cells selected in step (a) may be single cells. ) may also be used.

[0054] Specifically, as shown in Figure 2, cells that have olfactory receptors and express fluorescent proteins By repeatedly diluting the cell lineage population, a single or small number of cells are selected and cultured. The cell line may be established by culturing and growing the cells. Among the established cell lines, those that respond to odorants are A cell line whose response is equal to or greater than a predetermined standard value may be used as an odor detection cell. Among the multiple cell lines established, the cell line with the highest odorant response was selected for odor detection. It may also be used as an explant cell.

[0055] As shown in Figures 3 and 4, the established olfactory receptor-expressing cells were further transfected with other olfactory receptors. In other words, the first olfactory receptor established by the above method or the like may be expressed. A second olfactory receptor may be further expressed in the cells expressing the second olfactory receptor. The gene encoding the receptor was inserted into a vector, and the constructed vector was used to express the first olfactory receptor. The first olfactory receptor and the second olfactory receptor are transfected into cells expressing the It is possible to establish cells expressing olfactory receptors. The cells expressing the first olfactory receptor may also express multiple different olfactory receptors. The second olfactory receptor, Or-X, was introduced into cells expressing Or56a as a receptor. This figure shows an example of introducing a vector containing a gene and a vector containing an antibiotic resistance gene. Figure 4 shows the results of the transduction of a second olfactory receptor into cells expressing Or56a as the first olfactory receptor. We present an example of introducing a vector containing both the Or-X gene and an antibiotic resistance gene. are.

[0056] If the odor-detecting structure is an artificial structure, the artificial structure may be patterned with cells. The artificial structure may, for example, comprise a vesicle having a membrane, and an olfactory receptor disposed in the membrane. The artificial structure contains fluorescent proteins in the membrane that emit fluorescence depending on the concentration of ions. The artificial structure can be manufactured by referring to, for example, Non-Patent Document 1. It is Noh.

[0057] The odorant and each of the plurality of candidate inhibitor substances may be simultaneously applied to the odor detection structure. Alternatively, after applying the odorant to the odor detection structure, each of the multiple inhibitor candidate substances Alternatively, each of the plurality of candidate inhibitor substances may be applied to the odor-detecting structure. After the odorant is applied to the odor sensing structure, the odorant may be applied to the odor sensing structure. The candidate inhibitors that inhibited the influx of ions into the odor-detecting structure were found to be effective in the odor-sensing mechanisms of insects. are selected as inhibitors of the response.

[0058] The multiple inhibitor candidate substances screened using the odor detection structure were further screened. For example, screening of inhibitors of the odor response of insects according to the embodiment may be carried out. The stimulating method involves preparing an insect or a part of an insect and applying a stimulating agent to the insect or part of the insect. The stimulatory substance and the candidate inhibitor selected in the above screening are then injected into the insect or a part of the insect. and detecting a response from the insect or part of the insect.

[0059] Examples of insects include moths, cockroaches, mosquitoes, flies, bees, and lice. Examples of insects include the silkworm moth, the diamondback moth, the armyworm, and the pyralid moth. These include the Asian cockroach, the German cockroach, the American cockroach, and the American cockroach. Brown cockroach, Smoked cockroach, Japanese cockroach, Urushi cockroach, and Suzuki cockroach Examples of parts of insects include antennae, proboscises, legs, and wings.

[0060] Examples of irritants to insects or insect parts include bombykol, periplanone A, Periplanone B, and 1-octen-3-ol.

[0061] The stimulator and multiple inhibitor candidate substances were simultaneously administered to insects or a subset of insects. Alternatively, a stimulus may be administered to the insect or a part of the insect, and then multiple inhibitor candidate substances may be administered. Alternatively, multiple candidate inhibitors may be administered to the insect or a portion of the insect. After each of the above is given to the insect or a part of the insect, a stimulating substance is given to the insect or a part of the insect. Good too.

[0062] The behavior of the insects may be observed to detect their response. The candidate inhibitors that inhibited the insect's behavior were selected as inhibitors of the insect's odor response. Examples of insect behavior include flapping behavior, searching behavior, and mating behavior. In addition, the antennal potential may be detected in detecting the response of the insect's antennae. The candidate inhibitors that inhibited the changes in antennal potentials induced by stimulants were shown to have a significant effect on the insect's odor response. It is selected as an inhibitor of

[0063] In some embodiments, the inhibitor of insect odor response inhibits the influx of ions into the odor-sensing structure. less than 37%, 35% or less, 30% or less, 25% or less, 20% or less, or less than 20% For example, by observing the fluorescence intensity in the odor-sensing structure, the odor-sensing structure can be detected. When evaluating the influx of ions into the insect, an inhibitor of the insect's odor response according to an embodiment may be The fluorescence intensity is less than 37% of the fluorescence intensity generated by odor molecules in the absence of inhibitors. It can be 35% or less, 30% or less, 25% or less, 20% or less, or less than 20%. By observing the drain current of the transistor near the detection structure, the odor detection structure When evaluating the influx of ions into the insect, an inhibitor of the insect's odor response according to an embodiment may be In contrast to the modulation of drain current caused by odor molecules in the absence of inhibitors, the change in drain current The ratio is less than 37%, 35% or less, 30% or less, 25% or less, 20% or less, or less than 20%. The rate of decrease in ion inflow or drain current can be It may also be a value related to the rate of decrease in fluorescence intensity.

[0064] Inhibitors of insect odor responses include, for example, methylphenol derivatives. The inhibitor for the response is 4-isopropyl-3-methylphenol shown in the following chemical formula 1. The inhibitor of the insect odor response may be a 4-(te It may contain (rt-butyl)-2-methylphenol. Inhibitor of insect odor responses. The inhibitor may include 2-isopropyl-6-methylphenol shown in the following chemical formula 3. The inhibitor for the insect odor response is 2,4-diisopropylphenyl ether, shown in the following chemical formula 4. The inhibitor of the insect odor response may be a 2-ol represented by the following chemical formula 5. tert-butyl-4-methylphenol may be included.

[0065] [ka] [ka] [ka] [ka] [ka]

[0066] The inhibitor of the insect odor response may comprise a cinnamic acid derivative. Examples of cinnamates include methyl trans-cinnamate and tert-butyl trans-cinnamate. can be done.

[0067] Examples of solvents for inhibitors of insect odor responses include ethanol, hexane, and isopropyl alcohol. Examples of suitable insect odorants include alcohol, acetone, DMSO, and water. Inhibitors of the response can be used, for example, as inhibitors of the odor response of moths. The silkworm may be a silkworm moth.

[0068] As shown in FIG. 5, the insect inhibition system according to the embodiment inhibits the odor response of insects. The insect odor response inhibitor is a spraying unit 20 that sprays the agent. The spraying unit 20 may be, for example, an inhibitor of the odor response of insects. a reservoir 21 for storing a solution containing an insecticide; a solution containing an inhibitor for the insect's odor response in the form of microparticles; and a solution containing an inhibitor for the odor response of the atomized insects. The solvent of the solution may be any of the solvents described above.

[0069] The reservoir 21 is, for example, a tank, and includes a main body 21a and a lid 21b. is a two-fluid mixing device that mixes and atomizes two fluids, liquid and gas, arranged in the storage section 21. The blowing unit 23 may be provided on the cover 21b.

[0070] The two-fluid nozzle 25 has a gas inlet 25a through which gas flows in and a gas inlet 25b through which the odor of insects in the storage section 21 is detected. A solution inlet 25b is provided through which a solution containing an inhibitor for the gas is introduced. For example, the two-fluid nozzle 25 is provided with a spray nozzle 26. The solution containing the inhibitor against the insect's odor response in the form of a liquid film formed at the spray nozzle 26 is The particles are atomized by the shear force of the airflow and sprayed out from the spray nozzle 26. The solution containing the inhibitor against the granulated insect odor response is blown out of the reservoir 21 from the blowout section 23. is released to

[0071] The gas inlet 25a of the two-fluid nozzle 25 is connected to a gas inlet 25a via a connecting part 27 such as a pipe joint. A control unit 30 for supplying gas to the nozzle 25 is connected. The control unit 30 includes, for example, , a pump 32 for supplying air, and a gas supply pipe 3 connecting the pump 32 and the connection part 27. 4. A valve 33 such as an electromagnetic valve is provided on the gas supply pipe 34. The valve 33 is electrically connected to the controller 31. The controller 31 The pump 32 and the valve 33 are controlled to adjust the flow rate, pressure, etc. of the gas supplied to the two-fluid nozzle 25. Control.

[0072] Alternatively, an insect inhibiting system according to an embodiment may have the configuration shown in FIG. The spraying unit 120 of the insect inhibition system shown in FIG. 6 is, for example, an inhibitor of the odor response of insects. a storage section 41 for storing a solution containing an inhibitor for the insect's odor response; and a microparticulation unit 42 that sprays out a solution containing an inhibitor of the insect's odor response. It is equipped with a blowing section 43.

[0073] The reservoir 21 is, for example, a tank, and includes a main body 21a and a lid 21b. The granulation unit 42 is connected to a pipe for delivering a solution containing an inhibitor for the insect's odor response. The atomization unit 42 collects a solution containing an inhibitor for the odor response of insects, for example. a nozzle head 47 for receiving the piezoelectric element 4 such as a piezo element disposed in the nozzle head 47; The blowing unit 43 is provided in a nozzle head 47.

[0074] When a pulse voltage is applied to the piezoelectric element 45, it repeatedly deforms and restores its original shape. The volume of the nozzle head 47 repeatedly contracts and restores. The solution containing the inhibitor for the odor response is intermittently extruded from the blowout portion 43 and atomized. do.

[0075] The piezoelectric element 45 is connected to the control unit 130 via a wiring 48. The control unit 130 A voltage is applied to the piezoelectric element 45 to control the amount of deformation of the piezoelectric element 45 .

[0076] Alternatively, the insect inhibiting system according to the embodiment may have the configuration shown in FIG. The spraying unit 60 of the insect inhibition system shown in FIG. 7 sprays, for example, an inhibitor against the odor response of insects. a reservoir 61 for storing a solution containing an inhibitor for the insect's odor response; A microparticulation unit 62, which sprays out a solution containing atomized inhibitors of insect odor responses. It has a blowing section 63.

[0077] The storage section 61 is, for example, a flexible bag-shaped container. At least a part of the storage section 61 is The blowing section 43 is provided in the storage section 61. The storage section 61 is The particles are held in a vibration generating container 66 filled with working water 67. Equipped with a vibrator.

[0078] When a high-frequency AC voltage is applied to the ultrasonic vibrator, it vibrates ultrasonically. The vibration energy generated by the vibration is transferred to the reservoir 61 through the working water 67 and the ultrasonic transmission membrane 65. This allows the insects in the reservoir 61 to reach a solution containing an inhibitor for their odor response. A solution containing an inhibitor of the insect's odor response was vibrated, atomizing the solution surface and forming fine particles. A solution containing an inhibitor of the insect's odor response is blown out from blowout section 63.

[0079] The ultrasonic vibrator of the atomization unit 62 is connected to the control unit 230 via a wiring 70. The control unit 230 applies an AC voltage to the ultrasonic vibrator to control the vibration amount of the ultrasonic vibrator. do.

[0080] Alternatively, the insect inhibiting system according to the embodiment may have the configuration shown in FIG. The spraying unit 80 of the insect inhibition system shown in FIG. 8 sprays, for example, an inhibitor against the odor response of insects. a reservoir 81 for storing a solution containing an inhibitor for the insect's odor response; An electrostatic atomizer 82 and a solution containing an inhibitor of the insect's odor response that has been atomized. It is provided with a blowout part 83 for blowing out liquid.

[0081] The storage unit 81 is, for example, a tank, and includes a main body 81a and a lid 81b. The atomization unit 82 is disposed in the storage unit 81. The unit 82 includes an electrostatic spray nozzle 84 and a conveying unit 85 such as a pump. The spray nozzle 84 delivers a solution containing an inhibitor of the insect's odor response. A cylindrical member 86 is disposed around the periphery of the electrode 84. The upper end surface of the cylindrical member 86 is provided with a counter electrode or the like. A voltage application unit 87 is provided.

[0082] A high voltage is applied to the electrostatic spray nozzle 84 and the outside of the electrostatic spray nozzle 84 by the voltage application unit 87. Then, at the gas-liquid interface, the balance between the surface tension of the solution and the electrostatic force acting on the solution is The tip of the fine liquid thread is split into fine particles and ejected from the electrostatic spray nozzle 84. It is sprayed.

[0083] The voltage application unit 87 is connected to a high voltage control unit 330 via a wiring 71. The control unit 330 applies a high voltage to the voltage application unit 87 to control the electrostatic force acting on the solution. do.

[0084] Example 1: Establishment of a homogeneous odor-detecting cell line DmOr13a and DmOrco are expressed in Drosophila melanogaster. The olfactory receptors derived from the antennae of the genus Melanogaster are used to detect the target odor. GCaMP6s is an improved calcium-sensitive fluorescent protein that responds to 10-3-ol. The pIB vector containing DmOr13a and DmOrco, and GCaMP The pIZ vector containing 6s was transfected into Sf21 cells by lipofection. .

[0085] Grace's Insect Medium, Supplemented(116 05-094, Gibco) with a final concentration of 10% US Insect Cell Scr ed FBS (SH30070.03, GE Healthcare) and three antibiotics ( Gentamicin Reagent Solution (1 5710-064, Gibco), Blasticidin S at a final concentration of 10 μg / mL Zeocin HCl (A11139-03, Gibco), final concentration 100 μg / mL (R25001, Invitrogen) was added to prepare a subculture medium. Using the method, we identified the DmOr13a receptor and its co-receptor, DmOrco, and GCaMP6s. Sf21 cells expressing IgG were subcultured in flasks (353082, FALCON). The volume of the cell suspension at the time of subculture was 6 mL. When the cells became confluent, 6 mL of the supernatant was collected from the tube and placed in a 15 mL tube (91015, TPP). The 15 mL tube was centrifuged at 400 xg and 4°C for 3 minutes using a high-speed centrifuge.

[0086] After centrifugation, the supernatant was transferred into a 10 mL syringe (01007, TOP) and a 0.45 μm filter ( The sterilized supernatant was sterilized using an antibiotic (final concentration: 431220, Corning). 10 μg / mL Blasticidin S HCl, final concentration 100 μg / mL Ze Mix with an equal volume of fresh subculture medium containing ocin to make 10 mL of conditioned medium. The medium was prepared.

[0087] The cells attached to the bottom of the flask were removed from the supernatant and suspended in 1 mL of fresh medium. The cell suspension was collected in a 1.5 mL tube (MCT-150-C, AXYGEN). A cell suspension containing 40 cells was extracted and added to the conditioned medium described above, followed by thorough pipetting. The entire volume of the conditioned medium containing the cells was poured into a reservoir (BM-0850- 1, BMBio). Then, an 8-channel pipette (HT5123, HT Cells were added to a 96-well plate (3860-096, IWAKI) using a PBS (L). 100 μL of the conditioned medium was added dropwise to each well, and the cells were then cultured at 27°C. After the cells adhere to the wells, observe the wells with an inverted microscope to see if only single cells are present. The wells containing conditioned medium were identified.

[0088] The cells in the wells where single cells were confirmed at the time of seeding were approximately 80% to 90% confluent. The culture was continued until the cells were swelled. Then, the cells were transferred to a 24-well plate (3820-024, IWAKI) , 35mm dish (353801, CORNING), and T-25 flask in that order. The cells were scaled up to 24-well plates, 35 mm dishes, and T-25 flasks. For the culture in , adjust the volume of the medium so that the liquid volume is 500 μL, 2.5 mL, and 5 mL, respectively. The cells were cultured at 27°C and the odorant was scaled up to a T-25 flask. The response to α-glucan was investigated by calcium imaging, and the cell lines that showed good response were The lineage was obtained as a homogeneous odor-detecting cell line.

[0089] Example 2: Response of odor-detecting cells to inhibitors A 12 mm diameter cover glass (CS-12R: Warner Instruments) , LLC, Hamden, CT, USA) after seeding the odor-detecting cells obtained in Example 1. The cover glass was placed in an open bath chamber (RC-48LP: Warn The catheter was inserted into a catheter (Fer Instruments, LLC, Hamden, CT, USA).

[0090] To perfuse the solution into the odor detection cells, a peristaltic tube pump (MP-2 010:Tokyo Rikakikai Co. Ltd., Tokyo, Japan) Two silicone tubes with an inner diameter of 1 mm and an outer diameter of 3 mm were connected to the tube clamp. (CAT-1: NARISHIGE Co. Ltd., Tokyo, Japan) Therefore, they were connected to the inlet and outlet of an open-type bath chamber.

[0091] 20x water immersion objective lens (UMPlanFI 20x / 0.50W: Olympus, Tokyo, Japan) equipped with an upright fluorescent microscope (BX51WI: Olympus, T The upright fluorescence microscope was equipped with a fluorescent filter set for GFP. The NET (U-MGFPHQ: Olympus, Tokyo, Japan) was also installed. The upright fluorescence microscope was equipped with a 100W halogen lamp (TH4-100, Olympus) as a light source. The exposure time for fluorescence observation was set to 500 ms.

[0092] To measure changes in the fluorescence intensity of cells, an EM-CCD camera (DU-897E: Ando r Technology PLC, Belfast, UK). The camera is manufactured by AndoriQ (Andor Technology PLC, Belfas The EM-CCD camera captured 512 x 512 pixel images per second. was set to gain.

[0093] Perfusion with assay buffer was initiated. The flow rate was set to approximately 1.4 mL / min. The volume of the liquid inside the bar was set to approximately 230 μL. As shown in Figure 9, the odorant, 10 μmol When 1 / L of 1-octen-3-ol was poured together with the buffer for 15 seconds, the odor An increase in fluorescence intensity was observed in the exocytic cells.

[0094] 300 μmol / L geraniol, a candidate inhibitor, was injected for 60 seconds, followed by the odorant 10 μmol / L of 1-octen-3-ol is mixed with 300 μmol / L of geraniol. The test was then run with 300 μmol / L geraniol for 15 seconds, followed by 60 seconds of geraniol. The increase in fluorescence intensity in odor-detecting cells was suppressed compared to the absence of an odorant inhibitor. Controlled.

[0095] 300 μmol / L l-menthol, a candidate inhibitor, was injected for 60 seconds, followed by the odorant. The substance 10 μmol / L 1-octen-3-ol was added to 300 μmol / L l-menol. The solution was then washed with ethanol for 15 seconds, followed by 300 μmol / L l-menthol for 60 seconds. The increase in fluorescence intensity in odor-detecting cells was greater than that in the absence of odorant inhibitors. and was suppressed.

[0096] 300 μmol / L thymol, a candidate inhibitor, was injected for 60 seconds, followed by the odorant 10 μmol / L 1-octen-3-ol with 300 μmol / L thymol When 300 μmol / L thymol was poured into the filter for 15 seconds and then for another 60 seconds, the odor was detected. The increase in fluorescence intensity in the cells was suppressed compared to the case without the odorant inhibitor.

[0097] A 60-second flow of 300 μmol / L linalyl formate (LF), a known odorant inhibitor, was performed. Next, 300 μmol of 1-octen-3-ol (10 μmol / L) was added. LF was then passed for 15 seconds, and then 300 μmol / L LF was passed for another 60 seconds. The increase in fluorescence intensity in odor-detecting cells was suppressed compared to the absence of an odorant inhibitor. Controlled.

[0098] 300 μmol / L of 2-tert-butyl-6-methyl, a known odorant inhibitor, The odorant, 10 μmol / L of 1-octylphenol (BMP), was then injected for 60 seconds. Ten-3-ol was injected with 300 μmol / L BMP for 15 seconds, and then injected for another 60 seconds. When 00 μmol / L of BMP was injected, the increase in fluorescence intensity in the odor-detecting cells was The activity was suppressed compared to the absence of the inhibitor.

[0099] Finally, the odorant, 10 μmol / L 1-octen-3-ol, was added to the buffer. When both were flowed for 15 seconds, the increase in fluorescence intensity in the odor-detecting cells was restored.

[0100] The results of Example 2 show that odor-detecting cells are useful for screening inhibitors of odorants. This shows that...

[0101] Example 3: Response of odor detection cells depending on the concentration of fragrance components The inhibitor candidates are thymol, citral, 1-nonanol, and eugenol acetate. Inhibition of d-limonene, eugenol, geraniol, geranyl acetate, and known odorants The agents BMP and LF were prepared.

[0102] As in the measurement method of Figure 9, the odorant was 10 μmol / L 1- Octen-3-ol was applied to the odor detection cells obtained in Example 1, and the fluorescence in the odor detection cells was measured. Next, 10 μmol / L of the inhibitor candidate or inhibitor, 30 μmol / L of inhibitor candidate or inhibitor, 100 μmol / L of inhibitor candidate or inhibitor, 1 70 μmol / L of inhibitor candidate or inhibitor, 300 μmol / L of inhibitor candidate or 560 μmol / L of inhibitor candidate substance or 1 mmol / L of inhibitor candidate substance Supplementary substance or inhibitor, 3mmol / L inhibitor candidate substance or inhibitor, 10μmol / L sequentially The odor detection cells were given 1-octen-3-ol together with the Finally, without any inhibitor, 10 μmol / L of 1-octen-3- ol was applied to the odor detection cells, and the fluorescence intensity in the odor detection cells was measured.

[0103] 10 μmol / L of 1-octen-3-ol was administered to odor-detecting cells without any inhibitors. The average rate of change in fluorescence intensity in the odor-detecting cells when the inhibitors were administered was set at 100%. The relationship between the concentration of the inhibitor and the normalized intensity of fluorescence in the odor-detecting cells was obtained. The relationship is shown in the graph in Figure 10. As shown in Figure 10, the odorant activity in the odor detection cells The response to α-glucan was suppressed in a concentration-dependent manner by the inhibitor.

[0104] The results of Example 3 show that odor detection cells are useful for screening inhibitors of odorants. This shows that...

[0105] Example 4: Response of odor-detecting cells depending on the concentration of inhibitor candidate substances The inhibitor candidates, methyl trans-cinnamate and tert-butyl trans-cinnamate, We prepared a rule.

[0106] Without the inhibitor candidate, the odorant 10 μmol / L 1-octen-3- ol was applied to the odor detection cells obtained in Example 1, and the fluorescence intensity in the odor detection cells was measured. Next, we tested the inhibitor candidate at 10 μmol / L, 30 μmol / L, and 10 0 μmol / L of inhibitor candidate substance, 170 μmol / L of inhibitor candidate substance, 300 μmol l / L of the inhibitor candidate, 560 μmol / L of the inhibitor candidate, and 1 mmol / L of The inhibitor candidate substances were sequentially administered to the odor detection cell along with 10 μmol / L of 1-octen-3-ol. The intensity of fluorescence in the odor-detecting cells was measured. 10 μmol / L of 1-octen-3-ol was applied to the odor detection cells. The intensity of the fluorescence was measured.

[0107] Without the addition of inhibitor candidate substances, 10 μmol / L of 1-octen-3-ol was added to the odor detection cell. The average change in fluorescence intensity in the odor-detecting cells when the odorant was administered to the cells was set as 100%, and the percentage of each inhibitor was calculated. Normalization of the concentration of the inhibitor candidate and the fluorescence in the odor detection cells for the inhibitor candidate The resulting relationship between the measured intensity and the odor intensity is shown in the graph in Figure 11. As shown in Figure 11, The response of the detector cells to the odorant was suppressed depending on the concentration of the inhibitor candidate.

[0108] Example 5: Response of odor-detecting cells depending on the concentration of inhibitors and inhibitor candidate substances The inhibitor candidate, 2,4-diisopropylphenol, 4-isopropyl-3- Methylphenol, 2-tert-butyl-4-methylphenol, 4-tert-butyl 2-isopropyl-6-methylphenol and 2-isopropyl-6-methylphenol were prepared.

[0109] Without the inhibitor candidate, the odorant 10 μmol / L 1-octen-3- ol was applied to the odor detection cells obtained in Example 1, and the fluorescence intensity in the odor detection cells was measured. Next, we tested the inhibitor candidate at 10 μmol / L, 30 μmol / L, and 10 0 μmol / L of the inhibitor candidate, 170 μmol / L of the inhibitor candidate, and 300 μmol / L of the inhibitor candidate. mol / L of the inhibitor candidate substance was sequentially treated with 10 μmol / L of 1-octen-3-ol. The inhibitor was then administered to the odor detection cells, and the fluorescence intensity in the odor detection cells was measured. Without the presence of 10 μmol / L of 1-octen-3-ol, the odor detection cells were given 10 μmol / L of 1-octen-3-ol. The intensity of fluorescence in the transfected cells was measured.

[0110] Without the addition of inhibitor candidate substances, 10 μmol / L of 1-octen-3-ol was added to the odor detection cell. The average change in fluorescence intensity in the odor-detecting cells when the odorant was administered to the cells was set as 100%, and the percentage of each inhibitor was calculated. Normalization of the concentration of the inhibitor candidate and the fluorescence in the odor detection cells for the inhibitor candidate The resulting relationship between the measured intensity and the odor intensity is shown in the graph in Figure 12. As shown in Figure 12, The response of the detector cells to the odorant was suppressed depending on the concentration of the inhibitor candidate.

[0111] Example 6: Evaluation of inhibitors and inhibitor candidate substances by their inhibitory effects on odor detection cells depending on their concentration screening) Based on the results of Examples 3 to 5, DEET, d-limonene, citral, linalyl formate , eugenol, geraniol, 1-nonanol, geranyl acetate, thymol, eugenol acetate ethanol, methyl trans-cinnamate, tert-butyl trans-cinnamate, 2,4-diisopropyl 4-isopropyl-3-methylphenol, 2-isopropyl-6- Methylphenol, 2-tert-butyl-4-methylphenol, and 4-tert- The graph of the fluorescence intensity change rate when the concentration of butyl-2-methylphenol is 300 μM. The rough outline is shown in Figure 13.

[0112] Methyl trans-cinnamate, tert-butyl trans-cinnamate, 2,4-diisopropyl Phenol, 4-isopropyl-3-methylphenol, 2-isopropyl-6-methyl Phenol, 2-tert-butyl-4-methylphenol, and 4-tert-butyl -2-Methylphenol reacts with 10 μmol / L of 1-octen-3-ol in the odor detection cell. The change in fluorescence intensity in the odor-detecting cells when administered to the cells was less than 37%. As shown in Example 7, the inhibitor BMP and the inhibitor candidate 4-isopropyl 4-tert-butyl-3-methylphenol and 4-tert-butyl-2-methylphenol are In Example 6, the fluorescent intensity of these substances was The change in the degree of change was less than 25% (24.7%). The fluorescence intensity change rate was 37% or more for eugenol acetate, 1-nonanol, and thymol. In the case of Example 6, no inhibitory effect was observed in the electroantennogram. The fluorescence intensity change rate of these substances was 37% or more. Therefore, the fluorescence intensity change rate value was 25%. If the rate of change in fluorescence intensity is less than 37%, the inhibitory effect is confirmed. It was also shown that 2-tert-butyl-4-methylphenol and 4-tert-Butyl-2-methylphenol is 10 μmol / L of 1-octene- When 3-ol was administered to odor detection cells, the rate of change in fluorescence intensity in the odor detection cells was 20% or more. I put it down.

[0113] Example 7: Antennae response to inhibitors As shown in Figure 14, gel droplets (Spectra 36) were placed on the surfaces of two metal electrodes. 0 Electrode Gel (Parker Laboratories) was placed on the The antennae of a silkworm moth, the tip and base of which had been cut off, were placed in contact with a gel drop and the antennae were placed between two metal electrodes. In response to odorants, the potential between the tip and base of an insect's antenna changes. It is known that the potential changes as a result of electroantennogram (EE). It is called AG.

[0114] Bombykol (BOL), the sex pheromone of the silkworm moth, is an inhibitor of known odorants. The BMP, the inhibitor candidate 4-isopropyl-3-methylphenol, and the inhibitor The candidate substance, 4-tert-butyl-2-methylphenol, was prepared. 1000ng Filter paper with 1000ng of BOL dropped on it, and filter paper with 1000μg of BOL and 1000μg of BMP dropped on it. , 1000ng of BOL and 1000ng of 4-isopropyl-3-methylphenol were dropped. The filter paper and 1000ng of BOL and 1000ng of 4-tert-butyl-2-methyl- A filter paper with ethylphenol dropped on it was prepared. BOL was dropped into the first glass tube cartridge. Place the filter paper with BOL and BMP in the second glass tube cartridge. Add BOL and 4-isopropyl-3-methylphenol to the third glass tube cartridge. The filter paper was placed in the fourth glass tube cartridge, and BOL and 4-tert-butyl-2-methyl- A filter paper with methylphenol dropped on it was placed inside.

[0115] When gas containing BOL was sprayed onto the antennae from the first glass tube cartridge at 1 L / min. The potential between the tip and base of the antennae decreased as shown in the upper part of Figure 15(a). When gas containing BOL and BMP was sprayed onto the antennae from a glass tube cartridge at 1 L / min. As shown in the upper part of Figure 15(b), the potential drop between the tip and base of the antennae was observed when BMP was not present. The amount of contact from the first glass tube cartridge was reduced to less than half compared to when the contact was not applied. When gas containing BOL was sprayed onto the antennae at 1 L / min, the potential between the tip and base of the antennae was As shown in the top row of 15(c), it decreased.

[0116] When gas containing BOL was sprayed onto the antennae from the first glass tube cartridge at 1 L / min. The potential between the tip and base of the antennae decreased as shown in the middle of Figure 15(a). BOL and 4-isopropyl-3-methylphenyl ether were injected into the antennae at 1 L / min from the glass tube cartridge. When gas containing nitrite was sprayed, the potential drop between the tip and base of the antennae was as shown in Figure 15(b ) As shown in the middle section, compared to the case without 4-isopropyl-3-methylphenol, Again, B was introduced into the antennae from the first glass tube cartridge at 1 L / min. When gas containing OL was injected, the potential between the tip and base of the antennae was as shown in the middle of Figure 15(c). As shown, it decreased.

[0117] When gas containing BOL was sprayed onto the antennae from the first glass tube cartridge at 1 L / min. The potential between the tip and base of the antennae decreased as shown in the bottom of Figure 15(a). BOL and 4-tert-butyl-2-methyl ... When gas containing phenol was sprayed, the potential between the tip and base of the antennae decreased as shown in Figure 15. (b) As shown in the bottom row, when 4-tert-butyl-2-methylphenol is not included Compared to the previous case, the amount of serotonin was reduced by about half. Again, 1 L was poured into the antennae from the first glass tube cartridge. When gas containing BOL was injected at 1000 kJ / min, the potential between the tip and base of the antennae increased as shown in Figure 15(c ) As shown in the bottom row, it decreased.

[0118] Bombykol (BOL), a sex pheromone of the silkworm moth, and acetic acid, a candidate inhibitor, were Igenol, the inhibitor candidate 1-nonanol, and the inhibitor candidate thymol We prepared a filter paper with 1000 ng of BOL dropped on it, a filter paper with 1000 ng of BOL and 1000 μL of BOL. g of eugenol acetate dropped on the filter paper, 1000 ng of BOL and 1000 μg of 1-nonanoic acid Filter paper with 1000ng of BOL and 1000ng of thymol dropped on it. The filter paper with BOL dropped on it was placed in the fifth glass tube cartridge, and the sixth Place the filter paper with BOL and eugenol acetate in the glass tube cartridge, and then insert the seventh glass tube. The filter paper on which BOL and 1-nonanol had been dropped was placed in the cartridge, and the eighth glass tube cartridge Filter paper with drops of BOL and thymol was placed in the jar.

[0119] When gas containing BOL was sprayed onto the antennae from the fifth glass tube cartridge at 1 L / min. The potential between the tip and base of the antennae decreased as shown in the upper part of Figure 16(a). Gas containing BOL and eugenol acetate was sprayed onto the antennae from a glass tube cartridge at a rate of 1 L / min. As shown in the upper part of Figure 16(b), the potential drop between the tip and base of the antennae was The results were enhanced compared to the case without eugenol acid. When gas containing BOL was sprayed from the edge of the antenna at 1 L / min, the area between the tip and base of the antenna was The potential decreased as shown in the upper part of FIG. 16(c).

[0120] When gas containing BOL was sprayed onto the antennae from the fifth glass tube cartridge at 1 L / min. The potential between the tip and base of the antennae decreased as shown in the middle of Figure 16(a). Gas containing BOL and 1-nonanol was sprayed onto the antennae from a glass tube cartridge at 1 L / min. As a result, the potential drop between the tip and base of the antennae was 1- The results were enhanced compared to the case without nonanol. Again, the fifth glass tube cartridge When gas containing BOL was sprayed onto the antennae at 1 L / min, the potential between the tip and base of the antennae increased. As shown in the middle of Figure 16(c), it decreased.

[0121] When gas containing BOL was sprayed onto the antennae from the fifth glass tube cartridge at 1 L / min. The potential between the tip and base of the antennae decreased as shown in the bottom of Figure 16(a). When gas containing BOL and thymol was sprayed onto the antennae from a glass tube cartridge at a rate of 1 L / min, The decrease in the potential between the tip and base of the antennae is due to the addition of thymol, as shown in the bottom panel of Figure 16(b). Again, the fifth glass tube cartridge was inserted into the antennae. When gas containing BOL was injected at 1 L / min, the potential between the tip and base of the antennae increased as shown in Figure 16( c) Decreased as shown in the bottom row.

[0122] The results of Example 7 demonstrate that EAG is useful for screening inhibitors of odorants. It shows.

[0123] Example 8: Insect Response to Inhibitors Several transparent plastic containers with lids (Maru Cup 200MB, Mineron Chemical Industry Co., Ltd.) The cap of the plastic container was large enough to fit the tip of a Pasteur pipette. A small hole was made in each of the plastic containers. A male silkworm moth was placed in each of the containers. The lid was closed.

[0124] Bombykol (BOL), the sex pheromone of the silkworm moth, is an inhibitor of known odorants. BMP was prepared. Filter paper with 0.01ng of BOL dropped on it, and 0.1ng of BOL dropped on it. Filter paper with 1ng of BOL dropped on it, filter paper with 10ng of BOL dropped on it, filter paper with 100n g of BOL dropped on filter paper, 1000ng of BOL dropped on filter paper, and diluted with hexane A filter paper was prepared on which 1000 μg of BMP was dropped.

[0125] Place a filter paper with 0.01 ng of BOL in the first Pasteur pipette and place it in the second Pasteur pipette. Place a filter paper with 0.1 ng of BOL in the Pasteur pipette and a third Pasteur pipette. Place a filter paper with 1 ng of BOL in the first Pasteur pipette, and add 10 ng of BOL to the fourth Pasteur pipette. Put the filter paper with 100 ng of BOL in the fifth Pasteur pipette. Put 1000 ng of BOL into the sixth Pasteur pipette, add a filter paper with 1000 ng of BOL, and put 1000 ng of BOL into the seventh Pasteur pipette. A filter paper with BMP dropped into each Pasteur pipette was placed in each Pasteur pipette. The container was inserted into a plastic container, and the silkworm moth was stimulated with an air puff.

[0126] As shown in Figure 17, when silkworm moths were puffed three times with BOL at different concentrations, At BOL of 1000g, 3 out of 5 silkworm moths showed flapping behavior and 2 out of 5 silkworm moths showed exploratory behavior. At 10 ng of BOL, all silkworms showed exploratory behavior.

[0127] As shown in Figure 18, after three puffs of BMP, silkworm moths were treated with BOL at various concentrations. When the silkworms were puffed three times, 3 out of 5 showed no reaction at 1 ng BOL. At 10 ng BOL, 1 out of 5 silkworms showed flapping behavior. Moths showed no reaction, 2 out of 5 silkworm moths showed flapping behavior, 2 out of 5 silkworm moths At 100 ng of BOL, one out of three silkworm moths showed flapping behavior. At 1000 ng of BOL, the remaining silkworm moth showed exploratory behavior. A comparison of Figures 17 and 18 shows that BMP is a response of insects to odorants. By increasing the BOL concentration threshold that inhibits the response and induces flapping and exploratory behavior, It was shown that:

[0128] The results of Example 8 show that observing the response of insects can be used to screen for odorant inhibitors. This shows that it is useful for

[0129] Example 9: Screening of inhibitors using insects A plurality of plastic containers similar to those in Example 8 were prepared, and each of the plurality of plastic containers was A male silkworm moth was placed inside the container and the lid was closed.

[0130] Bombykol (BOL) diluted with hexane was prepared. 4-tert-butyl-2-methylphenol diluted in San, and diluted in ethanol 4-Isopropyl-3-methylphenol was prepared.

[0131] First, a Pasteur pipette was used to drop 0.01 mg of BOL diluted with hexane onto a filter paper. Then, 0.1 ng of BOL diluted with hexane was added dropwise to the second Pasteur pipette. A filter paper was placed inside, and 1 ng of BOL diluted with hexane was dropped into the third Pasteur pipette. Place the filter paper inside and drop 10 ng of BOL diluted with hexane into a fourth Pasteur pipette. Place filter paper on the fifth Pasteur pipette and drop 100 ng of BOL diluted in hexane. Place the filter paper under the pipette and add 1,000 mg of BO diluted in hexane to a sixth Pasteur pipette. A filter paper with L dropped on it was placed inside.

[0132] As shown in Figure 19, silkworm moths were puffed three times with BOL at various concentrations diluted with hexane. When stimulated with 0.01 mg of BOL diluted with hexane, two out of three silkworms reacted. The silkworm moths showed no reaction, and one silkworm moth showed flapping behavior. All silkworm moths exhibited exploratory behavior in the BOL.

[0133] 1000mg of 4-tert-butyl-2-methylphenol diluted in hexane was dropped. The filter paper was placed in a plastic container together with the silkworm moth for 10 minutes. As shown in the figure, silkworm moths were puffed three times with BOL diluted with hexane at various concentrations. However, none of the silkworms showed any reaction to 0.01 ng of BOL diluted in hexane. Two of three silkworms showed no reaction to 0.1 ng of BOL diluted in hexane, and one Silkworm moths showed wing flapping behavior. 2 out of 3 moths showed flapping behavior when 1ng of BOL was diluted with hexane. 100% of the silkworm moths showed no reaction, and 1 silkworm moth showed exploratory behavior. At 10 ng of BOL, one of two silkworm moths showed flapping behavior and one of them explored. The remaining silkworm moth explored 100 ng of BOL diluted in hexane. Comparison of Figures 19 and 20 shows that 4-tert-butyl-2-methylphenol The BOL inhibits insect responses to odorants and induces flapping and exploratory behavior. It has been shown to increase the concentration threshold.

[0134] The filter paper with ethanol dropped on it was placed in a plastic container together with the silkworm moth for 10 minutes. Then, as shown in Figure 21, silkworm moths were sprayed three times with BOL of each concentration diluted with hexane. When stimulated with 0.01 ng of BOL diluted with hexane, all silkworms responded. Two out of three silkworms reacted to 0.1 mg of BOL diluted in hexane. The silkworm moths showed no reaction, and one silkworm moth showed flapping behavior. All silkworm moths exhibited exploratory behavior in OL.

[0135] 1000ng of 4-isopropyl-3-methylphenol diluted with ethanol was added dropwise. The filter paper was placed in a plastic container together with the silkworm moth for 10 minutes. As shown in the figure, silkworm moths were puffed three times with BOL diluted with hexane at various concentrations. All silkworms responded to 0.01ng and 0.1ng of BOL diluted with hexane. Two out of three silkworms showed a reaction to 1 ng of BOL diluted in hexane. At first, one silkworm moth showed flapping behavior. Two of the three silkworm moths showed flapping behavior, and one silkworm moth showed exploratory behavior. All silkworms showed exploratory behavior when 100 ng of BOL was diluted in water. From a comparison of 22, 4-isopropyl-3-methylphenol significantly increased insect odorant responses. This inhibits flapping and exploratory behavior and increases the threshold concentration of BOL that induces flapping and exploratory behavior. was shown.

[0136] Example 10: Preparation of odor-sensing cells expressing Or56 The gene for the coreceptor DmOrco, derived from the Drosophila melanogaster antenna cDNA from the start codon to the stop codon using a primer containing the gene-specific sequence The DmOrco gene was obtained by PCR amplification. The obtained DmOrco gene was then inserted into pIZ NEBuilder was inserted into the multicloning site of a vector (Invitrogen). r HiFi DNA Assembly MasterMix(New English The pIZ-DmOrco vector was constructed using a DNA fragment inserted into the DNA fragment of the target gene (DNA fragment 1). Built it.

[0137] DmOrco: Forward: 5'-TTCGAATTTAAAGCTGCCGCCATGATGACAACCTCGATGCAGCC-3' Reverse: 5'-TTACCTTCGAACCGCTTACTTGAGCTGCACCAGCAC-3'

[0138] In addition, the constructed pIZ-DmOrco vector was amplified by PCR using the following primers: The NEBuilder was inserted into the Pci1 site of the pIB vector (Invitrogen). r HiFi DNA Assembly MasterMix(New English The pIB-DmOrco vector was constructed using a DNA fragment from a DNA fragment inserted into a DNA fragment from ... Built it.

[0139] pIB-Pci1: Forward: 5'-GCAGGAAAGAACATGCATGATGATAAACAATGTATGGTGCTAATG-3' Reverse: 5'-CCTTTTGCTCACATGGTTATCCCCTGATTCTGTGG-3

[0140] PCR amplification of the gene was performed using forward primers at a concentration of 100 pmol / μl each. and reverse primer, PrimeSTAR HS DNA polymerase (Takarabaya (O, R010A), using the reaction buffer and dNTPs provided with the polymerase. The PCR was carried out according to the protocol attached to the polymerase. The temperature conditions for PCR were 94°C for 1 min. 1 minute step, followed by 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 1.5 minutes. 30 cycles of 72°C, followed by a 5-minute step at 72°C. .

[0141] Next, the base sequence of the olfactory receptor DmOr56a was codon-converted into the insect cell Sf9. The following sequence was added as an adapter sequence to synthesize the gene (Integrated DNA Technologies).

[0142] Adapter sequence: Forward: 5'-CAGTGTGGTGGAATTGCCGCC-3' Reverse: 5'-GCCCTCTAGACTCGATTA-3'

[0143] The resulting synthetic gene of DmOr56a_Sf9 was cloned from the initiation codon to the termination codon as shown above. The adapter sequence was amplified by PCR using primers, and the DmOr56a_Sf9 gene was isolated. The resulting DmOr56a_Sf9 gene was then transferred to the constructed pIB-DmOrco NEBuilder HiFi DNA Assay for the multiple cloning site of the vector embly MasterMix(New England Biolabs Japa n) to construct the pIB-DmOr56a_Sf9-DmOrco vector. Ta.

[0144] PCR amplification of the gene was performed using forward primers at a concentration of 100 pmol / μl each. and reverse primer, PrimeSTAR HS DNA polymerase (Takarabaya (O, R010A), using the reaction buffer and dNTPs provided with the polymerase. The PCR was carried out according to the protocol attached to the polymerase. The temperature conditions for PCR were 98°C for 2 min. 1 minute step, followed by 98°C for 10 seconds, 55°C for 10 seconds, and 72°C for 1.5 minutes. 25 cycles of 72°C for 10 minutes followed by a 72°C step. Ta.

[0145] Similarly, a calcium-sensitive protein (GCaMP6s) expression vector was constructed. The CaMP6s gene was donated to Dr. Douglas Kim (Jan. 2011) via Addgene. elia Farm Research Campus, Howard Hughes The GCaMP6s gene was obtained from the National Institute of Medical Sciences. The region from the codon to the stop codon was amplified with primers containing the gene-specific sequence shown below, and GC The aMP6s gene was obtained. The obtained GCaMP6s gene was inserted into the pIZ vector (Inv NEBuilder HiFi D NA Assembly MasterMix(New England Biolab The vector pIZ-GCaMP6s was constructed using a recombinant DNA technology developed by the National Institute of Integrative Medicine (NII) Japan.

[0146] GCaMP6s: Forward: 5'-TTCGAATTTAAAGCTGCCGCCATGGGTTCTCATCATCATCATC-3' Reverse: 5'-TTACCTTCGAACCGCTCACTTCGCTGTCATCATTTGTAC-3'

[0147] The constructed olfactory receptor expression vector and calcium-sensitive protein expression vector were Transfection Reagent (TransIT-Insect Transfection Reagent (Mirus) was used to incubate Sf21 cells according to the attached manual. This resulted in the expression of the DmOr56a_Sf9 receptor, DmOrco, and GCaMP. Sf21 cells co-expressing 6s (hereafter referred to as "Or56-expressing odor-detecting cells"). .) was obtained.

[0148] Example 11: Establishment of a homogeneous odor-sensing cell line expressing Or56 As a preliminary preparation, 6 mL of Or56-expressing odor-detecting cells were cultured at the time of passage before the lineage was established. One flask (FALCON) of medium containing 100% ethanol was prepared. 6 mL of culture supernatant was collected from the tube and centrifuged at 400 × g for 3 minutes. The supernatant after centrifugation was collected in a 25 mL tube (IWAKI) and Sterilized using a mL syringe (TOP) and a 0.45 μm filter (CORNING). 5 mL of sterilized medium, 5 mL of new medium, and 1 mL of two antibiotics (Bla) sticidin (Life Technologies) and Zeocin (Life Technologies) were mixed to prepare a conditioned medium.

[0149] Next, the cells on the bottom of the flask were scraped off and suspended in 1 mL of fresh medium. The cells were collected in a 1.5 mL tube (AXYGEN). The cell number was measured, and 40 cells were added to the conditioned medium to obtain a cell suspension. The entire volume of the cell suspension was transferred to a reservoir (BMBio) and then pipetted using an 8-channel pipette. 100 μL of the solution was seeded into a 96-well plate (IWAKI) using a 27-well plate. After the cells had adhered to the wells, they were cultured in an incubator at 37°C. After the cells had adhered to the wells, single cells were observed under an inverted microscope. We checked the well containing the

[0150] In a 96-well plate, cells in the wells where single cells were confirmed were approximately 80% to 90% confluent. The culture was continued until the cells became confluent. Then, the cells were transferred to a 24-well plate (IWAKI), The culture was scaled up to a 100 mm dish (CORNING) and then to a T25 flask. The cells were scaled up to 25 flasks and showed fluorescence using calcium imaging. The response of the cells was confirmed, and non-responsive cells were discarded. Responding cells were frozen and stocked. The lineage was maintained through successive generations.

[0151] Example 12: Odor detection of Or56 expressing cells according to the concentration of inhibitors and inhibitor candidates cellular response) The inhibitor candidate substances, 2,4-diisopropylphenol and 2,5-diisopropyl Phenol, 4-isopropyl-3-methylphenol, 2-tert-butyl-4-methylphenol 4-(tert-butyl)-2-methylphenol, and 2-isopropyl We also prepared 2-tetramethylphenol, a known odorant inhibitor. rt-Butyl-6-methylphenol (BMP) was prepared.

[0152] The odorant, 1 μmol / L Geos, was tested without any inhibitor candidate or known inhibitor. The amine was administered to the odor detection cells expressing Or56 obtained in Example 11, and the The fluorescence intensity was measured. Next, 10 μmol / L of the inhibitor candidate or known inhibitor, 3 0 μmol / L of inhibitor candidate or known inhibitor, 100 μmol / L of inhibitor candidate 170 μmol / L of candidate inhibitors or known inhibitors, and 30 μmol / L of candidate inhibitors or known inhibitors. 0 μmol / L of inhibitor candidate or known inhibitor was sequentially treated with 1 μmol / L of geosmin. Both were administered to odor-detecting cells, and the intensity of fluorescence in the odor-detecting cells was measured. 1 μmol / L geosmin was administered to odor-detecting cells without any known inhibitors or drugs. The fluorescence intensity in the undetected cells was measured.

[0153] Geosmin was administered at 1 μmol / L without any inhibitor candidate or known inhibitor. The average change in fluorescence intensity in the odor-detecting cells when the odorant was administered to the cells was set as 100%, and the percentage of each inhibitor was calculated. Concentrations of candidate and known inhibitors and normalized fluorescence intensity in odor-detecting cells The obtained relationship between and is shown in the graph in Figure 23. As shown in Figure 23, The response of odorants to the odor-detecting cells was investigated by the inhibitor candidate and known inhibitors. was inhibited in a concentration-dependent manner.

[0154] Example 12: Insect Response to Inhibitors 4-Isopropyl-3-methylphenol in DMSO as a potential inhibitor (IPMP) and DMSO as a control were prepared as test drugs. 1 μL of crude extract (acetone solvent) of pheromones periplanone A and periplanone B Prepare the sample, attach it to a 15mm square piece of filter paper or aluminum piece, and allow the acetone solvent to evaporate sufficiently. Ta.

[0155] The lid of a container containing around 10 cockroaches was removed under red light in a dark room. The food dish and water cup were collected. Two petri dishes containing filter paper containing the test drug were placed in the container. The sample was placed in one of the four corners, one diagonally opposite the other. The container was then covered and left to stand for 30 minutes.

[0156] Start videotaping the container and place a piece of filter paper or a piece of a cockroach pheromone crude extract on it. The lumi piece was placed on one of the petri dishes containing filter paper containing the test drug. As shown in Figure 24, a transparent acrylic plate was placed on the container instead of the lid to make it easier to take photographs. I put it on and filmed a video for 10 minutes.

[0157] The video footage was analyzed, and the number of cockroaches was measured within five minutes of placing the transparent acrylic plate on the container. The total number of times cockroaches came into contact with filter paper or aluminum pieces to which crude extract of yellowtail pheromone had been applied. As a result, as shown in Figure 25, 4-isopropyl-3-methylphenol is transferred to a filter paper or aluminum foil containing the pheromone. As shown in Figure 26, the 4-isopropyl The higher the concentration of propyl-3-methylphenol, the more the pheromone-soaked filter paper or Cockroaches were prevented from approaching the aluminum pieces. [Explanation of symbols]

[0158] 20...spraying section, 21...storage section, 21a...main body, 21b...lid, 22... atomization section, 23 blowing section, 25 two-fluid nozzle, 25a gas inlet, 5b: Solution inlet; 26: Spray nozzle; 27: Connection portion; 30: Control portion; 31: Controller, 32 Pump, 33 Valve, 34 Gas supply pipe, 41 Storage section, 42 atomization section, 43 blowing section, 45 piezoelectric element, 47 Nozzle head, 48, wiring, 60, spraying section, 61, storage section, 62 Atomization section, 62...Atomization section, 63...Blowout section, 65...Ultrasonic transmission membrane, 66. Vibration generating vessel, 67 Working water, 70 Wiring, 71 Wiring, 80 Diffuser Fabric portion, 81...storage portion, 81a...main body, 81b...lid, 82...atomization portion, 83: blowing portion, 84: electrostatic spray nozzle, 85: conveying portion, 86: cylindrical member , 87... Voltage application unit, 120... Spray unit, 130... Control unit, 230... Control High voltage control section, 330

Claims

1. An odorant expressing an insect olfactory receptor and / or a co-receptor of said olfactory receptor. providing a detection structure; The odorant corresponding to the olfactory receptor and each of the plurality of inhibitor candidate substances are mixed with the odorant. providing a detection structure; The plurality of inhibitor candidate substances are screened based on the influx of ions into the odor detection structure. Leaning and A method for screening inhibitors of insect odor responses, comprising:

2. The olfactory receptor is a receptor for 1-octen-3-ol, and the odorant is 1-octen-3-ol.

10. The method for screening an inhibitor of an insect's odor response according to claim 1, wherein the inhibitor is 10-3-ol. How to do this.

3. 2. Screening of an inhibitor of an odor response of an insect according to claim 1, wherein the insect is a cockroach. Training method.

4. Providing insect antennae; A stimulating substance that stimulates the antennae of the insect and a candidate inhibitor selected by the screening. providing a substance to antennae of said insect; detecting an antennal response of the insect; The method for screening an inhibitor of an insect's odor response according to claim 1, further comprising: 。

5. The inhibitor of insect odor responses according to claim 4, wherein the stimulating substance is bombykol. Screening methods.

6. The inhibitor of the odor response of insects according to claim 4, wherein the antennae of the insect are the antennae of a moth. Screening methods.

7. 7. Screening of an inhibitor of an insect's odor response according to claim 6, wherein the moth is a silkworm moth. How to do this.

8. the olfactory receptor is a receptor for 1-octen-3-ol, The odorant is 1-octen-3-ol, the stimulant is bombykol, The insect antennae are moth antennae.

5. The method of claim 4 for screening inhibitors of the response to moth pheromones. A method for screening inhibitors of insect odor responses.

9. 5. The detecting of the antennal response of the insect comprises detecting an electrical potential of the antenna. A method for screening inhibitors of insect odor responses described in .

10. Preparing the insects, a stimulating substance that stimulates the insect and a candidate inhibitor selected by the screening; feeding said insects; detecting a response of the insect; The method for screening an inhibitor of an insect's odor response according to claim 1, further comprising: 。

11. The inhibition of an insect's odor response according to claim 10, wherein the stimulating substance is bombykol. Agent screening method.

12. 11. The method of claim 10, wherein the insect is a moth. How to do this.

13. The screen for an inhibitor of the odor response of an insect according to claim 12, wherein the moth is a silkworm moth. Training method.

14. The inhibitor of the odor response of insects according to claim 10, wherein the insect is a cockroach. Leaning method.

15. the olfactory receptor is a receptor for 1-octen-3-ol, The odorant is 1-octen-3-ol, the stimulant is bombykol, The insect is a moth. A method for screening for inhibitors of the response to moth pheromones, as claimed in claim 10. A method for screening inhibitors of insect odor responses.

16. 11. The method of claim 10, wherein detecting the insect's response comprises observing the behavior of the insect. A method for screening inhibitors of insect odor responses.

17. An insect obtained by the method for screening inhibitors of insect odor responses according to claim 1. An inhibitor of the odor response of ion influx into the odor sensing structure by less than 37% for insect odor responses. Inhibitors.

18. 18. The insect according to claim 17, wherein the influx of ions into the odor-sensing structure is 20% or less. Inhibitors of odor responses.

19. The inhibitor of insect odor responses according to claim 17, which is a methylphenol derivative.

20. 18. The insect odor response of claim 17, comprising 4-isopropyl-3-methylphenol. Inhibitors against

21. The insect of claim 17, comprising 4-(tert-butyl)-2-methylphenol. Inhibitors of odor responses.

22. The solvents are ethanol, hexane, isopropyl alcohol, acetone, DMSO, and 18. The odor response of the insect according to claim 17, wherein the odor response is at least one selected from the group consisting of water. Inhibitors against

23. 18. The inhibitor of insect odor responses according to claim 17, which is an inhibitor of moth odor responses. Agent.

24. The inhibitor of insect odor response according to claim 23, wherein the moth is a silkworm moth.

25. The compound according to claim 17, which is an inhibitor of the odor response of an insect, Inhibitors.

26. An insect obtained by the method for screening inhibitors of insect odor responses according to claim 1. An insect inhibition system comprising a spraying unit that sprays an inhibitor against the odor response of insects.

27. 27. The method according to claim 26, wherein the spraying unit microparticulates the inhibitor of the insect's odor response. Insect inhibition system.

28. The spraying unit atomizes the inhibitor against the insect's odor response. At least one selected from the group consisting of an ultrasonic vibrator, a piezoelectric element, and an electrostatic spray nozzle 27. The insect deterrent system of claim 26, comprising:

29. The solvent for the inhibitor of the insect's odor response is ethanol, hexane, isopropyl alcohol, or the like. At least one selected from the group consisting of alcohol, acetone, DMSO, and water.

27. The insect deterrent system of claim 26.

30. The inhibitor of the insect's odor response is 4-isopropyl-3-methylphenol or 27. The insect inhibitor of claim 26, comprising 4-(tert-butyl)-2-methylphenol. system.

31. An insect obtained by the method for screening inhibitors of insect odor responses according to claim 1.

10. A method of inhibiting insects comprising spraying an inhibiting solution of

32. In the spraying step, the inhibitor of the insect's odor response is micronized. Item 32. The insect inhibition method according to Item 31.

33. In the spraying, a two-fluid nozzle, an ultrasonic vibrator, a piezoelectric element, and an electrostatic spray nozzle and at least one selected from the group consisting of insects, insecticides, and insecticides.

32. The insect inhibiting method of claim 31, wherein the insect inhibiting method is micronized.

34. The solvent for the inhibitor of the insect's odor response is ethanol, hexane, isopropyl alcohol, or the like. At least one selected from the group consisting of alcohol, acetone, DMSO, and water.

32. The insect inhibiting method of claim 31.

35. The inhibitor of the insect's odor response is 4-isopropyl-3-methylphenol or 32. The insect inhibitor of claim 31, comprising 4-(tert-butyl)-2-methylphenol. method.

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