A screening method for inhibitors of insect odor responses, an inhibitor of insect odor responses, an insect inhibition system, and an insect inhibition method.

Abstract

To provide a screening method for an inhibitor of insect odor response.SOLUTION: 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: None

Description

Technical Field

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

Background Art

[0002] Olfaction is involved in many insect habits (see, for example, Patent Document 1 and Non-Patent Document 1). Olfaction is considered to play an important role in the feeding behavior of insects, the attraction between males and females for mating, and the selection of egg-laying sites. On the other hand, in order to repel harmful insects, it has been proposed to inhibit the olfaction of insects or to utilize the odors that insects avoid.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] There is a need for highly effective inhibitors of insect odor responses and methods for screening such inhibitors. Therefore, one of the objectives of the present invention is to provide a screening method for inhibitors of insect odor responses, an inhibitor of insect odor responses, an insect inhibition system, and an insect inhibition method. [Means for solving the problem]

[0006] A screening method for inhibitors of insect odor responses according to an aspect of the present invention includes: preparing an odor detection structure expressing at least one of an insect olfactory receptor and a co-receptor of the olfactory receptor; providing the odor substance corresponding to the olfactory receptor and each of a plurality of candidate inhibitor substances to the odor detection structure; and screening the plurality of candidate inhibitor substances based on the influx of ions into the odor detection structure. The insect olfactory receptor may be an ion channel type receptor. The odor detection structure may be a cell. The odor detection structure may be an artificial structure. The odor detection structure may express a co-receptor of the olfactory receptor.

[0007] In the above-described method for screening inhibitors of insect odor responses, the olfactory receptor may be a 1-octen-3-ol receptor, and the odorant may be 1-octen-3-ol.

[0008] In the above-described method for screening inhibitors for the odor response of insects, the insect may be a cockroach.

[0009] The above method for screening inhibitors for the odor response of insects may further include preparing insect antennae, applying an irritant to stimulate the insect antennae and the selected inhibitor candidates to the insect antennae, and detecting the response of the insect antennae. The irritant may be bombicol. The insect antennae may be those of a moth. The moth may be a silkworm moth.

[0010] In the above-described screening method for inhibitors of insect odor responses, the olfactory receptor may be a 1-octen-3-ol receptor, the odorant may be 1-octen-3-ol, the irritant may be bombicol, and the insect's antennae may be those of a moth. The above-described screening method for inhibitors of insect odor responses may also be a method for screening inhibitors of moth pheromone responses.

[0011] In the above-described screening method for inhibitors of the odor response of insects, the potential of the antennae may be detected when detecting the response of the insect's antennae.

[0012] The above method for screening inhibitors for the odor response of insects may further include preparing insects, providing the insects with an irritant that stimulates them and the candidate inhibitors selected by the screening, and detecting the insects' responses. The irritant may be bombicol. The insect may be a moth. The moth may be a silkworm moth. The insect may be a cockroach. The cockroach may be an American cockroach.

[0013] In the above-described screening method for inhibitors of insect odor responses, the olfactory receptor may be a 1-octen-3-ol receptor, the odorant may be 1-octen-3-ol, the irritant may be bombicol, and the insect may be a moth. The above-described screening method for inhibitors of insect odor responses may also be a method for screening inhibitors of moth pheromone responses.

[0014] In the above-described screening method for inhibitors of insect odor responses, the insect's behavior may be observed to detect the insect's response. The insect's behavior may be wing flapping. The insect's behavior may be exploratory behavior.

[0015] An inhibitor for the odor response of insects according to an aspect of the present invention is an inhibitor for the odor response of insects that reduces the influx of ions into the odor detection structure to less than 37%.

[0016] The inhibitor against the olfactory response of insects according to an aspect of the present invention is an inhibitor against the olfactory response of insects obtained by the screening method of the inhibitor against the olfactory response of the above insects, and is an inhibitor against the olfactory response of insects that reduces the influx of ions into the olfactory detection structure to less than 37%.

[0017] The above inhibitor against the olfactory response of insects may reduce the influx of ions into the olfactory detection structure to 20% or less.

[0018] The above inhibitor against the olfactory response of insects may be a methylphenol derivative.

[0019] The above inhibitor against the olfactory response of insects may contain 4-isopropyl-3-methylphenol.

[0020] The above inhibitor against the olfactory response of insects may contain 4-(tert-butyl)-2-methylphenol.

[0021] In the above inhibitor against the olfactory response of insects, the solvent may be at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water.

[0022] The above inhibitor against the olfactory response of insects may be an inhibitor against the olfactory response of termites. The termite may be a Formosan subterranean termite. The above inhibitor against the olfactory response of insects may be an inhibitor against the olfactory response of cockroaches. The cockroach may be a German cockroach.

[0023] The insect inhibition system according to an aspect of the present invention includes a spraying unit that sprays an inhibitor against the olfactory response of insects. The inhibitor against the olfactory response of insects may be the above inhibitor against the olfactory response of insects.

[0024] The insect inhibition system according to an aspect of the present invention includes a spraying unit that sprays an inhibitor against the olfactory response of the above-mentioned insects obtained by the screening method of the inhibitor against the olfactory response of insects.

[0025] In the above insect inhibition system, the spraying unit may atomize the inhibitor against the olfactory response of insects.

[0026] In the above insect inhibition system, the spraying unit may include at least one selected from the group consisting of a two-fluid nozzle, an ultrasonic oscillator, a piezoelectric element, and an electrostatic spray nozzle for atomizing the inhibitor against the olfactory response of insects.

[0027] In the above insect inhibition system, the solvent of the inhibitor against the olfactory response of insects may be at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water.

[0028] In the above insect inhibition system, the inhibitor against the olfactory response of insects may contain 4-isopropyl-3-methylphenol or 4-(tert-butyl)-2-methylphenol.

[0029] The insect inhibition method according to an aspect of the present invention includes spraying an insect inhibition solution. The inhibitor against the olfactory response of insects may be the inhibitor against the olfactory response of the above-mentioned insects.

[0030] The insect inhibition method according to an aspect of the present invention includes spraying an insect inhibition solution obtained by the screening method of the inhibitor against the olfactory response of the above-mentioned insects.

[0031] In the above insect inhibition method, in the spraying process, the inhibitor against the olfactory response of insects may be atomized.

[0032] In the insect inhibition method described above, the inhibitor of the insect's odor response may be atomized into fine particles using at least one selected from the group consisting of a two-fluid nozzle, an ultrasonic transducer, a piezoelectric element, and an electrostatic spray nozzle.

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

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

[0035] According to the present invention, it is possible to provide a screening method for inhibitors of insect odor responses, an inhibitor of insect odor responses, an insect inhibition system, and an insect inhibition method. [Brief explanation of the drawing]

[0036] [Figure 1] This is a schematic diagram illustrating a method for expressing olfactory receptors according to an embodiment in odor-detecting cells. [Figure 2] This is a schematic diagram illustrating a method for homogenizing odor-detecting cells according to an embodiment. [Figure 3] This is a schematic diagram illustrating a method for expressing multiple olfactory receptors according to an embodiment in odor-detecting cells. [Figure 4] This is a schematic diagram illustrating a method for expressing multiple olfactory receptors according to an embodiment in odor-detecting cells. [Figure 5] This is a schematic diagram of an insect inhibitory system according to an embodiment. [Figure 6] This is a schematic diagram of an insect inhibitory system according to an embodiment. [Figure 7] This is a schematic diagram of an insect inhibitory system according to an embodiment. [Figure 8]This is a schematic diagram of an insect inhibitory system according to an embodiment. [Figure 9] This graph shows the time-dependent change in the intensity of fluorescence emitted by odor-detecting cells in the example. [Figure 10] This graph shows the relationship between the concentrations of inhibitors and candidate inhibitors for the odor response of insects in the examples, and the normalized intensity of fluorescence emitted by odor-detecting cells. [Figure 11] This graph shows the relationship between the concentration of candidate inhibitors for the odor response of insects in the example and the normalized intensity of fluorescence emitted by odor-detecting cells. [Figure 12] This graph shows the relationship between the concentrations of inhibitors and candidate inhibitors for the odor response of insects in the examples, and the normalized intensity of fluorescence emitted by odor-detecting cells. [Figure 13] This graph shows the relationship between the inhibitors and candidate inhibitors for the odor response of insects in the examples, and the normalized intensity of fluorescence emitted by odor-detecting cells. [Figure 14] This is a photograph showing the EAG measurement method according to the example. [Figure 15] This graph shows the time change in the potential of the antennae in the example. [Figure 16] This graph shows the time change in the potential of the antennae in the example. [Figure 17] This table shows the insect responses to multiple concentrations of bombicol in the examples. [Figure 18] This table shows the responses of insects to multiple concentrations of bombicol after administering the inhibitors described in the examples. [Figure 19] This table shows the insect responses to multiple concentrations of bombicol in the examples. [Figure 20] This table shows the responses of insects to multiple concentrations of bombicol after administering the candidate inhibitor substances described in the examples to the insects. [Figure 21] This table shows the insect responses to multiple concentrations of bombicol in the examples. [Figure 22]This table shows the responses of insects to multiple concentrations of bombicol after administering the candidate inhibitor substances described in the examples to the insects. [Figure 23] This graph shows the relationship between the concentrations of inhibitors and candidate inhibitors for the odor response of insects in the examples, and the normalized intensity of fluorescence emitted by odor-detecting cells. [Figure 24] This is a photograph of a container used to observe the mating behavior of cockroaches in the example. [Figure 25] This graph shows the number of cockroach accesses to filter paper or aluminum pieces containing pheromones according to the examples. [Figure 26] This graph shows the number of cockroach accesses to filter paper or aluminum pieces containing pheromones according to the examples. [Modes for carrying out the invention]

[0037] A screening method for inhibitors of insect odor responses according to an embodiment includes: preparing an odor detection structure expressing at least one of an insect olfactory receptor and a co-receptor of the olfactory receptor; providing the odor substance corresponding to the olfactory receptor and each of a plurality of candidate inhibitor substances to the odor detection structure; and screening the plurality of candidate inhibitor substances based on the influx of ions into the odor detection structure.

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

[0039] If the odor detection structure is a cell, the odor detection cell expresses olfactory receptors on its cell membrane. In odor detection cells, olfactory receptors may be naturally expressed or expressed by a transgene. The olfactory receptors may also be insect olfactory receptors.

[0040] Odor-detecting cells may be insect cells. These insect cells may be derived from moths such as the armyworm (Spodoptera frugiperda) and the nettle moth (Trichoplusia ni). Examples of armyworm-derived cells include Sf21 and Sf9. Sf21 cells are derived from ovarian cells. Sf21 cells undergo indefinite division, making it possible to establish a stable expression line that permanently expresses the introduced gene. Furthermore, Sf21 cells can survive in a wide temperature range from 18°C ​​to 40°C and do not require carbon dioxide to adjust the culture medium pH. While Sf21 cells inherently lack olfactory receptors, it is possible to induce olfactory receptor expression by introducing the olfactory receptor gene. Sf9 cells are clones of Sf21. Examples of nettle moth-derived cells include High Five and Tni. Tni-derived cells are derived from ovarian cells.

[0041] Alternatively, the insect cells may be derived from fruit flies. An example of fruit fly-derived cells is Drosophila S2 cells. Or, the insect cells may be derived from cockroaches.

[0042] Olfactory receptors are ion channel receptors. Olfactory receptors may be G protein-coupled receptors or ion channel receptors. Ion channel receptors have a site that interacts with the ligand, which is an odor molecule, and a site into which ions flow. When an ion channel receptor in an odor-detecting cell binds to a ligand, cations such as sodium ions and calcium ions flow into the odor-detecting cell. In odor-detecting cells, ion influx can occur within tens of milliseconds from ligand binding. The amount of ions that flow in is large; for every ligand that binds, 10 ions flow into the cell. 7 It is also said to be individual.

[0043] Generally, certain types of olfactory receptors have specificity for specific odor molecules. In odor-detecting cells, only one type of olfactory receptor corresponding to one type of odor molecule may be expressed, or multiple types of olfactory receptors corresponding to multiple types of odor molecules may be expressed. The amount of olfactory receptors expressed may also be adjusted. Insect olfactory receptors have similar odor receptor reception mechanisms in many insect species, such as flies, mosquitoes, moths, bees, lice, and cockroaches. Therefore, an inhibitor of a particular insect's olfactory receptor may function as an inhibitor of insect olfactory receptors in general.

[0044] Odor detection structures may express olfactory receptors along with co-receptors of olfactory receptors. The co-receptors form heterocomplexes with olfactory receptors and function as receptors for odor molecules. Insect co-receptors also exhibit similar odor molecule reception mechanisms in many insect species, including flies, mosquitoes, moths, bees, lice, and cockroaches. Therefore, inhibitors of the co-receptors of olfactory receptors in a particular insect may function as inhibitors of the co-receptors of olfactory receptors in insects in general.

[0045] Examples of olfactory receptors include, but are not limited to, DmOr56a, a Drosophila receptor for geosmin, which has a musty odor; DmOr82a, a Drosophila receptor for geranyl acetate, which has an aromatic or fruity odor; DmOr49b, a Drosophila receptor for 2-methylphenol (o-cresol), which has the odor of human sweat; DmOr13a, a Drosophila receptor for 1-octen-3-ol, which has a musty odor; BmOR1, a Drosophila receptor for bombykol, a sex pheromone of the silkworm moth; BmOR3, a Drosophila receptor for bombykal, a minor component of the silkworm moth sex pheromone; DmOr85b, a general odor receptor of Drosophila melanogaster; and PxOR1, a sex pheromone receptor of the diamondback moth. Incidentally, "geosmin" is also called "geosmin."

[0046] When expressing olfactory receptors in odor-detecting cells using genetic engineering, for example, as shown in Figure 1, the gene encoding the olfactory receptor may be incorporated into a vector, and the constructed vector may be transfected into host cells. The gene encoding the olfactory receptor can be isolated, for example, by extracting mRNA from the olfactory organs of insects and synthesizing cDNA. From the isolated cDNA, it is possible to amplify a portion of the gene encoding the olfactory receptor using PCR with PCR primers.

[0047] Some genes encoding olfactory receptors can also be obtained by incorporating synthesized double-stranded cDNA into a suitable vector and transforming E. coli or other organisms using this vector to create a cDNA library. The cDNA can be incorporated into a vector by a conventional method using restriction enzymes and ligases, for example, by cleaving the obtained cDNA with a restriction enzyme and inserting it into the restriction enzyme sites of the vector DNA to ligate it into the vector.

[0048] Odor molecules corresponding to olfactory receptors are odor molecules that specifically react with olfactory receptors. The odor molecule corresponding to DmOr56a is geosmin. The odor molecule corresponding to DmOr82a is geranyl acetate. The odor molecule corresponding to DmOr49b is 2-methylphenol (o-cresol). The odor molecule corresponding to DmOr13a is 1-octen-3-ol. The odor molecule corresponding to BmOR1 is bombykol. The odor molecule corresponding to BmOR3 is bombykal. The odor molecule corresponding to PxOR1 is the sex pheromone of the diamondback moth.

[0049] In odor-detecting cells, fluorescent proteins whose fluorescence intensity changes in response to ions may be expressed. As mentioned above, when odor molecules bind to ion channel-type olfactory receptors in odor-detecting cells, ions flow into the odor-detecting cells. Therefore, by introducing a gene that expresses a fluorescent protein whose fluorescence intensity changes in response to ions into odor-detecting cells, it is possible to confirm whether or not the odor-detecting cells are detecting odor molecules from the change in fluorescence intensity or the rate of change in fluorescence intensity. Examples of fluorescent proteins include GCaMP3, GCaMP6s, and aequorin, which emit fluorescence in response to calcium ions.

[0050] When olfactory receptors react with odor molecules, the concentration of ions flowing into odor-detecting cells increases, causing many fluorescent proteins to fluoresce. Therefore, the fluorescence intensity emitted by odor-detecting cells increases. When the reaction between olfactory receptors and odor molecules is inhibited by an inhibitor, the concentration of ions flowing into odor-detecting cells decreases or becomes zero, resulting in fewer fluorescent proteins fluorescing or no fluorescence being emitted at all. Consequently, the fluorescence intensity emitted by odor-detecting cells weakens or disappears. Therefore, it is possible to evaluate whether the reaction between olfactory receptors and odor molecules is inhibited by an inhibitor based on the fluorescence intensity corresponding to the influx of ions into odor-detecting cells.

[0051] The influx of ions into odor-detecting cells may also be measured electrically. For example, a transistor comprising a source electrode, a drain electrode, and a gate electrode can be placed near the odor-detecting cells. When olfactory receptors react with odor molecules and ions flow into the odor-detecting cells, the gate potential of the gate electrode of the transistor is displaced, causing a modulation in the drain current flowing between the source and drain electrodes. If the reaction between olfactory receptors and odor molecules is inhibited by an inhibitor, the concentration of ions flowing into the odor-detecting cells decreases or becomes zero, and the modulation of the drain current decreases or disappears. Therefore, by detecting the modulation of the transistor's drain current 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.

[0052] Odor-detecting cells may be selected by performing multiple steps (a) through (c), which involve (a) selecting a portion of cells from a group of cells possessing olfactory receptors, (b) growing the selected cells, and (c) confirming the responsiveness of the grown cells to odor substances, and then selecting cells that have grown to a level equal to or higher than a standard value for responsiveness to odor substances. The cells selected in step (a) may be single cells.

[0053] Alternatively, odor-detecting cells may be selected by performing multiple steps (a) through (c), which involve (a) selecting a subset of cells from a group of cells possessing olfactory receptors, (b) growing the selected cells, and (c) confirming the responsiveness of the grown cells to odor substances, and then selecting the cells that exhibit the highest responsiveness to odor substances. The cells selected in step (a) may be single cells.

[0054] Specifically, as shown in Figure 2, a cell lineage may be established by repeatedly diluting a group of cell lines that possess olfactory receptors and express fluorescent proteins, selecting one or a small number of cells, and culturing and growing the selected cells. By performing this process multiple times, multiple cell lines can be established. Among the multiple established cell lines, a cell lineage whose responsiveness to odor substances is above a predetermined standard value may be used as an odor detection cell. Alternatively, among the multiple established cell lines, the cell lineage with the highest responsiveness to odor substances may be used as an odor detection cell.

[0055] As shown in Figures 3 and 4, cells expressing the established olfactory receptor may be further made to express another olfactory receptor. That is, cells expressing the first olfactory receptor established by the above method may be further made to express a second olfactory receptor. For example, by incorporating the gene encoding the second olfactory receptor into a vector and transfecting cells expressing the first olfactory receptor with the constructed vector, it is possible to establish cells expressing both the first and second olfactory receptors. Cells expressing the first olfactory receptor may be further made to express multiple different olfactory receptors. Figure 3 shows an example in which cells expressing Or56a as the first olfactory receptor are introduced into a vector containing the gene for the second olfactory receptor, Or-X, and a vector containing an antibiotic resistance gene. Figure 4 shows an example in which cells expressing Or56a as the first olfactory receptor are introduced into a vector containing both the gene for the second olfactory receptor, Or-X, and an antibiotic resistance gene.

[0056] If the odor detection structure is an artificial structure, the artificial structure may be modeled after a cell. The artificial structure may, for example, comprise a vesicle having a membrane and olfactory receptors arranged in the membrane. The artificial structure may also comprise a fluorescent protein within the membrane that emits fluorescence depending on the concentration of ions. The artificial structure can be manufactured, for example, by referring to Non-Patent Document 1.

[0057] The odorant and each of the multiple candidate inhibitors may be applied to the odor detection structure simultaneously. Alternatively, the odorant may be applied to the odor detection structure first, and then each of the multiple candidate inhibitors may be applied to the odor detection structure. Alternatively, each of the multiple candidate inhibitors may be applied to the odor detection structure first, and then the odorant may be applied to the odor detection structure. Candidate inhibitors that inhibit the influx of ions into the odor detection structure that may occur due to the odorant are selected as inhibitors of the insect's odor response.

[0058] Multiple candidate inhibitors screened using an odor detection structure may be screened further. For example, a screening method for inhibitors against the odor response of insects according to this embodiment further includes preparing an insect or a part of an insect, providing the insect or a part of an insect with an irritant that stimulates the insect or a part of an insect, and the candidate inhibitors selected in the above screening, and detecting the reaction of the insect or a part of an insect.

[0059] Examples of insects include moths, cockroaches, mosquitoes, flies, bees, and lice. Examples of moths include silkworms, diamondback moths, armyworms, and pygmy moths. Examples of cockroaches include the Eastern cockroach, German cockroach, American cockroach, small-spotted cockroach, brown cockroach, black cockroach, Japanese cockroach, lacquered cockroach, and Suzuki cockroach. Examples of some insect parts include antennae, rostrum, legs, and wings.

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

[0061] The irritant and each of the multiple candidate inhibitors may be administered simultaneously to an insect or a part of an insect. Alternatively, the irritant may be administered to an insect or a part of an insect, and then each of the multiple candidate inhibitors may be administered to the insect or a part of an insect. Or, each of the multiple candidate inhibitors may be administered to an insect or a part of an insect, and then the irritant may be administered to the insect or a part of an insect.

[0062] In detecting insect responses, insect behavior may be observed. Candidate inhibitors that inhibit insect behaviors that may be caused by stimulants are selected as inhibitors of the insect's odor response. Examples of insect behaviors include wing flapping, searching behavior, and mating behavior. In addition, in detecting the response of insect antennae, the potential of the antennae may be detected. Candidate inhibitors that inhibit changes in the potential of the antennae that may be caused by stimulants are selected as inhibitors of the insect's odor response.

[0063] The inhibitor for the odor response of insects according to the embodiment can reduce the influx of ions into the odor detection structure to less than 37%, 35% or less, 30% or less, 25% or less, 20% or less, or less than 20%. For example, when evaluating the influx of ions into the odor detection structure by observing the fluorescence intensity within the odor detection structure, the inhibitor for the odor response of insects according to the embodiment can reduce the fluorescence intensity to less than 37%, 35% or less, 30% or less, 25% or less, 20% or less, or less than 20% compared to the fluorescence intensity produced by odor molecules in the absence of the inhibitor. When evaluating the influx of ions into the odor detection structure by observing the drain current of a transistor near the odor detection structure, the inhibitor for the odor response of insects according to the embodiment can reduce the modulation of the drain current to less than 37%, 35% or less, 30% or less, 25% or less, 20% or less, or less than 20% compared to the modulation of the drain current produced by odor molecules in the absence of the inhibitor. Note that the rate of ion influx or the reduction in drain current may be a value related to the rate of reduction in fluorescence intensity according to the embodiment.

[0064] Inhibitors of insect odor responses include, for example, methylphenol derivatives. Inhibitors of insect odor responses may include 4-isopropyl-3-methylphenol, shown in Chemical Formula 1 below. Inhibitors of insect odor responses may include 4-(tert-butyl)-2-methylphenol, shown in Chemical Formula 2 below. Inhibitors of insect odor responses may include 2-isopropyl-6-methylphenol, shown in Chemical Formula 3 below. Inhibitors of insect odor responses may include 2,4-diisopropylphenol, shown in Chemical Formula 4 below. Inhibitors of insect odor responses may include 2-tert-butyl-4-methylphenol, shown in Chemical Formula 5 below.

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

[0066] Inhibitors of insect odor responses may include cinnamic acid derivatives. Examples of cinnamic acid derivatives include methyl transcinnamate and tert-butyl transcinnamate.

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

[0068] As shown in Figure 5, the insect inhibitor system according to the embodiment includes a spraying unit 20 for spraying an inhibitor of the insect's odor response. The inhibitor of the insect's odor response may be any of the above-mentioned inhibitors of the insect's odor response. The spraying unit 20 includes, for example, a storage unit 21 for storing a solution containing the inhibitor of the insect's odor response, a particle atomization unit 22 for atomizing the solution containing the inhibitor of the insect's odor response, and a blowing unit 23 for blowing out the atomized solution containing the inhibitor of the insect's odor response. The solvent of the solution may be any of the above-mentioned solvents.

[0069] The storage section 21 is, for example, a tank and comprises a main body 21a and a lid 21b. The atomization section 22 comprises, for example, a two-fluid nozzle 25 located inside the storage section 21 that mixes and atomizes two fluids, a liquid and a gas. The discharge section 23 may be provided on the lid 21b.

[0070] The two-fluid nozzle 25 is provided with a gas inlet 25a through which gas flows in, and a solution inlet 25b through which a solution containing an inhibitor for the insect's odor response in the storage section 21 flows in. The gas is, for example, high-pressure compressed air. The two-fluid nozzle 25 is also provided with a spray port 26. The liquid film-like solution containing the inhibitor for the insect's odor response that forms at the spray port 26 is atomized by the shear force of the airflow and ejected from the spray port 26. The atomized solution containing the inhibitor for the insect's odor response that is ejected from the spray port 26 is released outside the storage section 21 from the discharge section 23.

[0071] A control unit 30 for supplying gas to the two-fluid nozzle 25 is connected to the gas inlet 25a of the two-fluid nozzle 25 via a connection part 27 such as a pipe fitting. The control unit 30 includes, for example, a pump 32 for supplying air and a gas supply pipe 34 connecting the pump 32 to the connection part 27. A valve 33, such as a solenoid valve, is provided in the gas supply pipe 34. The pump 32 and the valve 33 are electrically connected to a controller 31. The controller 31 controls the flow rate and pressure of the gas supplied to the two-fluid nozzle 25 by controlling the pump 32 and the valve 33.

[0072] Alternatively, the insect inhibitor system according to the embodiment may have the configuration shown in Figure 6. The spraying unit 120 of the insect inhibitor system shown in Figure 6 includes, for example, a storage unit 41 for storing a solution containing an inhibitor for the insect's odor response, a particle atomization unit 42 for atomizing the solution containing the inhibitor for the insect's odor response, and a blowing unit 43 for blowing out the atomized solution containing the inhibitor for the insect's odor response.

[0073] The storage unit 21 is, for example, a tank and comprises a main body 21a and a lid 21b. The storage unit 21 and the atomization unit 42 are connected by piping for transporting a solution containing an inhibitor for the insect's odor response. The atomization unit 42 comprises, for example, a nozzle head 47 for containing the solution containing the inhibitor for the insect's odor response, and a piezoelectric element 45, such as a piezo element, located on the nozzle head 47. The discharge unit 43 is provided on the nozzle head 47.

[0074] When a pulse voltage is applied to the piezoelectric element 45, it repeatedly deforms and returns to its original shape. As a result, the volume of the nozzle head 47 repeatedly contracts and returns to its original shape. This causes the solution containing the inhibitor for the insect's odor response inside the nozzle head 47 to be intermittently pushed out from the discharge section 43 and atomized into fine particles.

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

[0076] Alternatively, the insect inhibitor system according to the embodiment may have the configuration shown in Figure 7. The spraying unit 60 of the insect inhibitor system shown in Figure 7 includes, for example, a storage unit 61 for storing a solution containing an inhibitor for the insect's odor response, a particle atomization unit 62 for atomizing the solution containing the inhibitor for the insect's odor response, and a blowing unit 63 for blowing out the atomized solution containing the inhibitor for the insect's odor response.

[0077] The storage section 61 is, for example, a flexible bag-shaped container. At least a portion of the storage section 61 is an ultrasonic permeable membrane 65. The discharge section 43 is provided in the storage section 61. The storage section 61 is held in a vibration generating container 66 filled with working water 67. The atomization section 62 is equipped with an ultrasonic transducer.

[0078] When a high-frequency AC voltage is applied to the ultrasonic transducer, it vibrates ultrasonically. The vibrational energy generated by the ultrasonic vibration reaches the solution containing the insect odor response inhibitor in the reservoir 61 via the working water 67 and the ultrasonic permeable membrane 65. As a result, the solution containing the insect odor response inhibitor in the reservoir 61 vibrates, the solution surface atomizes, and the finely atomized solution containing the insect odor response inhibitor is blown out from the discharge section 63.

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

[0080] Alternatively, the insect inhibitor system according to the embodiment may have the configuration shown in Figure 8. The spraying unit 80 of the insect inhibitor system shown in Figure 8 includes, for example, a storage unit 81 for storing a solution containing an inhibitor for the insect's odor response, an electrostatic spray type atomizing unit 82 for atomizing the solution containing the inhibitor for the insect's odor response, and a blowing unit 83 for blowing out the atomized solution containing the inhibitor for the insect's odor response.

[0081] The storage section 81 is, for example, a tank and comprises a main body 81a and a lid 81b. The discharge section 83 is provided in the storage section 81. The atomization section 82 is arranged inside the storage section 81. The atomization section 82 comprises an electrostatic spray nozzle 84 and a transport section 85 such as a pump. The transport section 85 transports a solution containing an inhibitor for the odor response of insects to the electrostatic spray nozzle 84. A cylindrical member 86 is arranged around the electrostatic spray nozzle 84. A voltage application section 87 such as a counter electrode is arranged on the upper end surface of the cylindrical member 86.

[0082] When 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, at the gas-liquid interface, the balance between the surface tension of the solution and the electrostatic force acting on the solution causes fine liquid threads to be drawn out, and the tips of these threads split into fine particles which are then sprayed from the electrostatic spray nozzle 84.

[0083] A high-voltage control unit 330 is connected to the voltage application unit 87 via wiring 71. The high-voltage control unit 330 applies a high voltage to the voltage application unit 87 to control electrostatic forces and other forces acting on the solution.

[0084] (Example 1: Establishment of a homogeneous odor detection cell line) DmOr13a and DmOrco are olfactory receptors derived from the antennae of Drosophila melanogaster, and they respond to the target odor, 1-octen-3-ol. GCaMP6s is an improved calcium-sensitive fluorescent protein. pIB vectors containing DmOr13a and DmOrco, and pIZ vectors containing GCaMP6s, were introduced into Sf21 cells by lipofection.

[0085] Grace's Insect Medium, Supplemented (11605-094, Gibco) is mixed with 10% US Insect Cell Screened FBS (SH30070.03, GE Healthcare) and three antibiotics (10 μg / mL Gentamicin Reagent Solution (15710-064, Gibco), 10 μg / mL Blasticidin S). Subculturing medium was prepared by adding HCl (A11139-03, Gibco) and Zeocin (R25001, Invitrogen) at a final concentration of 100 μg / mL. Sf21 cells expressing the DmOr13a receptor, co-receptor DmOrco, and GCaMP6s were subcultured in a flask (353082, FALCON) using this subculturing medium. The volume of the cell suspension at subculturing was 6 mL. Once the cells reached confluence, 6 mL of supernatant was collected from the flask and placed in a 15 mL tube (91015, TPP). The 15 mL tube was centrifuged at 400 × g at 4°C for 3 minutes using a micro-high-speed centrifuge.

[0086] After centrifugation, the supernatant was sterilized using a 10 mL syringe (01007, TOP) and a 0.45 μm filter (431220, CORNING). A 10 mL condition medium was prepared by mixing the sterilized supernatant with an equal volume of fresh subculture medium containing antibiotics (Blasticidin S HCl at a final concentration of 10 μg / mL and Zeocin at a final concentration of 100 μg / mL).

[0087] Cells adhering to the bottom of the flask from which the supernatant was collected were detached 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 above-mentioned condition medium, and thoroughly pipetted. The entire volume of the condition medium to which the cells were added was transferred to a reservoir (BM-0850-1, BMBio). Furthermore, using an 8-channel multichannel pipette (HT5123, HTL), 100 μL of the condition medium to which the cells were added was added dropwise to a 96-well plate (3860-096, IWAKI), and the cells were then cultured at 27°C. After the cells adhered to the wells, the wells were observed with an inverted microscope, and wells containing only single cells in the condition medium were confirmed.

[0088] Cells from wells where single cells were observed at seeding were cultured until approximately 80% to 90% of the cells reached confluence. Subsequently, the cells were scaled up in the following order: 24-well plates (3820-024, IWAKI), 35mm dishes (353801, CORNING), and T-25 flasks. For culture in 24-well plates, 35mm dishes, and T-25 flasks, the amount of culture medium was adjusted to 500 μL, 2.5 mL, and 5 mL, respectively, and cultured at 27°C. The responsiveness of cells that could be scaled up to T-25 flasks to odor substances was investigated by calcium imaging, and cell lines showing good responsiveness were obtained as homogeneous odor-detecting cell lines.

[0089] (Example 2: Response of odor-detecting cells to inhibitors) After seeding odor-detecting cells obtained in Example 1 onto a 12 mm diameter coverslip (CS-12R: Warner Instruments, LLC, Hamden, CT, USA), the coverslip was inserted into an open-type bath chamber for circular coverslips (RC-48LP: Warner Instruments, LLC, Hamden, CT, USA).

[0090] To perfusing the odor-detecting cells with solution, two silicone tubes, each with an inner diameter of 1 mm and an outer diameter of 3 mm, were connected to a peristaltic tube pump (MP-2010: Tokyo Rikakikai Co. Ltd., Tokyo, Japan). These tubes were then connected to the inlet and outlet of an open-type bath chamber, respectively, using tube clamps (CAT-1: NARISHIGE Co. Ltd., Tokyo, Japan).

[0091] An upright fluorescence microscope (BX51WI: Olympus, Tokyo, Japan) equipped with a 20x immersion objective lens (UMPlanFI 20x / 0.50W: Olympus, Tokyo, Japan) was prepared. A fluorescence filter set for GFP (U-MGFPHQ: Olympus, Tokyo, Japan) was placed on the upright fluorescence microscope. In addition, a 100W halogen lamp (TH4-100: Olympus, Japan) was placed on the upright fluorescence microscope as a light source. The exposure time for fluorescence observation was set to 500 milliseconds.

[0092] An EM-CCD camera (DU-897E: Andor Technology PLC, Belfast, UK) was prepared to measure changes in cell fluorescence intensity. The EM-CCD camera was operated using AndoriQ (Andor Technology PLC, Belfast, UK). The EM-CCD camera was configured to acquire 512 x 512 pixels of images per second.

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

[0094] When 300 μmol / L geraniol, a candidate inhibitor, was passed through the cells for 60 seconds, then 10 μmol / L 1-octen-3-ol, an odorant, was passed through the cells together with 300 μmol / L geraniol for 15 seconds, and finally 300 μmol / L geraniol was passed through the cells for another 60 seconds, the increase in fluorescence intensity in odor-detecting cells was suppressed compared to the case without the odorant inhibitor.

[0095] When 300 μmol / L l-menthol, a candidate inhibitor, was flowed for 60 seconds, then 10 μmol / L 1-octen-3-ol, an odorant, was flowed together with 300 μmol / L l-menthol for 15 seconds, and then 300 μmol / L l-menthol was flowed again for 60 seconds, the increase in fluorescence intensity in odor-detecting cells was suppressed compared to when no odorant inhibitor was present.

[0096] When 300 μmol / L thymol, a candidate inhibitor, was flowed for 60 seconds, then 10 μmol / L 1-octen-3-ol, an odorant, was flowed together with 300 μmol / L thymol for 15 seconds, and then 300 μmol / L thymol was flowed again for 60 seconds, the increase in fluorescence intensity in odor-detecting cells was suppressed compared to when no odorant inhibitor was used.

[0097] When 300 μmol / L linalyl formate (LF), an inhibitor of a known odor substance, was passed through cells for 60 seconds, followed by 10 μmol / L 1-octen-3-ol, an odor substance, being passed through cells together with 300 μmol / L LF for 15 seconds, and then 300 μmol / L LF being passed through cells for another 60 seconds, the increase in fluorescence intensity in odor-detecting cells was suppressed compared to the case without an odor substance inhibitor.

[0098] When 300 μmol / L of 2-tert-butyl-6-methylphenol (BMP), an inhibitor of a known odor substance, was passed through the cells for 60 seconds, followed by 10 μmol / L of 1-octen-3-ol, an odor substance, being passed through the cells together with 300 μmol / L of BMP for 15 seconds, and then 300 μmol / L of BMP being passed through the cells for another 60 seconds, the increase in fluorescence intensity in odor-detecting cells was suppressed compared to the case without the odor substance inhibitor.

[0099] Finally, when 10 μmol / L of 1-octen-3-ol, an odorant, was passed through the cells with buffer for 15 seconds, the increase in fluorescence intensity in the odor-detecting cells was restored.

[0100] The results of Example 2 demonstrate that odor-detecting cells are useful for screening for odor-causing inhibitors.

[0101] (Example 3: Response of odor-detecting cells in response to the concentration of fragrance components) We prepared candidate inhibitors such as thymol, citral, 1-nonanol, eugenol acetate, d-limonene, eugenol, geraniol, geranyl acetate, and BMP and LF, which are inhibitors of known odor substances.

[0102] Similar to the measurement method in Figure 9, 10 μmol / L of 1-octen-3-ol, an odorant, was administered to the odor-detecting cells obtained in Example 1 without the presence of an inhibitor, and the fluorescence intensity in the odor-detecting cells was measured. Next, 10 μmol / L of a candidate inhibitor or inhibitor, 30 μmol / L of a candidate inhibitor or inhibitor, 100 μmol / L of a candidate inhibitor or inhibitor, 170 μmol / L of a candidate inhibitor or inhibitor, 300 μmol / L of a candidate inhibitor or inhibitor, 560 μmol / L of a candidate inhibitor or inhibitor, 1 mmol / L of a candidate inhibitor or inhibitor, and 3 mmol / L of a candidate inhibitor or inhibitor were sequentially administered to the odor-detecting cells along with 10 μmol / L of 1-octen-3-ol, and the fluorescence intensity in the odor-detecting cells was measured. Finally, 10 μmol / L of 1-octen-3-ol was administered to the odor-detecting cells without the presence of an inhibitor, and the fluorescence intensity in the odor-detecting cells was measured.

[0103] Figure 10 shows the relationship between the concentration of each inhibitor and the normalized fluorescence intensity in odor-detecting cells, with the average percentage change in fluorescence intensity in odor-detecting cells set as 100% when 10 μmol / L of 1-octen-3-ol was administered to odor-detecting cells without the inhibitor. As shown in Figure 10, the response of odor-detecting cells to odor substances was suppressed in proportion to the concentration of the inhibitor.

[0104] The results of Example 3 demonstrate that odor-detecting cells are useful for screening for odor-causing inhibitors.

[0105] (Example 4: Response of odor-detecting cells in response to the concentration of candidate inhibitor substances) We prepared methyl transcinnamate and tert-butyl transcinnamate, which are candidate inhibitors.

[0106] Without adding the candidate inhibitor, 10 μmol / L of 1-octen-3-ol, an odorant, was administered to the odor-detecting cells obtained in Example 1, and the fluorescence intensity in the odor-detecting cells was measured. Next, 10 μmol / L, 30 μmol / L, 100 μmol / L, 170 μmol / L, 300 μmol / L, 560 μmol / L, and 1 mmol / L of the candidate inhibitor were sequentially administered to the odor-detecting cells along with 10 μmol / L of 1-octen-3-ol, and the fluorescence intensity in the odor-detecting cells was measured. Finally, without adding the inhibitor, 10 μmol / L of 1-octen-3-ol was administered to the odor-detecting cells, and the fluorescence intensity in the odor-detecting cells was measured.

[0107] Figure 11 shows the relationship between the concentration of each candidate inhibitor and the normalized fluorescence intensity in odor-detecting cells, with the average percentage change in fluorescence intensity in odor-detecting cells set as 100% when 10 μmol / L of 1-octen-3-ol was administered to odor-detecting cells without the candidate inhibitor. As shown in Figure 11, the response of odor-detecting cells to odor substances was suppressed in proportion to the concentration of the candidate inhibitor.

[0108] (Example 5: Response of odor-detecting cells according to the concentration of inhibitors and candidate inhibitors) We prepared candidate inhibitors: 2,4-diisopropylphenol, 4-isopropyl-3-methylphenol, 2-tert-butyl-4-methylphenol, 4-tert-butyl-2-methylphenol, and 2-isopropyl-6-methylphenol.

[0109] Without adding the candidate inhibitor, 10 μmol / L of 1-octen-3-ol, an odorant, was administered to the odor-detecting cells obtained in Example 1, and the fluorescence intensity in the odor-detecting cells was measured. Next, 10 μmol / L, 30 μmol / L, 100 μmol / L, 170 μmol / L, and 300 μmol / L of the candidate inhibitor were sequentially administered to the odor-detecting cells along with 10 μmol / L of 1-octen-3-ol, and the fluorescence intensity in the odor-detecting cells was measured. Finally, without adding the inhibitor, 10 μmol / L of 1-octen-3-ol was administered to the odor-detecting cells, and the fluorescence intensity in the odor-detecting cells was measured.

[0110] Figure 12 shows the relationship between the concentration of each candidate inhibitor and the normalized fluorescence intensity in odor-detecting cells, with the average percentage change in fluorescence intensity in odor-detecting cells set as 100% when 10 μmol / L of 1-octen-3-ol was administered to odor-detecting cells without the candidate inhibitor. As shown in Figure 12, the response to odor substances in odor-detecting cells was suppressed in proportion to the concentration of the candidate inhibitor.

[0111] (Example 6: Screening of inhibitors and candidate inhibitors based on their inhibitory effect on odor-detecting cells according to concentration) Based on the results of Examples 3 to 5, Figure 13 shows graphs of the percentage change in fluorescence intensity when the concentration of DEET, d-limonene, citral, linalyl formate, eugenol, geraniol, 1-nonanol, geranyl acetate, thymol, eugenol acetate, methyl transcinnamate, tert-butyl transcinnamate, 2,4-diisopropylphenol, 4-isopropyl-3-methylphenol, 2-isopropyl-6-methylphenol, 2-tert-butyl-4-methylphenol, and 4-tert-butyl-2-methylphenol is 300 μM.

[0112] Transmethyl cinnamate, trans tert-butyl cinnamate, 2,4-diisopropylphenol, 4-isopropyl-3-methylphenol, 2-isopropyl-6-methylphenol, 2-tert-butyl-4-methylphenol, and 4-tert-butyl-2-methylphenol reduced the fluorescence intensity change rate in odor-detecting cells to less than 37% when 10 μmol / L of 1-octen-3-ol was administered to odor-detecting cells. As shown in Example 7 described later, the inhibitor BMP, and the candidate inhibitors 4-isopropyl-3-methylphenol and 4-tert-butyl-2-methylphenol, showed inhibitory effects in the electroanthenogram. In Example 6, the fluorescence intensity change rate of these substances was less than 25% (24.7%). On the other hand, as shown in Example 7 described later, eugenol acetate, 1-nonanol, and thymol, which showed fluorescence intensity change rates of 37% or more, did not show inhibitory effects in the electroanthenogram. In Example 6, the fluorescence intensity change rate of these substances was 37% or more. Therefore, it was shown that substances with a fluorescence intensity change rate of less than 25% showed an inhibitory effect, and that an inhibitory effect could be obtained if the fluorescence intensity change rate was less than 37%. In addition, 2-tert-butyl-4-methylphenol and 4-tert-butyl-2-methylphenol reduced the fluorescence intensity change rate in odor-detecting cells to 20% or less when 10 μmol / L of 1-octen-3-ol was administered to odor-detecting cells.

[0113] (Example 7: Tactile response to the inhibitor) As shown in Figure 14, gel droplets (Spectra 360 Electrode Gel, Parker Laboratories) were placed on the surfaces of two metal electrodes, and silkworm antennae, with their tips and bases removed, were brought into contact with the gel droplets, arranging the antennae in an arch shape between the two metal electrodes. It is known that the potential between the tip and base of insect antennae changes in response to odor substances, and recording this potential is called electroanthenogram (EAG).

[0114] We prepared bombicol (BOL), a sex pheromone of the silkworm moth; BMP, an inhibitor of a known odor substance; 4-isopropyl-3-methylphenol, a candidate inhibitor; and 4-tert-butyl-2-methylphenol, another candidate inhibitor. We prepared filter paper with 1000 ng of BOL, 1000 ng of BOL and 1000 μg of BMP, 1000 ng of BOL and 1000 ng of 4-isopropyl-3-methylphenol, and 1000 ng of BOL and 1000 ng of 4-tert-butyl-2-methylphenol. A filter paper with BOL added was placed in the first glass tube cartridge, a filter paper with BOL and BMP added was placed in the second glass tube cartridge, a filter paper with BOL and 4-isopropyl-3-methylphenol added was placed in the third glass tube cartridge, and a filter paper with BOL and 4-tert-butyl-2-methylphenol added was placed in the fourth glass tube cartridge.

[0115] When a gas containing BOL was injected into the antennae at a rate of 1 L / min from the first glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the upper part of Figure 15(a). Next, when a gas containing BOL and BMP was injected into the antennae at a rate of 1 L / min from the second glass tube cartridge, the decrease in potential between the tip and base of the antennae was suppressed to less than half compared to the case without BMP, as shown in the upper part of Figure 15(b). Again, when a gas containing BOL was injected into the antennae at a rate of 1 L / min from the first glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the upper part of Figure 15(c).

[0116] When a gas containing BOL was injected into the antennae at a rate of 1 L / min from the first glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the middle of Figure 15(a). Next, when a gas containing BOL and 4-isopropyl-3-methylphenol was injected into the antennae at a rate of 1 L / min from the third glass tube cartridge, the decrease in potential between the tip and base of the antennae was suppressed to about half compared to the case without 4-isopropyl-3-methylphenol, as shown in the middle of Figure 15(b). Again, when a gas containing BOL was injected into the antennae at a rate of 1 L / min from the first glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the middle of Figure 15(c).

[0117] When a gas containing BOL was injected into the antennae at a rate of 1 L / min from the first glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the lower part of Figure 15(a). Next, when a gas containing BOL and 4-tert-butyl-2-methylphenol was injected into the antennae at a rate of 1 L / min from the fourth glass tube cartridge, the decrease in potential between the tip and base of the antennae was suppressed to about half compared to the case without 4-tert-butyl-2-methylphenol, as shown in the lower part of Figure 15(b). Again, when a gas containing BOL was injected into the antennae at a rate of 1 L / min from the first glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the lower part of Figure 15(c).

[0118] We prepared bombicol (BOL), a sex pheromone of the silkworm moth, as well as candidate inhibitors: eugenol acetate, 1-nonanol, and thymol. We prepared filter paper with 1000 ng of BOL, 1000 ng of BOL and 1000 μg of eugenol acetate, 1000 ng of BOL and 1000 μg of 1-nonanol, and 1000 ng of BOL and 1000 ng of thymol. We placed the filter paper with BOL in the fifth glass tube cartridge, the filter paper with BOL and eugenol acetate in the sixth glass tube cartridge, the filter paper with BOL and 1-nonanol in the seventh glass tube cartridge, and the filter paper with BOL and thymol in the eighth glass tube cartridge.

[0119] When a gas containing BOL was injected into the antennae at a rate of 1 L / min from the fifth glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the upper part of Figure 16(a). Next, when a gas containing BOL and eugenol acetate was injected into the antennae at a rate of 1 L / min from the sixth glass tube cartridge, the decrease in potential between the tip and base of the antennae was enhanced compared to the case without eugenol acetate, as shown in the upper part of Figure 16(b). Again, when a gas containing BOL was injected into the antennae at a rate of 1 L / min from the fifth glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the upper part of Figure 16(c).

[0120] When a gas containing BOL was injected into the antennae at a rate of 1 L / min from the fifth glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the middle of Figure 16(a). Next, when a gas containing BOL and 1-nonanol was injected into the antennae at a rate of 1 L / min from the seventh glass tube cartridge, the decrease in potential between the tip and base of the antennae was enhanced compared to the case without 1-nonanol, as shown in the middle of Figure 16(b). Again, when a gas containing BOL was injected into the antennae at a rate of 1 L / min from the fifth glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the middle of Figure 16(c).

[0121] When a gas containing BOL was injected into the antennae at a rate of 1 L / min from the fifth glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the lower part of Figure 16(a). Next, when a gas containing BOL and thymol was injected into the antennae at a rate of 1 L / min from the eighth glass tube cartridge, the decrease in potential between the tip and base of the antennae was enhanced compared to the case without thymol, as shown in the lower part of Figure 16(b). Again, when a gas containing BOL was injected into the antennae at a rate of 1 L / min from the fifth glass tube cartridge, the potential between the tip and base of the antennae decreased as shown in the lower part of Figure 16(c).

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

[0123] (Example 8: Insect response to inhibitor) Several transparent plastic containers with lids (MaruCup 200MB, Mineron Chemical Industry Co., Ltd.) were prepared. Small holes were made in the lids of the plastic containers to allow the tip of a Pasteur pipette to be inserted. One male silkworm moth was placed in each of the plastic containers and the lids were closed.

[0124] We prepared bombicol (BOL), a sex pheromone of the silkworm moth, and BMP, an inhibitor of known odor substances. Filter papers were prepared with 0.01 ng of BOL, 0.1 ng of BOL, 1 ng of BOL, 10 ng of BOL, 100 ng of BOL, 1000 ng of BOL, and 1000 μg of BMP diluted with hexane.

[0125] Filter paper with 0.01 ng of BOL was placed in the first Pasteur pipette, then in the second Pasteur pipette, then in the third Pasteur pipette, then in the fourth Pasteur pipette, then in the fourth Pasteur pipette, then in the fifth Pasteur pipette, then in the fifth Pasteur pipette, then in the sixth Pasteur pipette, then in the sixth Pasteur pipette, then in the sixth Pasteur pipette, then in the seventh

[0126] As shown in Figure 17, when silkworms were puff-stimulated three times with BOL at sequential concentrations, 3 out of 5 silkworms exhibited flapping behavior and 2 exhibited exploratory behavior at 1 ng of BOL. At 10 ng of BOL, all silkworms exhibited exploratory behavior.

[0127] As shown in Figure 18, after three puff stimuli with BMP, silkworms were sequentially stimulated three times with BOL at each concentration. With 1 ng of BOL, 3 out of 5 silkworms showed no response, and 2 showed flapping behavior. With 10 ng of BOL, 1 out of 5 silkworms showed no response, 2 out of 5 showed flapping behavior, and 2 out of 5 showed exploratory behavior. With 100 ng of BOL, 1 out of 3 silkworms showed flapping behavior, and 2 showed exploratory behavior. With 1000 ng of BOL, the remaining 1 silkworm showed exploratory behavior. A comparison of Figures 17 and 18 shows that BMP inhibits the insect's response to odor substances and raises the threshold concentration of BOL that elicits flapping and exploratory behavior.

[0128] The results of Example 8 demonstrate that observing insect responses is useful for screening odor inhibitors.

[0129] (Example 9: Screening of inhibitors using insects) Multiple plastic containers, similar to those used in Example 8, were prepared, and one male silkworm moth was placed in each of them before closing the lids.

[0130] Bombicol (BOL) diluted with hexane was prepared. As candidate inhibitors, 4-tert-butyl-2-methylphenol diluted with hexane and 4-isopropyl-3-methylphenol diluted with ethanol were prepared.

[0131] A filter paper with 0.01 ng of BOL diluted with hexane added was placed in the first Pasteur pipette, a filter paper with 0.1 ng of BOL diluted with hexane added was placed in the second Pasteur pipette, a filter paper with 1 ng of BOL diluted with hexane added was placed in the third Pasteur pipette, a filter paper with 10 ng of BOL diluted with hexane added was placed in the fourth Pasteur pipette, a filter paper with 100 ng of BOL diluted with hexane added was placed in the fifth Pasteur pipette, and a filter paper with 1000 ng of BOL diluted with hexane added was placed in the sixth Pasteur pipette.

[0132] As shown in Figure 19, when silkworms were puffed three times with BOL at various concentrations sequentially diluted with hexane, two out of three silkworms showed no reaction to BOL diluted with 0.01 ng of hexane, while one silkworm exhibited flapping behavior. All silkworms exhibited exploratory behavior to BOL diluted with 0.1 ng of hexane.

[0133] Filter paper to which 1000 ng of 4-tert-butyl-2-methylphenol diluted with hexane was dropped was placed in a plastic container with silkworms for 10 minutes. Then, as shown in Figure 20, the silkworms were puffed three times with BOL of various concentrations diluted with hexane. None of the silkworms reacted to 0.01 ng of BOL diluted with hexane. Two out of three silkworms did not react to 0.1 ng of BOL diluted with hexane, and one silkworm showed flapping behavior. Two out of three silkworms did not react to 1 ng of BOL diluted with hexane, and one silkworm showed exploratory behavior. One out of two silkworms showed flapping behavior to 10 ng of BOL diluted with hexane, and one silkworm showed exploratory behavior. The remaining silkworm showed exploratory behavior to 100 ng of BOL diluted with hexane. A comparison of Figure 19 and Figure 20 shows that 4-tert-butyl-2-methylphenol inhibits the insect's response to odor molecules and increases the threshold concentration of BOL that elicits flapping and searching behaviors.

[0134] Filter paper with ethanol drops was placed in a plastic container with silkworms for 10 minutes. Then, as shown in Figure 21, the silkworms were puffed three times with BOL of various concentrations sequentially diluted with hexane. None of the silkworms reacted to BOL diluted with 0.01 ng of hexane. Two out of three silkworms did not react to BOL diluted with 0.1 ng of hexane, and one silkworm showed flapping behavior. All of the silkworms showed exploratory behavior to BOL diluted with 1 ng of hexane.

[0135] Filter paper to which 1000 ng of 4-isopropyl-3-methylphenol diluted with ethanol was dropped was placed in a plastic container with silkworms for 10 minutes. Then, as shown in Figure 22, the silkworms were puffed three times with BOL of various concentrations sequentially diluted with hexane. None of the silkworms reacted to 0.01 ng and 0.1 ng of BOL diluted with hexane. Two out of three silkworms did not react to 1 ng of BOL diluted with hexane, and one silkworm showed flapping behavior. Two out of three silkworms showed flapping behavior to 10 ng of BOL diluted with hexane, and one silkworm showed exploratory behavior. All of the silkworms showed exploratory behavior to 100 ng of BOL diluted with hexane. A comparison of Figure 21 and Figure 22 shows that 4-isopropyl-3-methylphenol inhibits the insect's response to odor molecules and increases the threshold concentration of BOL that elicits flapping and searching behaviors.

[0136] (Example 10: Preparation of odor-detecting cells expressing Or56) The DmOrco gene, a co-receptor derived from the antennal cDNA of Drosophila melanogaster, was amplified by PCR from the start codon to the stop codon using primers containing the gene-specific sequence described below to obtain the DmOrco gene. The obtained DmOrco gene was inserted into the multi-cloning site of a pIZ vector (Invitrogen) using NEBuilder HiFi DNA Assembly MasterMix (New England Biolabs Japan) to construct the pIZ-DmOrco vector.

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

[0138] Furthermore, the constructed pIZ-DmOrco vector was amplified by PCR using the following primers, and then inserted into the Pci1 site of the pIB vector (Invitrogen) using NEBuilder HiFi DNA Assembly MasterMix (New England Biolabs Japan) to construct the pIB-DmOrco vector.

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

[0140] Gene amplification by PCR was performed using forward and reverse primers at concentrations of 100 pmol / μl each, PrimeSTAR HS DNA polymerase (Takara Bio, R010A), the reaction buffer provided with the polymerase, and dNTPs, following the protocol provided with the polymerase. The PCR temperature conditions were as follows: a step of 1 minute at 94°C, followed by 30 cycles of temperature cycling: 10 seconds at 98°C, 15 seconds at 55°C, and 1.5 minutes at 72°C, followed by a step of 5 minutes at 72°C.

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

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

[0143] The start and stop codons of the obtained DmOr56a_Sf9 synthetic gene were amplified by PCR using the above-mentioned adapter sequence primers to obtain the DmOr56a_Sf9 gene. The obtained DmOr56a_Sf9 gene was inserted into the multi-cloning site of the constructed pIB-DmOrco vector using NEBuilder HiFi DNA Assembly MasterMix (New England Biolabs Japan) to construct the pIB-DmOr56a_Sf9-DmOrco vector.

[0144] Gene amplification by PCR was performed using forward and reverse primers at concentrations of 100 pmol / μl each, PrimeSTAR HS DNA polymerase (Takara Bio, R010A), the reaction buffer provided with the polymerase, and dNTPs, following the protocol provided with the polymerase. The PCR temperature conditions were as follows: a step of 2 minutes at 98°C, followed by 25 cycles of temperature cycling: 10 seconds at 98°C, 10 seconds at 55°C, and 1.5 minutes at 72°C, followed by a step of 10 minutes at 72°C.

[0145] Similarly, a calcium-sensitive protein (GCaMP6s) expression vector was constructed. The GCaMP6s gene was obtained via Addgene from Dr. Douglas Kim (Janelia Farm Research Campus, Howard Hughes Medical Institute). The GCaMP6s gene was amplified from the start codon to the stop codon using primers containing the gene-specific sequence described below to obtain the GCaMP6s gene. The obtained GCaMP6s gene was inserted into the multi-cloning site of a pIZ vector (Invitrogen) using NEBuilder HiFi DNA Assembly MasterMix (New England Biolabs Japan) to construct the pIZ-GCaMP6s vector.

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

[0147] The constructed olfactory receptor expression vector and calcium-sensitive protein expression vector were introduced into Sf21 cells using a transfection reagent (TransIT-Insect Transfection Reagent: Mirus) according to the attached manual. This resulted in Sf21 cells co-expressing the DmOr56a_Sf9 receptor, DmOrco, and GCaMP6s (hereinafter referred to as "Odor-Detecting Cells Expressing Or56").

[0148] (Example 11: Establishment of a homogeneous odor detection cell line expressing Or56) As a preliminary step, one flask (FALCON) was prepared containing 6 mL of Or56-expressing odor-detecting cells during passaging before phylogenetic development. 6 mL of the culture supernatant was collected from the confluent cell flask into a 15 mL tube (TPP) and centrifuged at 400 × g for 3 minutes at 4°C. The supernatant after centrifugation was collected into a 25 mL tube (IWAKI) and sterilized using a 10 mL syringe (TOP) and a 0.45 μm filter (CORNING). Conditioned medium was prepared by mixing 5 mL of the sterilized medium, 5 mL of fresh medium, and two antibiotics (Blasticidin (Life Technologies) and Zeocin (Life Technologies)) equivalent to the amount of fresh medium.

[0149] Next, the cells were detached from the bottom of the flask, suspended in 1 mL of fresh medium, and collected in a 1.5 mL tube (AXYGEN). The number of cells collected in the 1.5 mL tube was counted, and 40 cells were added to the conditioning medium to obtain a cell suspension. The entire volume of the obtained cell suspension was transferred to a reservoir (BMBio), and 100 μL was seeded into each well of a 96-well plate (IWAKI) using an 8-multichannel pipette (HTL), and cultured in a 27°C incubator. After the cells had adhered to the wells, wells containing single cells were confirmed using an inverted microscope.

[0150] Cells in wells where single cells were confirmed in a 96-well plate were cultured until approximately 80% to 90% confluence. Subsequently, the culture was scaled up in the following order: 24-well plate (IWAKI), 35mm dish (CORNING), and T25 flask. Cells that were scaled up to the T25 flask were checked for fluorescence response using calcium imaging. Cells that did not respond were discarded, and cells that responded were frozen to create stocks, and the lineage was maintained by repeatedly subculturing.

[0151] (Example 12: Response of odor-detecting cells expressing Or56 in accordance with the concentration of inhibitors and candidate inhibitors) We prepared candidate inhibitors: 2,4-diisopropylphenol, 2,5-diisopropylphenol, 4-isopropyl-3-methylphenol, 2-tert-butyl-4-methylphenol, 4-(tert-butyl)-2-methylphenol, and 2-isopropyl-6-methylphenol. We also prepared 2-tert-butyl-6-methylphenol (BMP), an inhibitor of a known odor substance.

[0152] Without adding any candidate inhibitors or known inhibitors, 1 μmol / L geosmin, an odorant, was administered to odor-detecting cells expressing Or56 obtained in Example 11, and the fluorescence intensity in the odor-detecting cells was measured. Next, 10 μmol / L of a candidate inhibitor or known inhibitor, 30 μmol / L of a candidate inhibitor or known inhibitor, 100 μmol / L of a candidate inhibitor or known inhibitor, 170 μmol / L of a candidate inhibitor or known inhibitor, and 300 μmol / L of a candidate inhibitor or known inhibitor were sequentially administered to odor-detecting cells along with 1 μmol / L geosmin, and the fluorescence intensity in the odor-detecting cells was measured. Finally, without adding any inhibitors or known inhibitors, 1 μmol / L geosmin was administered to odor-detecting cells, and the fluorescence intensity in the odor-detecting cells was measured.

[0153] Figure 23 shows the relationship between the concentration of each candidate inhibitor and a known inhibitor and the normalized fluorescence intensity in odor-detecting cells, with the average percentage change in fluorescence intensity in odor-detecting cells set as 100% when 1 μmol / L geosmin was administered to odor-detecting cells without any candidate inhibitors or known inhibitors. As shown in Figure 23, the response to odor substances in odor-detecting cells expressing Or56 was suppressed according to the concentration of the candidate inhibitor and the known inhibitor.

[0154] (Example 1) 3 (Insect response to inhibitors) As a candidate inhibitor, 4-isopropyl-3-methylphenol (IPMP) was prepared using DMSO as the solvent, and DMSO was prepared as the control agent. 1 μL of crude extracts (acetone solvent) of the cockroach pheromones periplanone A and periplanone B were prepared and attached to a 15 mm square piece of filter paper or aluminum foil, and the acetone solvent was allowed to evaporate completely.

[0155] The lid of a container holding approximately 10 cockroaches was removed under red light in a dark room, and the food dish and water cup were collected. Two petri dishes, each containing filter paper soaked in the test insecticide, were placed in diagonal corners of the container. In addition, water-soaked Kimwipes were placed in the container to maintain humidity, and the lid was then closed. The container was then left undisturbed for 30 minutes.

[0156] Video recording of the container was started, and a piece of filter paper or aluminum coated with crude cockroach pheromone extract was placed on top of one of the petri dishes containing filter paper soaked in the test agent. Then, as shown in Figure 24, a transparent acrylic plate was placed on the container in place of a lid to facilitate filming, and video was recorded for 10 minutes.

[0157] The recorded video was analyzed, and the total number of times cockroaches came into contact with filter paper or aluminum pieces coated with crude cockroach pheromone extract was measured in the 5 minutes immediately following the placement of the transparent acrylic plate in the container. As shown in Figure 25, 300 mmol / L of 4-isopropyl-3-methylphenol with DMSO as the solvent prevented cockroaches from approaching and coming into contact with the pheromone-containing filter paper or aluminum pieces. Furthermore, as shown in Figure 26, the higher the concentration of 4-isopropyl-3-methylphenol, the more the cockroaches were suppressed from approaching the pheromone-containing filter paper or aluminum pieces. [Explanation of Symbols]

[0158] 20...Spraying section, 21...Storage section, 21a...Main body, 21b...Lid, 22...Atomization section, 23...Blow-out section, 25...Two-fluid nozzle, 25a...Gas inlet, 25b...Solution inlet, 26...Spray nozzle, 27...Connection section, 30...Control unit, 31...Controller, 32...Pump, 33...Valve, 34...Gas supply pipe, 41...Storage section, 42...Microparticle section, 43...Blow-out section, 45...Piezoelectric element, 47...Nozzle head, 48...Wiring, 60...Spraying section, 6 1...Storage section, 62...Atomization section, 62...Microparticle section, 63...Blow-out section, 65...Ultrasonic permeable membrane, 66...Vibration generating container, 67...Water, 70...Wiring, 71...Wiring, 80...Spraying section, 81...Storage section, 81a...Main body, 81b...Lid, 82...Microparticle section, 83...Blow-out section, 84...Electrostatic spray nozzle, 85...Transportation section, 86...Cylindrical member, 87...Voltage application section, 120...Spraying section, 130...Control section, 230...Control section, 330...High voltage control section

Claims

1. Inhibitors of insect odor response, including 4-isopropyl-3-methylphenol.

2. Inhibitors of insect odor response, including 4-(tert-butyl)-2-methylphenol.

3. Inhibitors of insect odor response, including 2-isopropyl-6-methylphenol.

4. Inhibitors of insect odor response, including methyl transcinnamate.

5. An inhibitor of insect odor response, containing tert-butyl transcinnamate.

6. An inhibitor for the odor response of insects according to any one of claims 1 to 5, wherein the solvent is at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water.

7. An inhibitor of the odor response of insects according to any one of claims 1 to 5, which is an inhibitor of the odor response of moths.

8. The inhibitor for the odor response of insects according to claim 7, wherein the moth is a silkworm moth.

9. An inhibitor of the odor response of an insect, as described in any one of claims 1 to 5, which is an inhibitor of the odor response of a cockroach.

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