JP2024012184A - Screening method for inhibitor of insect odor response, inhibitor of insect odor response, insect inhibition system, and insect inhibition method - Google Patents

Abstract

PROBLEM TO BE SOLVED: 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

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention relates to environmental technology, and relates to a screening method for an inhibitor of insect odor response, an inhibitor of insect odor response, an insect inhibition system, and an insect inhibition method.

The sense of smell is involved in many of the habits of insects (see, for example, Patent Document 1 and Non-Patent Document 1). The sense of smell is thought to play an important role in the feeding behavior of insects, the attraction between males and females for the purpose of mating, and the selection of spawning sites. On the other hand, in order to repel harmful insects, it has been proposed to inhibit the insect's sense of smell or to utilize odors that insects repel.

International Publication No. 2021/045233

Satoshi Hamada et al., "Giant vesicles functionally expressing membrane receptors for an insect pheromone," Chem. Commun., 2014,50, 2958

Highly effective inhibitors of insect odor responses and methods for screening such inhibitors are desired. Therefore, one of the objects of the present invention is to provide a screening method for an inhibitor of insect odor response, an inhibitor of insect odor response, an insect inhibition system, and an insect inhibition method.

A method of screening for an inhibitor of an insect odor response according to an aspect of the present invention includes providing an odor detection structure expressing at least one of an insect olfactory receptor and an olfactory receptor co-receptor; A plurality of inhibitor candidate substances are screened based on providing an odorant corresponding to a receptor and a plurality of inhibitor candidate substances to an odor detection structure, and on the influx of ions into the odor detection structure. Including. Insect olfactory receptors may be ionotropic receptors. The odor detection structure may be a cell. The odor detection structure may be a man-made structure. The odor detection structure may express coreceptors for olfactory receptors.

In the above method for screening an inhibitor of insect odor response, the olfactory receptor may be a 1-octen-3-ol receptor, and the odorant may be 1-octen-3-ol.

In the above method for screening for inhibitors of insect odor responses, the insect may be a cockroach.

The above-mentioned method for screening for inhibitors against the odor response of insects includes preparing insect antennae, applying a stimulating substance that stimulates the insect antennae, and an inhibitor candidate substance selected by screening to the insect antennae. and detecting a response of the antennae of the insect. The irritant may be bombykol. The antennae of the insect may be the antennae of a moth. The moth may be a silk moth.

In the above method for screening inhibitors of insect odor responses, the olfactory receptor is a 1-octen-3-ol receptor, the odorant is 1-octen-3-ol, and the stimulant is bombycol. Yes, the antennae of the insect may be the antennae of a moth. The above-described method of screening for an inhibitor against the odor response of an insect may also be a method for screening for an inhibitor against the response of a moth to a pheromone.

In the above-described method of screening for an inhibitor against the odor response of an insect, the electric potential of the antennae may be detected in detecting the response of the antennae of the insect.

The above-mentioned method for screening for inhibitors against odor responses in insects involves preparing insects, giving the insects a stimulating substance that stimulates the insects, and candidate inhibitor substances selected through screening, and monitoring the insects' responses. The method may further include detecting. The irritant may be bombykol. The insect may be a moth. The moth may be a silk moth. The insect may be a cockroach. The cockroach may be an American cockroach.

In the above method for screening inhibitors of insect odor responses, the olfactory receptor is a 1-octen-3-ol receptor, the odorant is 1-octen-3-ol, and the stimulant is bombycol. Yes, the insect may be a moth. The above-described method of screening for an inhibitor against the odor response of an insect may also be a method for screening for an inhibitor against the response of a moth to a pheromone.

In the above-described method of screening for inhibitors of insect odor responses, insect behavior may be observed in detecting insect responses. The behavior of the insect may be flapping behavior. The behavior of the insect may be exploratory behavior.

Inhibitors of insect odor responses according to embodiments of the present invention are inhibitors of insect odor responses that result in less than 37% influx of ions into the odor sensing structure.

An inhibitor of insect odor response according to an aspect of the present invention is an inhibitor of insect odor response obtained by the above-described method for screening an inhibitor of insect odor response, and is an inhibitor of insect odor response that is obtained by the above-mentioned method for screening an inhibitor of insect odor response. It is an inhibitor of the insect odor response, resulting in an influx of less than 37%.

The inhibitor of the insect odor response described above may reduce the influx of ions into the odor detection structure by 20% or less.

The above-mentioned inhibitor of the insect odor response may be a methylphenol derivative.

The inhibitor of the insect odor response described above may include 4-isopropyl-3-methylphenol.

The inhibitor of the insect odor response described above may include 4-(tert-butyl)-2-methylphenol.

In the above inhibitor of insect odor response, the solvent may be at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water.

The above-mentioned inhibitor of insect odor response may be an inhibitor of moth odor response. The moth may be a silk moth. The above-mentioned inhibitor of the insect odor response may be an inhibitor of the cockroach odor response. The cockroach may be an American cockroach.

An insect inhibition system according to an aspect of the present invention includes a sprayer that sprays an inhibitor of insect odor responses. The inhibitor of insect odor response may be an inhibitor of insect odor response as described above.

An insect inhibition system according to an aspect of the present invention includes a spraying unit that sprays an inhibitor against an insect odor response obtained by the above-described method for screening an inhibitor against an insect odor response.

In the insect inhibition system described above, the dispensing unit may micronize the inhibitor against the insect's odor response.

In the above insect inhibition system, the dispersion unit is at least one selected from the group consisting of a two-fluid nozzle, an ultrasonic vibrator, a piezoelectric element, and an electrostatic spray nozzle that atomizes the inhibitor to the insect's odor response. may be provided.

In the above insect inhibition system, the solvent for the inhibitor of insect odor response may be at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water.

In the insect inhibition system described above, the inhibitor of insect odor response may include 4-isopropyl-3-methylphenol or 4-(tert-butyl)-2-methylphenol.

A method of inhibiting insects according to an embodiment of the invention includes applying an insect inhibiting solution. The inhibitor of insect odor response may be an inhibitor of insect odor response as described above.

An insect inhibition method according to an embodiment of the present invention includes spraying an insect inhibition solution obtained by the above-described method for screening an inhibitor of insect odor response.

In the above method for inhibiting insects, the inhibitor to the odor response of insects may be made into fine particles during spraying.

In the above method for inhibiting insects, in the spraying, at least one selected from the group consisting of a two-fluid nozzle, an ultrasonic vibrator, a piezoelectric element, and an electrostatic spray nozzle is used to apply an inhibitor to the odor response of insects in fine particles. may be converted into

In the above method for inhibiting insects, the solvent for the inhibitor of insect odor responses may be at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water.

In the insect inhibition method described above, the inhibitor of insect odor response may include 4-isopropyl-3-methylphenol or 4-(tert-butyl)-2-methylphenol.

According to the present invention, it is possible to provide a method of screening for an inhibitor of insect odor response, an inhibitor of insect odor response, an insect inhibition system, and an insect inhibition method.

FIG. 2 is a schematic diagram showing a method for expressing an olfactory receptor in an odor detection cell according to an embodiment. FIG. 2 is a schematic diagram showing a method for homogenizing odor detection cells according to an embodiment. FIG. 2 is a schematic diagram showing a method for expressing multiple olfactory receptors in odor detection cells according to an embodiment. FIG. 2 is a schematic diagram showing a method for expressing multiple olfactory receptors in odor detection cells according to an embodiment. FIG. 1 is a schematic diagram of an insect inhibition system according to an embodiment. FIG. 1 is a schematic diagram of an insect inhibition system according to an embodiment. FIG. 1 is a schematic diagram of an insect inhibition system according to an embodiment. FIG. 1 is a schematic diagram of an insect inhibition system according to an embodiment. 2 is a graph showing temporal changes in the intensity of fluorescence emitted by odor detection cells according to Examples. 2 is a graph showing the relationship between the concentration of an inhibitor and an inhibitor candidate substance for the odor response of an insect and the normalized intensity of fluorescence emitted by an odor detection cell according to an example. 2 is a graph showing the relationship between the concentration of an inhibitor candidate substance and the normalized intensity of fluorescence emitted by odor detection cells with respect to the odor response of insects according to an example. 2 is a graph showing the relationship between the concentration of an inhibitor and an inhibitor candidate substance for the odor response of an insect and the normalized intensity of fluorescence emitted by an odor detection cell according to an example. 3 is a graph showing the relationship between inhibitors and inhibitor candidate substances for insect odor responses and the normalized intensity of fluorescence emitted by odor detection cells according to Examples. It is a photograph which shows the measuring method of EAG based on an Example. It is a graph showing the time change of the electric potential of the antenna according to the example. It is a graph showing the time change of the electric potential of the antenna according to the example. 1 is a table showing insect responses to multiple concentrations of bombykol according to an example. Figure 2 is a table showing insect responses to multiple concentrations of bombykol after feeding the insects with an example inhibitor. 1 is a table showing insect responses to multiple concentrations of bombykol according to an example. FIG. 2 is a table showing the response of insects to multiple concentrations of bombykol after administering the inhibitor candidate substance according to the example to the insects. FIG. 1 is a table showing insect responses to multiple concentrations of bombykol according to an example. FIG. 2 is a table showing the response of insects to multiple concentrations of bombykol after administering the inhibitor candidate substance according to the example to the insects. FIG. 2 is a graph showing the relationship between the concentration of an inhibitor and an inhibitor candidate substance for the odor response of an insect and the normalized intensity of fluorescence emitted by an odor detection cell according to an example. It is a photograph of a container for observing mating behavior of cockroaches according to an example. It is a graph showing the number of accesses of cockroaches to a filter paper piece or an aluminum piece containing a pheromone according to an example. It is a graph showing the number of accesses of cockroaches to a filter paper piece or an aluminum piece containing a pheromone according to an example.

A method of screening for an inhibitor of an insect odor response according to an embodiment includes preparing an odor detection structure expressing at least one of an insect olfactory receptor and a co-receptor for the olfactory receptor; providing an odorant corresponding to the odorant and a plurality of inhibitor candidate substances to an odor detection structure, and screening a plurality of inhibitor candidate substances based on the influx of ions into the odor detection structure. ,including.

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

When the odor detection structure is a cell, the odor detection cell expresses an olfactory receptor on its cell membrane. In odor detection cells, olfactory receptors may be expressed naturally or by transgenes. The olfactory receptor may be an insect olfactory receptor.

The odor detection cell may be an insect cell. The insect cells may be cells derived from moths such as Spodoptera frugiperda and Trichoplusia ni. Examples of cells derived from Spodoptera include Sf21 and Sf9. Sf21 cells are derived from ovarian cells. Sf21 cells divide indefinitely, making it possible to establish a stable expression line that permanently expresses the introduced gene. Moreover, Sf21 cells can survive in a wide temperature range from 18° C. to 40° C., and carbon dioxide is not required to adjust the pH of the culture solution. Although Sf21 cells originally do not have olfactory receptors, it is possible to express olfactory receptors by introducing an olfactory receptor gene. Sf9 cells are a clone of Sf21. Examples of cells derived from Stinging Urticaria include High Five and Tni. Tni-derived cells are derived from ovarian cells.

Alternatively, the insect cell may be a cell derived from Drosophila. Examples of Drosophila-derived cells include Drosophila S2 cells. Alternatively, the insect cell may be a cockroach-derived cell.

Olfactory receptors are ionotropic receptors. The olfactory receptor may be a G protein-coupled receptor or an ionotropic receptor. Ionotropic receptors have a site that interacts with a ligand, which is an odor molecule, and a site into which ions flow. When the ion channel type receptor of the odor detection cell binds to a ligand, cations such as sodium ions and calcium ions flow into the odor detection cell. In odor-sensing cells, ion influx can occur on the order of tens of milliseconds after ligand binding. The amount of ions that flow into the cell is large, and it is said that 10 ⁷ ions flow into the cell for each binding of one ligand.

Generally, certain types of olfactory receptors have specificity for particular odor molecules. In odor detection 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. Furthermore, the amount of olfactory receptors to be expressed may be adjusted. Insect olfactory receptors have similar odorant reception mechanisms in many insect species such as flies, mosquitoes, moths, bees, lice, and cockroaches. Thus, an inhibitor of a particular insect's olfactory receptors can function as an inhibitor of insect olfactory receptors in general.

The odor detection structure may express an olfactory receptor as well as a co-receptor for the olfactory receptor. Coreceptors form heterocomplexes with olfactory receptors and function as receptors for odorants. Insect co-receptors also have similar odorant reception mechanisms in many insect species such as flies, mosquitoes, moths, bees, lice, and cockroaches. Thus, an inhibitor of a coreceptor of a particular insect olfactory receptor may function as an inhibitor of coreceptors of olfactory receptors of insects in general.

Examples of olfactory receptors include DmOr56a, which is a Drosophila receptor for geosmin, which has a musty odor, and DmOr82a, which is a Drosophila receptor, which is a receptor for geranyl acetate, which has an aromatic or fruity odor. , DmOr49b, which is a Drosophila receptor for 2-methylphenol (o-cresol), which has the odor of human sweat, and 1-octene-3-, which is a Drosophila receptor and has a musty odor. DmOr13a, which is a receptor for 1-octen-3-ol, BmOR1, which is a receptor for Bombykol, a sex pheromone of silkworm moths, and reception of Bombykal, a subcomponent of silkworm sex pheromone. Examples include, but are not limited to, BmOR3, which is a common odor receptor of Drosophila melanogaster, and PxOR1, which is a sex pheromone receptor of diamondback moth. Note that "Geosmin" is also called "Geosmin".

When expressing olfactory receptors in odor detection cells by genetic engineering, for example, as shown in Figure 1, a gene encoding an olfactory receptor may be inserted into a vector, and the constructed vector may be transfected into host cells. good. Genes encoding olfactory receptors can be isolated by, for example, extracting mRNA from the olfactory organs of insects and synthesizing cDNA. From the isolated cDNA, it is possible to amplify a part of the gene encoding the olfactory receptor by PCR using PCR primers.

A part of the gene encoding the olfactory receptor can also be obtained by inserting a synthesized double-stranded cDNA into an appropriate vector and transforming Escherichia coli etc. with the vector to create a cDNA library. can. cDNA can be integrated into a vector by a conventional method using restriction enzymes and ligase, for example, by cutting the obtained cDNA with a restriction enzyme, inserting it into the restriction enzyme site of the vector DNA, and ligating it to the vector.

An odorant corresponding to an olfactory receptor is an odorant that specifically reacts with the olfactory receptor. The odorant corresponding to DmOr56a is geosmin. The odorant corresponding to DmOr82a is geranyl acetate. The odorant corresponding to DmOr49b is 2-methylphenol (o-cresol). The odorant corresponding to DmOr13a is 1-octen-3-ol. The odorant corresponding to BmOR1 is Bombykol. The odorant corresponding to BmOR3 is Bombykal. The odorant corresponding to PxOR1 is the sex pheromone of the diamondback moth.

A fluorescent protein whose fluorescence intensity changes depending on the ion may be expressed within the odor detection cell. As described above, when an odor molecule binds to an ion channel type olfactory receptor in an odor detection cell, ions flow into the odor detection cell. Therefore, by introducing a gene that expresses a fluorescent protein whose fluorescence intensity changes depending on the ion into odor detection cells, it is possible to determine whether the odor detection cells are detecting odor molecules based on the change in fluorescence intensity or the rate of change in fluorescence intensity. It is possible to check whether this is the case. Examples of fluorescent proteins include GCaMP3, GCaMP6s, and aequorin, which emit fluorescence upon reaction with calcium ions.

When olfactory receptors and odor molecules react, the concentration of ions flowing into odor detection cells increases, causing many fluorescent proteins to emit fluorescence. Therefore, the intensity of fluorescence emitted by odor detection cells increases. When the reaction between olfactory receptors and odor molecules is inhibited by an inhibitory substance, the concentration of ions flowing into odor detection cells becomes low or zero, and fewer fluorescent proteins emit fluorescence or no fluorescence is emitted. . Therefore, the fluorescence intensity emitted by odor detection cells becomes weaker or disappears. Therefore, it is possible to evaluate whether the reaction between olfactory receptors and odorant molecules is inhibited by an inhibitory substance, based on the fluorescence intensity corresponding to the influx of ions into odor detection cells.

The influx of ions into the odor detection cell may be measured electrically. For example, a transistor including a source electrode, a drain electrode, and a gate electrode is placed near the odor detection cell. When olfactory receptors and odor molecules react and ions flow into odor detection cells, the gate potential of the gate electrode of the transistor shifts, causing modulation in the drain current flowing between the source and drain electrodes. When the reaction between olfactory receptors and odor molecules is inhibited by an inhibitor, the concentration of ions flowing into the odor-detecting cell becomes low or zero, and the modulation of the drain current becomes low or absent. Therefore, by detecting the modulation of the drain current of the transistor corresponding to the influx of ions into the odor detection cell, it is possible to evaluate whether the reaction between the olfactory receptor and the odor molecule is inhibited by an inhibitory substance. It is.

Odor detection cells are obtained by (a) selecting some cells from a group of cells that have olfactory receptors, (b) proliferating the selected cells, and (c) confirming the responsiveness of the proliferated cells to odorants. Steps (a) to (c) may be performed a plurality of times, and cells may be selected from cells that have proliferated and have a responsiveness to odorants equal to or higher than a reference value. The cell selected in step (a) may be a single cell.

Alternatively, the odor detection cells (a) select some cells from a group of cells having olfactory receptors, (b) proliferate the selected cells, and (c) determine the responsiveness of the proliferated cells to odorants. Steps (a) to (c) of confirming the odorant may be performed multiple times, and the proliferated cells with the highest responsiveness to the odorant may be selected. The cell selected in step (a) may be a single cell.

Specifically, as shown in Figure 2, single cells or a small number of cells are selected by repeatedly diluting a group of cell lines that have olfactory receptors and express fluorescent proteins. Cell lines may be established by culturing and propagating. By performing the steps multiple times, multiple cell lines are established. Among the plurality of established cell lines, a cell line whose responsiveness to an odorant is equal to or higher than a predetermined reference value may be used as an odor detection cell. Alternatively, among the plurality of established cell lines, the cell line with the highest responsiveness to an odorant may be used as the odor detection cell.

As shown in FIGS. 3 and 4, cells expressing established olfactory receptors may be further made to express another olfactory receptor. That is, cells expressing the first olfactory receptor established by the above method or the like may be further made to express the second olfactory receptor. For example, by incorporating a gene encoding the second olfactory receptor into a vector and transfecting the constructed vector into cells expressing the first olfactory receptor, It is possible to establish cells expressing olfactory receptors. The cells expressing the first olfactory receptor may further express a plurality of different olfactory receptors. Figure 3 shows the introduction of a vector containing the gene for Or-X, the second olfactory receptor, and a vector containing the antibiotic resistance gene into cells expressing Or56a as the first olfactory receptor. An example is shown. Figure 4 shows an example in which a vector containing both the gene for Or-X, a second olfactory receptor, and an antibiotic resistance gene is introduced into cells expressing Or56a as a first olfactory receptor. There is.

When the odor detection structure is an artificial structure, the artificial structure may be patterned with cells. The artificial structure comprises, for example, a vesicle with a membrane and olfactory receptors arranged in the membrane. The artificial structure may include a fluorescent protein within the membrane that emits fluorescence depending on the concentration of ions. The artificial structure can be manufactured with reference to, for example, Non-Patent Document 1.

The odorant and each of the plurality of inhibitor candidate substances may be applied to the odor detection structure simultaneously. Alternatively, after applying the odorant to the odor detection structure, each of the plurality of inhibitor candidate substances may be applied to the odor detection structure. Alternatively, the odorant may be applied to the odor detection structure after each of the plurality of inhibitor candidate substances is applied to the odor detection structure. Candidate inhibitor substances that inhibit the influx of ions into the odor detection structure that can be caused by odorants are selected as inhibitors of insect odor responses.

Multiple inhibitor candidate substances screened using the odor detection structure may be further screened. For example, the method of screening for an inhibitor of the odor response of an insect according to an embodiment includes preparing an insect or a part of the insect, an irritant substance that stimulates the insect or the part of the insect, and a substance selected by the above screening. The method further includes providing an inhibitor candidate substance to the insect or part of the insect and detecting a response of the insect or part of the insect.

Examples of insects include moths, cockroaches, mosquitoes, flies, bees, and lice. Examples of moths include silkworm moth, diamondback moth, fall armyworm, and armyworm moth. Examples of cockroaches include the European cockroach, the German cockroach, the American cockroach, the little cockroach, the brown cockroach, the black cockroach, the Japanese cockroach, the Japanese cockroach, and the Japanese cockroach. Examples of insect parts include antennae, proboscis, legs, and wings.

Examples of irritants to insects or parts of insects include bombycol, peripranone A, peripranone B, and 1-octen-3-ol.

The stimulant and each of the plurality of inhibitor candidate substances may be provided simultaneously to the insect or part of the insect. Alternatively, each of the plurality of inhibitor candidate substances may be provided to the insect or part of the insect after the stimulating substance is provided to the insect or part of the insect. Alternatively, the stimulating substance may be provided to the insect or part of the insect after each of the plurality of inhibitor candidate substances is provided to the insect or part of the insect.

In detecting the insect's response, the behavior of the insect may be observed. Candidate inhibitor substances that inhibit insect behavior that can be caused by stimulants are selected as inhibitors of insect odor responses. Examples of insect behaviors include flapping behavior, exploratory behavior, and mating behavior. Furthermore, in detecting the response of the antennae of an insect, the electric potential of the antennae may be detected. Candidate inhibitor substances that inhibit changes in antennal potential caused by stimulants are selected as inhibitors of insect odor responses.

Inhibitors of insect odor responses according to embodiments may reduce the influx of ions into the odor detection structure by less than 37%, less than 35%, less than 30%, less than 25%, less than 20%, or less than 20%. For example, when evaluating the influx of ions into an odor detection structure by observing the fluorescence intensity within the odor detection structure, an inhibitor of an insect odor response according to an embodiment may be used to detect odor molecules in the absence of an inhibitor. The fluorescence intensity can be less than 37%, 35% or less, 30% or less, 25% or less, 20% or less, or less than 20% with respect to the fluorescence intensity generated by. When evaluating the influx of ions into the odor detection structure by observing the drain current of a transistor in the vicinity of the odor detection structure, the inhibitor of the insect odor response according to the embodiment may be used to detect the odor in the absence of the inhibitor. For the modulation of drain current caused by molecules, the modulation of drain current can be less than 37%, less than 35%, less than 30%, less than 25%, less than 20%, or less than 20%. Note that the rate of decrease in ion influx or drain current may be a value related to the rate of decrease in fluorescence intensity according to the embodiment.

Inhibitors of insect odor responses are, for example, methylphenol derivatives. The inhibitor of insect odor response may include 4-isopropyl-3-methylphenol shown in Formula 1 below. The inhibitor of insect odor response may include 4-(tert-butyl)-2-methylphenol shown in Formula 2 below. The inhibitor of insect odor response may include 2-isopropyl-6-methylphenol shown in Formula 3 below. The inhibitor of insect odor response may include 2,4-diisopropylphenol shown in Formula 4 below. The inhibitor of insect odor response may include 2-tert-butyl-4-methylphenol shown in Formula 5 below.

Inhibitors of insect odor responses may include cinnamic acid derivatives. Examples of cinnamic acid derivatives include methyl transcinnamate and tertbutyl transcinnamate.

Examples of solvents for inhibitors of insect odor responses include ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water. Inhibitors of insect odor responses according to embodiments can be used, for example, as inhibitors of moth odor responses. The moth may be a silk moth.

As shown in FIG. 5, the insect inhibition system according to the embodiment includes a spraying unit 20 that sprays an inhibitor against the odor response of insects. The inhibitor of insect odor response may be an inhibitor of insect odor response as described above. The dispersion unit 20 includes, for example, a storage unit 21 that stores a solution containing an inhibitor to the insect odor response, an atomization unit 22 that atomizes the solution containing the inhibitor to the insect odor response, and a micronization unit 22 that atomizes the solution containing the inhibitor to the insect odor response. A blowout section 23 is provided that blows out a solution containing an inhibitor against odor response. The solvent of the solution may be the solvent described above.

The storage section 21 is, for example, a tank, and includes a main body 21a and a lid 21b. The atomization unit 22 includes, for example, a two-fluid nozzle 25 disposed within the storage unit 21 that mixes two fluids, a liquid and a gas, and atomizes the mixture. The blowing part 23 may be provided on the lid 21b.

The two-fluid nozzle 25 is provided with a gas inlet 25a through which gas flows, and a solution inlet 25b through which a solution containing an inhibitor to the odor response of insects in the reservoir 21 flows. The gas is, for example, high pressure compressed air. Further, the two-fluid nozzle 25 is provided with a spray port 26 . A liquid film-like solution containing an inhibitor to the odor response of insects is formed at the spray nozzle 26 and is atomized by the shearing force of the airflow, and is ejected from the spray nozzle 26 . The solution containing the atomized insect odor response inhibitor ejected from the spray nozzle 26 is discharged from the ejection section 23 to the outside of the storage section 21 .

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 connecting portion 27 such as a pipe joint. The control unit 30 includes, for example, a pump 32 for supplying air, and a gas supply pipe 34 that connects the pump 32 and the connection unit 27. The gas supply pipe 34 is provided with a valve 33 such as a solenoid valve. Pump 32 and valve 33 are electrically connected to controller 31. The controller 31 controls the pump 32 and the valve 33 to control the flow rate, pressure, etc. of the gas supplied to the two-fluid nozzle 25.

Alternatively, the insect inhibition system according to the embodiment may have the configuration shown in FIG. 6 . The dispersion unit 120 of the insect inhibition system shown in FIG. 6 includes, for example, a storage unit 41 that stores a solution containing an inhibitor to insect odor responses, and an atomization unit 42 that atomizes the solution containing an inhibitor to insect odor responses. , and a blowing section 43 for blowing out a solution containing an atomized insect odor response inhibitor.

The storage section 21 is, for example, a tank, and includes a main body 21a and a lid 21b. The storage section 21 and the atomization section 42 are connected by piping for delivering a solution containing an inhibitor to the odor response of insects. The atomization unit 42 includes, for example, a nozzle head 47 that accommodates a solution containing an inhibitor to the odor response of insects, and a piezoelectric element 45 such as a piezo element disposed in the nozzle head 47. The blowing section 43 is provided in the nozzle head 47.

When a pulse voltage is applied to the piezoelectric element 45, the piezoelectric element 45 repeats deformation and restoration. Therefore, the volume of the nozzle head 47 repeatedly contracts and restores. As a result, the solution containing the inhibitor against the odor response of insects in the nozzle head 47 is intermittently extruded from the blowing portion 43 and becomes fine particles.

A control section 130 is connected to the piezoelectric element 45 via a 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 and the like.

Alternatively, the insect inhibition system according to the embodiment may have the configuration shown in FIG. 7 . The dispersion unit 60 of the insect inhibition system shown in FIG. 7 includes, for example, a storage unit 61 that stores a solution containing an inhibitor to insect odor responses, and an atomization unit 62 that atomizes the solution containing an inhibitor to insect odor responses. , and a blowing section 63 for blowing out a solution containing an atomized insect odor response inhibitor.

The storage section 61 is, for example, a flexible bag-shaped container. At least a portion of the reservoir 61 is an ultrasound-transmissive membrane 65. The blowout section 43 is provided in the storage section 61 . The storage portion 61 is held in a vibration generating container 66 filled with working water 67 . The atomization unit 62 includes an ultrasonic vibrator.

The ultrasonic vibrator vibrates ultrasonically when a high-frequency alternating current voltage is applied to it. The vibrational energy generated by the ultrasonic vibration reaches the solution containing the inhibitor against the odor response of insects in the reservoir 61 via the working water 67 and the ultrasonic permeable membrane 65 . As a result, the solution containing the inhibitor against the odor response of insects in the storage part 61 is vibrated, the surface of the solution is atomized, and the finely divided solution containing the inhibitor against the odor response of insects is blown out from the blowing part 63. be done.

A control unit 230 is connected to the ultrasonic transducer of the atomization 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.

Alternatively, the insect inhibition system according to the embodiment may have the configuration shown in FIG. 8 . The spraying unit 80 of the insect inhibition system shown in FIG. 8 includes, for example, a storage unit 81 that stores a solution containing an inhibitor to the odor response of insects, and an electrostatic spray type that atomizes the solution containing an inhibitor to the odor response of insects. and a blowing part 83 for blowing out a solution containing an atomized insect odor response inhibitor.

The storage section 81 is, for example, a tank, and includes a main body 81a and a lid 81b. The blowing section 83 is provided in the storage section 81 . A micronization unit 82 is arranged within the storage unit 81 . The atomization unit 82 includes an electrostatic spray nozzle 84 and a transport unit 85 such as a pump. The conveying unit 85 conveys a solution containing an inhibitor to 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 applying section 87 such as a counter electrode is arranged on the upper end surface of the cylindrical member 86 .

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, fine particles are generated at the gas-liquid interface due to the balance between the surface tension of the solution and the electrostatic force acting on the solution. The liquid thread is pulled out, and its tip is split into fine particles and sprayed from the electrostatic spray nozzle 84.

A high voltage control section 330 is connected to the voltage application section 87 via a wiring 71. The high voltage control section 330 applies a high voltage to the voltage application section 87 to control electrostatic force and the like acting on the solution.

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

Grace's Insect Medium, Supplemented (11605-094, Gibco) was supplemented with US Insect Cell Screened FBS (SH30070.03, GE Healthcare) at a final concentration of 10% and three types of antibiotics (final concentration of 10 μg/mL). Gentamicin Reagent Solution (15710-064, Gibco), Blasticidin S HCl (A11139-03, Gibco) with a final concentration of 10 μg/mL, and Zeocin (R25001, Invitrogen) with a final concentration of 100 μg/mL were added for passage. Prepared medium Using the passage medium, Sf21 cells expressing the DmOr13a receptor, co-receptor DmOrco, and GCaMP6s were subcultured in a flask (353082, FALCON). The liquid volume was 6 mL. When the cells became confluent, 6 mL of supernatant was collected from the flask and placed in a 15 mL tube (91015, TPP). Using a high-speed microcentrifuge, the 15 mL tube was centrifuged at 400 x g. Centrifugation was performed at 4°C for 3 minutes.

After centrifugation, the supernatant was sterilized using a 10 mL syringe (01007, TOP) and a 0.45 μm filter (431220, CORNING). 10 mL of conditioned 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, Zeocin at a final concentration of 100 μg/mL). .

Cells adhering to the bottom of the flask from which the supernatant was collected were peeled off and suspended in 1 mL of fresh medium, and the cell suspension was collected into a 1.5 mL tube (MCT-150-C, AXYGEN). A cell suspension containing 40 cells was extracted, added to the above conditioned medium, and pipetted well. The entire volume of conditioned medium with added cells was transferred to a reservoir (BM-0850-1, BMBio). Furthermore, using an 8 multichannel pipette (HT5123, HTL), 100 μL of the conditioned medium containing the cells was added dropwise to a 96-well plate (3860-096, IWAKI), and then the cells were cultured at 27°C. After the cells adhered to the wells, the wells were observed under an inverted microscope to identify wells in which only a single cell was present in the conditioned medium.

Cells in wells in which single cells were confirmed at the time of seeding were continued to be cultured until they reached about 80% to about 90% confluence. Thereafter, cells were scaled up in the following order: 24-well plate (3820-024, IWAKI), 35 mm dish (353801, CORNING), and T-25 flask. For culturing in a 24-well plate, 35 mm dish, and T-25 flask, the volume of the medium was adjusted to 500 μL, 2.5 mL, and 5 mL, respectively, and the culture was performed at 27°C. The responsiveness to odorants of the cells scaled up to T-25 flasks was investigated by calcium imaging, and cell lines that showed good responsiveness were obtained as homogeneous odor detection cell lines.

(Example 2: Response of odor detection cells to inhibitors)
After seeding the odor detection cells obtained in Example 1 on a 12 mm diameter cover glass (CS-12R: Warner Instruments, LLC, Hamden, CT, USA), the cover glass was placed in an open bath chamber for circular cover glasses (RC). -48LP: Warner Instruments, LLC, Hamden, CT, USA).

In order to perfuse the odor detection cells with the solution, two silicone tubes 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), respectively. It was connected to the inlet and outlet of the open bath chamber by clamps (CAT-1: NARISHIGE Co. Ltd., Tokyo, Japan), respectively.

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

An EM-CCD camera (DU-897E: Andor Technology PLC, Belfast, UK) was prepared to measure changes in fluorescence intensity of cells. The EM-CCD camera was operated with an AndoriQ (Andor Technology PLC, Belfast, UK). The EM-CCD camera was set to acquire images at 512 x 512 pixels per second.

Perfusion with assay buffer was started. The flow rate was set to about 1.4 mL/min, and the internal liquid volume of the chamber was set to about 230 μL. As shown in FIG. 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 the odor detection cells.

300 μmol/L geraniol, an inhibitor candidate substance, was flowed for 60 seconds, then 10 μmol/L 1-octen-3-ol, an odorant, was flowed together with 300 μmol/L geraniol for 15 seconds, and 300 μmol/L was flowed for another 60 seconds. When geraniol was applied, the increase in fluorescence intensity in odor-detecting cells was suppressed compared to when no odorant inhibitor was present.

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 60 μmol/L l-menthol was flowed for 60 seconds. When l-menthol was flowed at 300 μmol/L per second, the increase in fluorescence intensity in the odor detection cells was suppressed compared to the case without the odorant inhibitor.

Thymol at 300 μmol/L, an inhibitor candidate substance, 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 300 μmol/L was flowed for another 60 seconds. When thymol was applied, the increase in fluorescence intensity in odor-detecting cells was suppressed compared to when no odorant inhibitor was present.

Linalyl formate (LF) at 300 μmol/L, a known odorant inhibitor, was flowed for 60 seconds, then 10 μmol/L 1-octen-3-ol, an odorant, was injected with 300 μmol/L LF for 15 seconds. When 300 μmol/L LF was further flowed for 60 seconds, the increase in fluorescence intensity in the odor detection cells was suppressed compared to the case without the odorant inhibitor.

300 μmol/L of 2-tert-butyl-6-methylphenol (BMP), a known odorant inhibitor, was flowed for 60 seconds, and then 300 μmol of 10 μmol/L of 1-octen-3-ol, an odorant. When the cells were flown together with BMP/L for 15 seconds and then 300 μmol/L BMP was flowed for an additional 60 seconds, the increase in fluorescence intensity in the odor detection cells was suppressed compared to the case without the odorant inhibitor.

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

The results of Example 2 demonstrate that odor detection cells are useful for screening inhibitors of odorants.

(Example 3: Response of odor detection cells according to the concentration of fragrance components)
Inhibitor candidate substances such as thymol, citral, 1-nonanol, eugenol acetate, d-limonene, eugenol, geraniol, and geranyl acetate, and known odorant inhibitors BMP and LF were prepared.

Similar to the measurement method in FIG. 9, 10 μmol/L of 1-octen-3-ol, an odorant, was given to the odor detection cells obtained in Example 1 without containing an inhibitor, and the fluorescence in the odor detection cells was The strength was measured. Next, 10 μmol/L inhibitor candidate substance or inhibitor, 30 μmol/L inhibitor candidate substance or inhibitor, 100 μmol/L inhibitor candidate substance or inhibitor, 170 μmol/L inhibitor candidate substance or inhibitor , 300 μmol/L inhibitor candidate substance or inhibitor, 560 μmol/L inhibitor candidate substance or inhibitor, 1 mmol/L inhibitor candidate substance or inhibitor, 3 mmol/L inhibitor candidate substance or inhibitor in sequence. It was given to odor detection cells together with 10 μmol/L of 1-octen-3-ol, and the intensity of fluorescence in the odor detection cells was measured. Finally, 10 μmol/L of 1-octen-3-ol without an inhibitor was given to the odor detection cells, and the intensity of fluorescence in the odor detection cells was measured.

For each inhibitor, the average value of the fluorescence intensity change rate in the odor detection cells when 10 μmol/L of 1-octen-3-ol is given to the odor detection cells without containing the inhibitor is 100%. The obtained relationship between the concentration of 0 and the normalized intensity of fluorescence in odor detection cells is shown in the graph of FIG. As shown in FIG. 10, the response to odorants in odor detection cells was suppressed depending on the concentration of the inhibitor.

The results of Example 3 demonstrate that odor detection cells are useful for screening for inhibitors of odorants.

(Example 4: Response of odor detection cells according to concentration of inhibitor candidate substance)
Methyl transcinnamate and tert-butyl transcinnamate, which are candidate inhibitor substances, were prepared.

10 μmol/L of 1-octen-3-ol, which is an odorant, was given to the odor detection cells obtained in Example 1 without containing an inhibitor candidate substance, and the intensity of fluorescence in the odor detection cells was measured. Next, 10 μmol/L inhibitor candidate substance, 30 μmol/L inhibitor candidate substance, 100 μmol/L inhibitor candidate substance, 170 μmol/L inhibitor candidate substance, 300 μmol/L inhibitor candidate substance, 560 μmol/L L of the inhibitor candidate substance and 1 mmol/L of the inhibitor candidate substance were sequentially given to the odor detection cells together with 10 μmol/L of 1-octen-3-ol, and the intensity of fluorescence in the odor detection cells was measured. Finally, 10 μmol/L of 1-octen-3-ol without an inhibitor was given to the odor detection cells, and the intensity of fluorescence in the odor detection cells was measured.

For each inhibitor candidate substance, the average value of the fluorescence intensity change rate in the odor detection cells when 10 μmol/L of 1-octen-3-ol is given to the odor detection cells without containing the inhibitor candidate substance is 100%. The obtained relationship between the concentration of the inhibitor candidate substance and the normalized intensity of fluorescence in the odor detection cells is shown in the graph of FIG. As shown in FIG. 11, the response to the odorant in the odor detection cells was suppressed depending on the concentration of the inhibitor candidate substance.

(Example 5: Response of odor detection cells according to the concentration of inhibitor and inhibitor candidate substance)
Inhibitor candidate substances 2,4-diisopropylphenol, 4-isopropyl-3-methylphenol, 2-tert-butyl-4-methylphenol, 4-tert-butyl-2-methylphenol, and 2-isopropylphenol 6-Methylphenol was prepared.

10 μmol/L of 1-octen-3-ol, which is an odorant, was given to the odor detection cells obtained in Example 1 without containing an inhibitor candidate substance, and the intensity of fluorescence in the odor detection cells was measured. Next, 10 μmol/L inhibitor candidate substance, 30 μmol/L inhibitor candidate substance, 100 μmol/L inhibitor candidate substance, 170 μmol/L inhibitor candidate substance, and 300 μmol/L inhibitor candidate substance were sequentially added. It was given to odor detection cells together with 10 μmol/L of 1-octen-3-ol, and the intensity of fluorescence in the odor detection cells was measured. Finally, 10 μmol/L of 1-octen-3-ol without an inhibitor was given to the odor detection cells, and the intensity of fluorescence in the odor detection cells was measured.

For each inhibitor candidate substance, the average value of the fluorescence intensity change rate in the odor detection cells when 10 μmol/L of 1-octen-3-ol is given to the odor detection cells without containing the inhibitor candidate substance is 100%. The obtained relationship between the concentration of the inhibitor candidate substance and the normalized intensity of fluorescence in the odor detection cells is shown in the graph of FIG. As shown in FIG. 12, the response to the odorant in the odor detection cells was suppressed depending on the concentration of the inhibitor candidate substance.

(Example 6: Screening of inhibitors and inhibitor candidate substances by inhibitory effect on odor detection cells depending on concentration)
Based on the results of Examples 3 to 5, 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 concentrations. FIG. 13 shows a graph of the fluorescence intensity change rate when is 300 μM.

Trans-methyl 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 caused the fluorescence intensity change rate in the odor detection cells to be less than 37% when 10 μmol/L of 1-octen-3-ol was given to the odor detection cells. As shown in Example 7 below, BMP, which is an inhibitor, and 4-isopropyl-3-methylphenol and 4-tert-butyl-2-methylphenol, which are inhibitor candidate substances, have the following electroanthenograms: An inhibitory effect was observed. In the case of Example 6, the fluorescence intensity change rate of those substances was less than 25% (24.7%). On the other hand, as shown in Example 7 below, eugenol acetate, 1-nonanol, and thymol, which had a fluorescence intensity change rate of 37% or more, had no inhibitory effect in the electroantenogram. In the case of Example 6, the rate of change in fluorescence intensity of these substances was 37% or more. Therefore, it was shown that a substance with a fluorescence intensity change rate of less than 25% has an inhibitory effect, and a fluorescence intensity change rate of less than 37% shows an inhibitory effect. In addition, 2-tert-butyl-4-methylphenol and 4-tert-butyl-2-methylphenol were found in odor detection cells when 10 μmol/L of 1-octen-3-ol was given to them. The rate of change in fluorescence intensity was set to 20% or less.

Example 7: Antenna response to inhibitors
As shown in Figure 14, a gel droplet (Spectra 360 Electrode Gel, Parker Laboratories) was placed on each of the surfaces of two metal electrodes, and a silk moth antennae with its tip and base cut off was brought into contact with the gel droplet. The antennae were placed in an arch shape between the metal electrodes. It is known that the electric potential between the tip and base of insect antennae changes in response to odorants, and recording this electric potential is called an electroantenogram (EAG).

Bombykol (BOL), a silk moth sex pheromone, BMP, a known odorant inhibitor, 4-isopropyl-3-methylphenol, a candidate inhibitor, and 4-tert-butyl, a candidate inhibitor. -2-Methylphenol was prepared. Filter paper with 1000 ng of BOL dropped, filter paper with 1000 ng of BOL and 1000 μg of BMP dropped, filter paper with 1000 ng of BOL and 1000 ng of 4-isopropyl-3-methylphenol dropped, and 1000 ng of BOL and 1000 ng of 4-tert- A filter paper onto which butyl-2-methylphenol was dripped was prepared. Put the filter paper with BOL dripped into the first glass tube cartridge, put the filter paper with BOL and BMP dripped into the second glass tube cartridge, and put BOL and 4-isopropyl-3-methylphenol into the third glass tube cartridge. The filter paper on which BOL and 4-tert-butyl-2-methylphenol had been dropped was placed in the fourth glass tube cartridge.

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

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

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

Bombykol (BOL), a silk moth sex pheromone, eugenol acetate, a candidate inhibitor, 1-nonanol, a candidate inhibitor, and thymol, a candidate inhibitor were prepared. Filter paper on which 1000 ng of BOL was dropped, filter paper on which 1000 ng of BOL and 1000 μg of eugenol acetate were dropped, filter paper on which 1000 ng of BOL and 1000 μg of 1-nonanol were dropped, and filter paper on which 1000 ng of BOL and 1000 ng of thymol were dropped were prepared. . A filter paper on which BOL has been dropped is placed in the fifth glass tube cartridge, a filter paper on which BOL and eugenol acetate have been dropped is placed in the sixth glass tube cartridge, and a filter paper on which BOL and 1-nonanol have been dropped is placed in the seventh glass tube cartridge. Then, the filter paper on which BOL and thymol had been dropped was placed in the eighth glass tube cartridge.

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

When a gas containing BOL was injected into 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 row of FIG. 16(a). Next, when a gas containing BOL and 1-nonanol was injected from the seventh glass tube cartridge into the antennae at 1 L/min, the decrease in potential between the tip and base of the antennae was shown in the middle row of Figure 16(b). This was enhanced compared to the case without 1-nonanol. When the gas containing BOL was again injected into 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 row of FIG. 16(c).

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

The results of Example 7 demonstrate that EAG is useful for screening odorant inhibitors.

Example 8: Insect response to inhibitors
A plurality of transparent plastic containers with lids (Maru Cup 200MB, Mineron Chemical Industries, Ltd.) were prepared. A small hole was made in the lid of the plastic container into which the tip of a Pasteur pipette could be inserted. A male silkworm moth was placed in each of several plastic containers and the lids were closed.

Bombykol (BOL), a silk moth sex pheromone, and BMP, a known odorant inhibitor, were prepared. Filter paper on which 0.01 ng of BOL was dropped, filter paper on which 0.1 ng of BOL was dropped, filter paper on which 1 ng of BOL was dropped, filter paper on which 10 ng of BOL was dropped, filter paper on which 100 ng of BOL was dropped, filter paper on which 1000 ng of BOL was dropped. A filter paper and a filter paper onto which 1000 μg of BMP diluted with hexane were dropped were prepared.

Put a filter paper with 0.01 ng of BOL dropped into the first Pasteur pipette, put a filter paper with 0.1 ng of BOL dropped into the second Pasteur pipette, and drop 1 ng of BOL into the third Pasteur pipette. Place the filter paper with 10 ng of BOL dropped into the fourth Pasteur pipette, put the filter paper with 100 ng of BOL dropped into the fifth Pasteur pipette, and add 1000 ng of BOL into the sixth Pasteur pipette. The filter paper on which BMP had been dropped was placed in the seventh Pasteur pipette. Each Pasteur pipette was inserted into a plastic container and the silk moths were stimulated with air-puff.

As shown in FIG. 17, when silkworm moths were puff-stimulated three times with each concentration of BOL, three out of five silkworm moths showed flapping behavior and two silkworm moths showed exploratory behavior at 1 ng of BOL. At 10 ng BOL, all silk moths showed exploratory behavior.

As shown in Figure 18, after puff stimulation with BMP three times, silkworm moths were puff stimulated three times with each concentration of BOL in sequence; three out of five silkworm moths showed no response to 1 ng of BOL; Two silk moths showed flapping behavior. At 10 ng of BOL, 1 out of 5 silkworms showed no response, 2 out of 5 silkworms showed flapping behavior, and 2 out of 5 silkworms showed exploratory behavior. At 100 ng of BOL, one out of three silkworm moths showed flapping behavior and two silkmoths showed exploratory behavior. At 1000 ng BOL, one remaining silk moth showed exploratory behavior. Comparison of FIGS. 17 and 18 shows that BMP inhibits the response of insects to odorants and increases the threshold concentration of BOL that causes flapping and exploratory behaviors.

The results of Example 8 demonstrate that observing insect responses is useful for screening odorant inhibitors.

(Example 9: Screening for inhibitors using insects)
A plurality of plastic containers similar to those in Example 8 were prepared, one male silkworm moth was placed in each of the plurality of plastic containers, and the lids were closed.

Bombykol (BOL) diluted with hexane was prepared. As inhibitor candidate substances, 4-tert-butyl-2-methylphenol diluted with hexane and 4-isopropyl-3-methylphenol diluted with ethanol were prepared.

Put a filter paper onto which 0.01 ng of BOL diluted with hexane has been dropped into the first Pasteur pipette, put a filter paper onto which 0.1 ng of BOL diluted with hexane has been dropped into the second Pasteur pipette, and perform the third pass. Put a filter paper with 1 ng of BOL diluted with hexane dropped into the tool pipette, put a filter paper with 10 ng of BOL diluted with hexane dropped into the fourth Pasteur pipette, and put 100 ng of BOL diluted with hexane into the fifth Pasteur pipette. 1000 ng of BOL diluted with hexane was added to the sixth Pasteur pipette.

As shown in Figure 19, when silkworm moths were puff stimulated three times with BOL of each concentration sequentially diluted with hexane, 2 out of 3 silkworms showed no response to BOL diluted with 0.01 ng of hexane. One silk moth showed flapping behavior. All silkmoths showed exploratory behavior in BOL diluted with 0.1 ng hexane.

A filter paper onto which 1000 ng of 4-tert-butyl-2-methylphenol diluted with hexane was dropped was placed in a plastic container with silkworm moths for 10 minutes. Thereafter, as shown in FIG. 20, silkworm moths were puff-stimulated three times with BOL of each concentration sequentially diluted with hexane, and none of the silkmoths showed a reaction to 0.01 ng of BOL diluted with hexane. Two out of three silkworm moths showed no response to 0.1 ng of BOL diluted with hexane, and one silkworm moth showed flapping behavior. Two out of three silkworm moths showed no response to 1 ng of BOL diluted in hexane, and one silkworm moth showed exploratory behavior. At 10 ng of BOL diluted with hexane, 1 out of 2 silkworms showed flapping behavior and 1 silkworm showed exploratory behavior. One silkworm remaining with 100 ng of BOL diluted in hexane showed exploratory behavior. Comparison of Figures 19 and 20 shows that 4-tert-butyl-2-methylphenol inhibits insect responses to odorants and increases the threshold concentration of BOL that produces flapping and exploratory behaviors. It was done.

The filter paper dripped with ethanol was placed in a plastic container with the silkworm moth for 10 minutes. Thereafter, as shown in FIG. 21, silkworm moths were puff-stimulated three times with BOL of each concentration sequentially diluted with hexane, and none of the silkmoths showed a reaction with BOL diluted with 0.01 ng of hexane. Two out of three silkworms showed no response to BOL diluted with 0.1 ng of hexane, and one silkworm showed flapping behavior. All silk moths showed exploratory behavior in BOL diluted with 1 ng of hexane.

A filter paper onto which 1000 ng of 4-isopropyl-3-methylphenol diluted with ethanol was dropped was placed in a plastic container with silkworm moths for 10 minutes. Thereafter, as shown in Figure 22, when the silkworms were puff-stimulated three times with BOL of each concentration diluted with hexane, all of the silkmoths showed a response to 0.01 ng and 0.1 ng of BOL diluted with hexane. There wasn't. Two out of three silkworm moths showed no response to 1 ng of BOL diluted in hexane, and one silkworm moth showed flapping behavior. At 10 ng of BOL diluted in hexane, 2 out of 3 silk moths showed flapping behavior and 1 silk moth showed exploratory behavior. All silkmoths showed exploratory behavior at 100 ng BOL diluted in hexane. Comparison of Figures 21 and 22 shows that 4-isopropyl-3-methylphenol inhibits the response of insects to odorants and increases the threshold concentration of BOL that causes flapping and exploratory behaviors. .

(Example 10: Production of odor detection cells expressing Or56)
The region from the start codon to the stop codon of the gene for DmOrco, a co-receptor, derived from Drosophila melanogaster tactile cDNA was amplified by PCR using primers containing the gene-specific sequence shown below to obtain the DmOrco gene. The obtained DmOrco gene was inserted into the multi-cloning site of the pIZ vector (manufactured by Invitrogen) using NEBuilder HiFi DNA Assembly MasterMix (New England Biolabs Japan), and the pIZ-DmOrco vector was Built.

DmOrco:
Forward: 5'-TTCGAATTTAAAGCTGCCGCCATGATGACAACCTCGATGCAGCC-3'
Reverse: 5'-TTACCTTCGAACCGCTTACTTGAGCTGCACCAGCAC-3'

In addition, the constructed pIZ-DmOrco vector was amplified by PCR using the following primers, and inserted into the Pci1 site of the pIB vector (manufactured by Invitrogen) using NEBuilder HiFi DNA Assembly MasterMix (New England Biolabs Japan). and pIB- A DmOrco vector was constructed.

pIB-Pci1:
Forward: 5'-GCAGGAAAGAACATGCATGATGATAAACAATGTATGGTGCTAATG-3'
Reverse: 5'-CCTTTTGCTCACATGGTTATCCCTGATTCTGTGG-3

Gene amplification by PCR uses forward and reverse primers each at a concentration of 100 pmol/μl, PrimeSTAR HS DNA polymerase (Takara Bio, R010A), the reaction buffer attached to the polymerase, and dNTP, and according to the protocol attached to the polymerase. I followed. The temperature conditions for PCR were a step of 94°C for 1 minute, followed by 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 1.5 minutes, followed by a step of 72°C for 1.5 minutes. The step was 5 minutes.

Next, the base sequence of DmOr56a, which is an olfactory receptor, was codon-converted to insect cell Sf9, and the following sequence was added as an adapter sequence to synthesize a gene (Integrated DNA Technologies).

Adapter array:
Forward: 5'-CAGTGTGGTGGAATTGCCGCC-3'
Reverse: 5'-GCCCTCTAGACTCGATTA-3'

The region from the start codon to the stop codon of the obtained DmOr56a_Sf9 synthetic gene was amplified by PCR using the primer of the above adapter sequence 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 create pIB-DmOr56a_Sf9- A DmOrco vector was constructed.

Gene amplification by PCR uses forward and reverse primers each at a concentration of 100 pmol/μl, PrimeSTAR HS DNA polymerase (Takara Bio, R010A), the reaction buffer attached to the polymerase, and dNTP, and according to the protocol attached to the polymerase. I followed. The temperature conditions for PCR were a step of 98°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds, 55°C for 10 seconds, and 72°C for 1.5 minutes, followed by 72°C for 1.5 minutes. The step was 10 minutes.

Similarly, a calcium sensitive protein (GCaMP6s) expression vector was constructed. The GCaMP6s gene was obtained from Dr. Douglas Kim (Janelia Farm Research Campus, Howard Hughes Medical Institute) via Addgene. The region from the start codon to the stop codon of the GCaMP6s gene was amplified using primers containing the gene-specific sequence shown below to obtain the GCaMP6s gene. The obtained GCaMP6s gene was inserted into the multi-cloning site of pIZ vector (manufactured by Invitrogen) using NEBuilder HiFi DNA Assembly MasterMix (New England Biolabs Japan) to create pIZ-GCaMP6. s vector was constructed.

GCaMP6s:
Forward: 5'-TTCGAATTTAAAGCTGCCGCCATGGGTTCTCATCATCATCATC-3'
Reverse: 5'-TTACCTTCGAACCGCTCACTTCGCTGTCATCATTTGTAC-3'

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: manufactured by Mirus) according to the attached manual. Thereby, Sf21 cells co-expressing the DmOr56a_Sf9 receptor, DmOrco, and GCaMP6s (hereinafter referred to as "odor detection cells expressing Or56") were obtained.

(Example 11: Establishment of a homogeneous odor detection cell line expressing Or56)
As a preliminary preparation, one flask (FALCON) of a medium containing 6 mL of odor detection cells expressing Or56 was prepared during passage before lineage. 6 mL of culture supernatant was collected from the confluent cell flask into a 15 mL tube (TPP) and centrifuged at 400 xg 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). A conditioned medium was prepared by mixing 5 mL of sterilized medium, 5 mL of new medium, and two antibiotics (Blasticidin (Life Technologies) and Zeocin (Life Technologies)) for the new medium.

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

Cells in wells in which single cells were confirmed in a 96-well plate were continued to be cultured until they reached approximately 80% to 90% confluence. Thereafter, the scale-up was performed in the following order: 24-well plate (IWAKI), 35 mm dish (CORNING), and T25 flask. For cells that were scaled up to T25 flasks, fluorescence response was confirmed using calcium imaging, cells that did not respond were discarded, cells that responded were made into frozen stocks, and the lineage was maintained through repeated subculture. .

(Example 12: Response of odor detection cells expressing Or56 depending on the concentration of inhibitor and inhibitor candidate substance)
Inhibitor candidate substances 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 were prepared. In addition, 2-tert-butyl-6-methylphenol (BMP), a known odorant inhibitor, was prepared.

1 μmol/L of the odorant geosmin was given to the odor detection cells expressing Or56 obtained in Example 11 without containing an inhibitor candidate substance or a known inhibitor, and the intensity of fluorescence in the odor detection cells was measured. . Next, 10 μmol/L inhibitor candidate substance or known inhibitor, 30 μmol/L inhibitor candidate substance or known inhibitor, 100 μmol/L inhibitor candidate substance or known inhibitor, 170 μmol/L inhibition The drug candidate substance or known inhibitor and 300 μmol/L of the inhibitor candidate substance or known inhibitor were sequentially given to the odor detection cells together with 1 μmol/L of geosmin, and the intensity of fluorescence in the odor detection cells was measured. Finally, 1 μmol/L of geosmin was given to the odor detection cells without inhibitors or known inhibitors, and the intensity of fluorescence in the odor detection cells was measured.

The average value of the fluorescence intensity change rate in odor detection cells when 1 μmol/L of geosmin is given to odor detection cells without containing inhibitor candidate substances or known inhibitors is set as 100%, and each inhibitor candidate substance and known inhibitor The obtained relationship between the concentration of the inhibitor and the normalized intensity of fluorescence in the odor detection cells is shown in the graph of FIG. As shown in FIG. 23, the response to odorants in odor detection cells expressing Or56 was suppressed depending on the concentration of the inhibitor candidate substance and known inhibitor.

Example 12: Insect response to inhibitors
4-isopropyl-3-methylphenol (IPMP) using DMSO as a solvent was prepared as an inhibitor candidate substance, and DMSO was prepared as a control as a test drug. 1 μL of crude extracts (acetone solvent) of cockroach pheromones periplanone A and periplanone B were prepared and attached to a 15 mm square filter paper piece or aluminum piece, and the acetone solvent was sufficiently volatilized.

The lid of the container housing approximately 10 cockroaches was removed under red lighting in a dark room, and the feeding tray and water cup were collected. Two petri dishes carrying filter paper impregnated with the test agent were placed at diagonal locations among the four corners of the container. In addition, to moisturize, Kimwipe moistened with water was placed in the container and the container was covered. Thereafter, it was allowed to stand for 30 minutes.

Video recording of the container was started, and a piece of filter paper or an aluminum piece to which the cockroach pheromone crude extract was attached was placed on top of one of the petri dishes on which the filter paper impregnated with the test agent was placed. Thereafter, as shown in FIG. 24, a transparent acrylic plate was placed on the container instead of the lid to make it easier to take pictures, and a video was taken for 10 minutes.

The video footage was analyzed and the total number of times a cockroach came into contact with a piece of filter paper or aluminum coated with crude cockroach pheromone extract was measured for 5 minutes immediately after the transparent acrylic board was placed on the container. As a result, as shown in FIG. 25, 300 mmol/L of 4-isopropyl-3-methylphenol using DMSO as a solvent prevented cockroaches from approaching and coming into contact with the pheromone-impregnated filter paper piece or aluminum piece. Furthermore, as shown in FIG. 26, the higher the concentration of 4-isopropyl-3-methylphenol, the more inhibited cockroaches were from approaching the pheromone-impregnated filter paper piece or aluminum piece.

20... Spraying part, 21... Storage part, 21a... Main body, 21b... Lid, 22... Atomization part, 23... Blowout part, 25... Two-fluid nozzle, 25a ... Gas inlet, 25b... Solution inlet, 26... Spray port, 27... Connection section, 30... Control section, 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 . 66... Vibration generating container, 67... Working water, 70... Wiring, 71... Wiring, 80... Spraying part, 81... Storage part, 81a... Main body, 81b... - Lid, 82... Atomization section, 83... Blowing section, 84... Electrostatic spray nozzle, 85... Conveyance section, 86... Cylindrical member, 87... Voltage application section, 120... Spraying section, 130... Control section, 230... Control section, 330... High voltage control section

Claims (35)

providing an odor detection structure expressing at least one of an insect olfactory receptor and a co-receptor for the olfactory receptor;
providing each of an odorant corresponding to the olfactory receptor and a plurality of inhibitor candidate substances to the odor detection structure;
Screening the plurality of inhibitor candidate substances based on the influx of ions into the odor detection structure;
A method for screening inhibitors of insect odor responses, including: 2. The method of screening for an inhibitor of insect odor response according to claim 1, wherein the olfactory receptor is a 1-octen-3-ol receptor, and the odorant is 1-octen-3-ol. The method of screening for an inhibitor of insect odor response according to claim 1, wherein the insect is a cockroach. preparing insect antennae;
providing the antennae of the insect with a stimulating substance that stimulates the antennae of the insect and a candidate inhibitor substance selected in the screening;
detecting a response of the antennae of the insect;
The method of screening for an inhibitor of insect odor response according to claim 1, further comprising: 5. The method of screening for an inhibitor of insect odor response according to claim 4, wherein the stimulant is bombycol. 5. The method of screening for an inhibitor of insect odor response according to claim 4, wherein the insect antennae are moth antennae. 7. The method of screening for an inhibitor against the odor response of an insect according to claim 6, wherein the moth is a silk moth. the olfactory receptor is a 1-octen-3-ol receptor,
the odorant is 1-octen-3-ol,
the irritant substance is bombykol;
the insect antennae are moth antennae;
5. The method of screening for an inhibitor of an insect odor response according to claim 4, which is for screening an inhibitor of a moth response to a pheromone. 5. The method of screening for an inhibitor against the odor response of an insect according to claim 4, wherein in detecting the response of the antennae of the insect, the electric potential of the antennae is detected. preparing insects;
providing the insect with a stimulating substance that stimulates the insect and an inhibitor candidate substance selected in the screening;
detecting a reaction of the insect;
The method of screening for an inhibitor of insect odor response according to claim 1, further comprising: 11. The method of screening for an inhibitor of insect odor response according to claim 10, wherein the stimulant is bombycol. 11. The method of screening for an inhibitor against the odor response of an insect according to claim 10, wherein the insect is a moth. 13. The method of screening for an inhibitor of insect odor response according to claim 12, wherein the moth is a silk moth. 11. The method of screening for an inhibitor of insect odor response according to claim 10, wherein the insect is a cockroach. the olfactory receptor is a 1-octen-3-ol receptor,
the odorant is 1-octen-3-ol,
the irritant substance is bombykol;
the insect is a moth;
11. The method for screening an inhibitor of insect odor response according to claim 10, for screening an inhibitor of moth response to pheromone. 11. The method of screening for an inhibitor against the odor response of an insect according to claim 10, wherein in detecting the response of the insect, the behavior of the insect is observed. An inhibitor of insect odor response obtained by the method for screening an inhibitor of insect odor response according to claim 1, comprising:
An inhibitor of insect odor response that causes less than 37% ion influx into the odor detection structure. 18. The inhibitor of insect odor response according to claim 17, wherein the influx of ions into the odor detection structure is less than 20%. 18. An inhibitor of insect odor response according to claim 17, which is a methylphenol derivative. 18. An inhibitor of insect odor response according to claim 17, comprising 4-isopropyl-3-methylphenol. 18. An inhibitor of insect odor response according to claim 17, comprising 4-(tert-butyl)-2-methylphenol. 18. The inhibitor of insect odor response according to claim 17, wherein the solvent is at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water. 18. The inhibitor of insect odor response according to claim 17, which is an inhibitor of moth odor response. 24. An inhibitor of insect odor response according to claim 23, wherein the moth is a silk moth. 18. The inhibitor of insect odor response according to claim 17, which is an inhibitor of cockroach odor response. An insect inhibition system comprising a spraying section that sprays an inhibitor against an insect odor response obtained by the method for screening an inhibitor against an insect odor response according to claim 1. 27. The insect inhibition system of claim 26, wherein the dispensing section micronizes the inhibitor of the insect's odor response. 5. The spraying unit comprises at least one selected from the group consisting of a two-fluid nozzle, an ultrasonic vibrator, a piezoelectric element, and an electrostatic spray nozzle that atomizes the inhibitor to the insect's odor response. Insect inhibition system according to 26. 27. The insect inhibition system of claim 26, wherein the solvent for the inhibitor of insect odor response is at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water. 27. The insect inhibition system of claim 26, wherein the inhibitor of insect odor response comprises 4-isopropyl-3-methylphenol or 4-(tert-butyl)-2-methylphenol. A method for inhibiting insects, comprising spraying an insect inhibition solution obtained by the method for screening inhibitors for insect odor responses according to claim 1. 32. The method of inhibiting insects according to claim 31, wherein in the spraying, the inhibitor to the odor response of the insect is atomized. 2. The spraying step comprises atomizing the insect odorant response inhibitor using at least one selected from the group consisting of a two-fluid nozzle, an ultrasonic vibrator, a piezoelectric element, and an electrostatic spray nozzle. 32. The method for inhibiting insects according to 31. 32. The method of inhibiting insects according to claim 31, wherein the solvent for the inhibitor of insect odor response is at least one selected from the group consisting of ethanol, hexane, isopropyl alcohol, acetone, DMSO, and water. 32. The method of inhibiting insects according to claim 31, wherein the inhibitor of insect odor response comprises 4-isopropyl-3-methylphenol or 4-(tert-butyl)-2-methylphenol.

Images