Insect repellent and insect repelling method

Abstract

To provide a porous silica material having a structure in which silicon atoms are crosslinked via an acetylene linker.SOLUTION: An insect repellent comprises one or more insect-repelling compounds selected from compounds represented by the general formulas (1) to (3) in the figure (where R1 and R2 are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, or a C1-C5 alkylthio group).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an insect repellent and a method for repelling insects. [Background technology]

[0002] Damage caused by pests is a serious social issue that affects human health and food security, including the spread of infectious diseases and damage to agricultural crops. Current pest control is mainly carried out using insecticides. However, there are concerns that using insecticides has a significant impact on the environment. For this reason, insect repellents are becoming increasingly important as an alternative to insecticides. Until now, the mechanisms of insect repellency have not been well understood. This has delayed the development of insect repellents. However, in recent years, many receptors involved in insect repellency have been identified, creating an environment that makes it easier to develop insect repellents that target them.

[0003] Conventionally, insect repellents contain DEET (N,N-diethyl-3-methylbenzamide (DEET)). DEET acts on insects' olfactory receptors and is a typical insect repellent that has been used on the market for a long time. However, DEET has the drawback of having a short repellent action time and not being able to achieve sufficient repellency unless used in high concentrations. An insect repellent that can compensate for the drawbacks of DEET is a repellent ingredient called icaridin, which was developed in the 1980s.

[0004] Further, development of animal repellents containing thiazoline compounds has been underway. For example, Patent Document 1 describes an animal repellent composition containing a thiazoline compound as an active ingredient. Additionally, some animal repellents contain plant compositions or extracts thereof as plant-derived ingredients, including at least one of chili pepper, herb, bracken, and cypress (see, for example, Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2022-138619 [Patent Document 2] Japanese Patent Application Publication No. 2023-98711 Summary of the Invention [Problem to be solved by the invention]

[0006] Currently, in order to suppress damage caused by pests, there is a demand for insect repellents and methods that exhibit stronger repellent effects against insects. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an insect repellent and an insect repelling method that exhibit a strong repellent effect against insects. [Means for solving the problem]

[0007] The present inventors have focused on the physiological responses of insects in order to solve the above problems and realize an insect repellent that exhibits a strong repellent effect against insects, and have conducted extensive research into compounds that induce a repellent effect as a physiological response of insects. As a result, they found that some compounds among thiazoline compounds used as repellents for animals such as mice exhibit a strong repellent effect against insects, and thus arrived at the present invention. That is, the present invention relates to the following items.

[0008] [1] An insect repellent comprising one or more insect repellent compounds selected from the compounds represented by the following general formulas (1) to (3):

[0009] [ka] (In formulas (1) to (3), R1 and R2 each independently represent a hydrogen atom, a halogen atom, an alkyl group having 1 to 6 carbon atoms, or an alkylthio group having 1 to 5 carbon atoms.)

[0010] [2] The insect repellent according to [1], wherein R1 in the formulae (1) to (3) is a methyl group or an ethyl group. [3] The insect repellent according to [1], wherein the insect repellent compound is a compound represented by formula (1).

[0011] [4] An insect repelling method, comprising the step of diffusing the insect repellent compound contained in the insect repellent according to any one of [1] to [3] into a space where insects are to be repelled. [Effects of the Invention]

[0012] The insect repellent of the present invention contains one or more insect repellent compounds selected from the compounds represented by general formulas (1) to (3), and therefore exhibits a strong repellent effect against insects. In addition, in the insect repellent method of the present invention, one or more insect repellent compounds selected from the compounds represented by general formulas (1) to (3) contained in the insect repellent are diffused into a space where insects are desired to be repelled. Therefore, according to the insect repellent method of the present invention, insects can be effectively repelled from a space where insects are desired to be repelled. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1(a) is a schematic cross-sectional view of the experimental setup, and Figure 1(b) is a photograph of the assay plate removed from the experimental setup shown in Figure 1(a). [Figure 2] Figure 2(a) is a graph showing the relationship between the time elapsed after releasing Drosophila melanogaster onto the assay plate and the preference index (PI). Figure 2(b) is a graph showing the preference index (PI) of Drosophila melanogaster at 60 minutes after the time elapsed in the graph shown in Figure 1(a). Figure 2(c) is a graph showing the relationship between the concentration of 2-methyl-2-thiazoline (2MT) and the preference index (PI). [Figure 3] FIG. 3 is a graph showing the time course of preference index (PI) for 100 μM quinine in control flies (w1118). [Figure 4]FIG. 4 is a graph showing the preference index (PI) for 100 μM quinine after 60 minutes in control flies (w1118). [Figure 5] FIG. 5 is a graph showing the time course of preference index (PI) for 1 mM 2MT in control flies (w1118). [Figure 6] FIG. 6 is a graph showing the preference index (PI) for 1 mM 2MT in control flies (w1118) after 60 minutes. [Figure 7] Figure 7 is a graph showing the time course of preference index (PI) for 1 mM 2MT in control (w1118), TrpA11, and Orco2 flies. [Figure 8] FIG. 8 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in control (w1118), TrpA11, and Orco2 flies. [Figure 9] FIG. 9 is a graph showing the time course of preference index (PI) for 1 mM 2MT in control (w1118), pain4, and wtrw2 flies. [Figure 10] FIG. 10 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in control (w1118), pain4, and wtrw2 flies. [Figure 11] FIG. 11 is a graph showing the time course of preference index (PI) for 1 mM 2MT in control (w1118), TrpMB03672 (TrpMB), TrplMB10553 (TrplMB), and TrpγG4 flies. [Figure 12] FIG. 12 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in control (w1118), TrpMB03672 (TrpMB), TrplMB10553 (TrplMB), and TrpγG4 flies. [Figure 13] FIG. 13 is a graph showing the time course of preference index (PI) for 100 μM 2MT in control (w1118), TrpA11, and Orco2 flies. [Figure 14] FIG. 14 is a graph showing the preference index (PI) for 100 μM 2MT in control (w1118), TrpA11, Orco2, and flies after 60 minutes. [Figure 15] Figure 15 is a graph showing the preference index (PI) for 2MT (10 μM to 10 mM) after 60 minutes in control (w1118), TrpA11, and Orco2 flies. [Figure 16] FIG. 16 is a graph showing the egg-laying index (OI) in control (w1118), TrpA11, Orco2, and flies at 1 mM 2MT. [Figure 17] FIG. 17 is a graph showing the time course of preference index (PI) for 300 μM 4E2MT in control (w1118), TrpA11, and Orco2 flies. [Figure 18] FIG. 18 is a graph showing the preference index (PI) for 300 μM 4E2MT in control (w1118), TrpA11, and Orco2 flies after 60 minutes. [Figure 19] FIG. 19 is a graph showing the time course of preference index (PI) for 100 μM TMO in control (w1118) and TrpA11 flies. [Figure 20] FIG. 20 is a graph showing preference index (PI) for 100 μM TMO after 60 minutes in control (w1118) and TrpA11 flies. [Figure 21] FIG. 21 is a graph showing the time course of preference index (PI) for 1 mM 2MO in control (w1118) and TrpA11 flies. [Figure 22] FIG. 22 is a graph showing the preference index (PI) for 1 mM 2MO in control (w1118) and TrpA11 flies after 60 minutes. [Figure 23]Figure 23 is a graph showing the time course of preference index (PI) for 1 mM 2MT in Gr66a-GAL4 / + (Gr66a-G4 / +), + / UAS-dicer2; + / UAS-TrpA1 RNAi (TrpA1 RNAi / +), and Gr66a-GAL4 / UAS-dicer2; + / UAS-TrpA1 RNAi (Gr66a>TrpA1 RNAi) flies. [Figure 24] Figure 24 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in Gr66a-GAL4 / + (Gr66a-G4 / +), + / UAS-dicer2; + / UAS-TrpA1 RNAi (TrpA1 RNAi / +), and Gr66a-GAL4 / UAS-dicer2; + / UAS-TrpA1 RNAi (Gr66a>TrpA1 RNAi) flies. [Figure 25] Figure 25 is a graph showing the time course of preference index (PI) for 1 mM 2MT in ppk-GAL4 / + (ppk-G4 / +), TrpA1 RNAi / +, and + / UAS-dicer2; ppk-GAL4 / UAS-TrpA1 RNAi (ppk>TrpA1 RNAi) flies. [Figure 26] Figure 26 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in ppk-GAL4 / + (ppk-G4 / +), TrpA1 RNAi / +, + / UAS-dicer2; ppk-GAL4 / UAS-TrpA1 RNAi (ppk>TrpA1 RNAi) flies. [Figure 27] Figure 27 is a graph showing the time course of preference index (PI) for 1 mM 2MT in Orco-GAL4 / + (Orco-G4 / +), TrpA1 RNAi / +, and Orco-GAL4 / UAS-dicer2; + / UAS-TrpA1 RNAi (Orco>TrpA1 RNAi) flies. [Figure 28]Figure 28 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in Orco-GAL4 / + (Orco-G4 / +), TrpA1 RNAi / +, and Orco-GAL4 / UAS-dicer2; + / UAS-TrpA1 RNAi (Orco>TrpA1 RNAi) flies. [Figure 29] Figure 29 is a graph showing the time course of preference index (PI) for 100 μM 2MT in Orco-GAL4 / + (Orco-G4 / +), + / UAS-Kir2.1::GFP (Kir2.1 / +), and Orco-GAL4 / UAS-Kir2.1::GFP (Orco>Kir2.1) flies. [Figure 30] Figure 30 is a graph showing the preference index (PI) for 100 μM 2MT after 60 minutes in Orco-GAL4 / + (Orco-G4 / +), + / UAS-Kir2.1::GFP (Kir2.1 / +), and Orco-GAL4 / UAS-Kir2.1::GFP (Orco>Kir2.1) flies. [Figure 31] Figure 31 is a graph showing the time course of preference index (PI) for 1 mM 2MT in Orco-GAL4 / + (Orco-G4 / +), + / UAS-Kir2.1::GFP (Kir2.1 / +), and Orco-GAL4 / UAS-Kir2.1::GFP (Orco>Kir2.1) flies. [Figure 32] Figure 32 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in Orco-GAL4 / + (Orco-G4 / +), + / UAS-Kir2.1::GFP (Kir2.1 / +), and Orco-GAL4 / UAS-Kir2.1::GFP (Orco>Kir2.1) flies. [Figure 33]Figure 33 is a graph showing the time course of preference index (PI) for 1 mM 2MT in TrpA11 / TrpA1-T2A-GAL4 (TrpA1-KI), TrpA11 / TrpA1-KO (TrpA1-KO), TrpA11 / TrpA1-CKI-T2A-GAL4 (TrpA1-CKI), and TrpA11 / TrpA1-DKI-T2A-GAL4 (TrpA1-DKI) flies. [Figure 34] Figure 34 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in TrpA11 / TrpA1-T2A-GAL4 (TrpA1-KI), TrpA11 / TrpA1-KO (TrpA1-KO), TrpA11 / TrpA1-CKI-T2A-GAL4 (TrpA1-CKI), and TrpA11 / TrpA1-DKI-T2A-GAL4 (TrpA1-DKI) flies. [Figure 35] Figure 35 is a graph showing the time course of preference index (PI) for 1 mM 2MT in TrpA1-KI, TrpA1-KO, TrpA11 / TrpA1-AKI-T2A-GAL4 (TrpA1-AKI), TrpA11 / TrpA1-BKI-T2A-GAL4 (TrpA1-BKI), and TrpA11 / TrpA1-EKI-T2A-GAL4 (TrpA1-EKI) flies. [Figure 36] Figure 36 is a graph showing the preference index (PI) for 1 mM 2MT after 60 minutes in TrpA1-KI, TrpA1-KO, TrpA11 / TrpA1-AKI-T2A-GAL4 (TrpA1-AKI), TrpA11 / TrpA1-BKI-T2A-GAL4 (TrpA1-BKI), and TrpA11 / TrpA1-EKI-T2A-GAL4 (TrpA1-EKI) flies. [Figure 37] Figure 37 shows TrpA1-T2A-GAL4 / UAS-mCD8::GFP. [Figure 38] Figure 38 shows TrpA1-CKI-T2A-GAL4 / UAS-mCD8::GFP. [Figure 39] Figure 39 shows TrpA1-DKI-T2A-GAL4 / UAS-mCD8::GFP. [Figure 40] Figure 40 shows Gr66a-GAL4 / UAS-mCD8::GFP. [Figure 41] Figure 41 shows ppk-GAL4 / UAS-mCD8::GFP. [Figure 42] Figure 42 shows mCherry (left), GFP (middle), and a merged image (right) of the leg. [Figure 43] FIG. 43 shows a representative Fura-2 response to 2MT in cells expressing TrpA1-C. [Figure 44] FIG. 44 shows a representative Fura-2 response to 2MT in cells expressing TrpA1-D. [Figure 45] FIG. 45 shows Fura-2 dose-response curves for TrpA1-C, TrpA1-D, and mock control (cells expressing nothing, Mock) to 2MT. [Figure 46] FIG. 46 shows a representative Fura-2 response to 4E2MT in cells expressing TrpA1-C. [Figure 47] FIG. 47 shows a representative Fura-2 response to 4E2MT in cells expressing TrpA1-D. [Figure 48] FIG. 48 shows Fura-2 dose-response curves for TrpA1-C, TrpA1-D, and mock control (cells expressing nothing, Mock) against 4E2MT. [Figure 49] Figure 49 shows the genomic structure of the TrpA1-C and TrpA1-D isoforms. [Figure 50] Figure 50. Quantification of the maximum Ca2+ i increase (Ca2+ i max) in response to 10 mM 2MT in cells expressing TrpA1-C (left, WT), TrpA1-C C480S (left), TrpA1-D (middle, WT), TrpA1-D C480S (middle), TrpA1-D C729S (middle), TrpA1-D C480S / C729S (middle), and mock control (right, Mock). [Figure 51] Figure 51 is the cover of the supplementary material. [Figure 52]Figure 52 is Supplementary Figure 1. [Figure 53] Figure 53 is Supplementary Figure 2. [Figure 54] Figure 54 is Supplementary Figure 3. [Figure 55] Figure 55 is Supplementary Figure 4. [Figure 56] Figure 56 is a legend to Supplementary Figure 5. [Figure 57] Figure 57 is Supplementary Figure 5. [Figure 58] Figure 58 is Supplementary Figure 6. [Figure 59] Figure 59 is Supplementary Figure 7. [Figure 60] Figure 60 is Supplementary Figure 8. [Figure 61] Figure 61 is Supplementary Figure 9. [Figure 62] Figure 62 is Supplementary Figure 10. [Figure 63] Figure 63 is Supplementary Figure 11. [Figure 64] Figure 64 is Supplementary Figure 12. [Figure 65] Figure 65 is Supplementary Figure 13. [Figure 66] Figure 66 is Supplementary Figure 14 (cDNA sequence of TrpA1-C). [Figure 67] Figure 67 is Supplementary Figure 14 (cDNA sequence of TrpA1-D). [Figure 68] Figure 68 is a description of Supplementary Figure 14. [Figure 69] Figure 69 is Supplementary Table 1 and Supplementary Table 2. [Figure 70] Figure 70 is a reference for the present invention. [Figure 71] FIG. 71 is a reference for the present invention. [Figure 72] FIG. 72 is a reference for the present invention. [Figure 73] FIG. 73 is a reference for the present invention. [Figure 74] Figure 74 is a reference for the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] The insect repellent and insect repellent method of the present invention will be described in detail below. (insect repellent) In the insect repellent of this embodiment, the type of insect to which the insect repellent is applied is not particularly limited, and examples thereof include harmful insects that spread infectious diseases or cause damage to agricultural crops, such as Drosophila melanogaster, Drosophila suzukii, and Aedes aegypti.

[0015] The insect repellent of this embodiment contains one or more insect repellent compounds selected from the compounds represented by the following general formulas (1) to (3). The insect repellent compound contained in the insect repellent of this embodiment exhibits a strong repellent effect against flies such as Drosophila melanogaster and Drosophila suzukii, and therefore the insect repellent of this embodiment can be suitably used as an insect repellent for flies.

[0016] [ka] (In formulas (1) to (3), R1 and R2 each independently represent a hydrogen atom, a halogen atom, an alkyl group having 1 to 6 carbon atoms, or an alkylthio group having 1 to 5 carbon atoms.)

[0017] In the formulas (1) to (3), R1 and R2 may be the same or different. When R1 and / or R2 are halogen atoms, examples of R1 and / or R2 include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0018] When R1 and / or R2 are alkyl groups having 1 to 6 carbon atoms, the alkyl groups having 1 to 6 carbon atoms may be linear or branched. When R1 and / or R2 are alkyl groups having 1 to 6 carbon atoms, the number of carbon atoms is preferably 1 to 4, and is more preferably a methyl group or an ethyl group, as this facilitates the synthesis of the insect repellent compound.

[0019] When R1 and / or R2 are alkylthio groups having 1 to 5 carbon atoms, the alkylthio groups may be linear or branched. When R1 and / or R2 are alkylthio groups having 1 to 5 carbon atoms, the number of carbon atoms is preferably 1 to 4, and is more preferably a methylthio group or an ethylthio group, as this facilitates the synthesis of the insect repellent compound.

[0020] The insect repellent compound in this embodiment preferably contains one or more compounds represented by formula (1), and may contain only the compound represented by formula (1). The compound represented by formula (1) acts on a TRP (transient receptor potential) channel, which is an insect nociceptive receptor, and exhibits a strong repellent effect against insects. Therefore, an insect repellent containing the compound represented by formula (1) exhibits a strong repellent effect against insects.

[0021] The compound represented by formula (1) preferably includes 2-methyl-2-thiazoline (2MT) in which R1 is a methyl group and R2 is a hydrogen atom, and / or 2-methyl-4-ethylthiazoline (4E2MT) in which R1 is a methyl group and R2 is an ethyl group, and particularly preferably includes 2-methyl-2-thiazoline.

[0022] 2-Methyl-2-thiazoline is a compound that exhibits particularly strong insect repellency by activating multiple sensory pathways mediated by insect TRP channels and odorant receptors (ORs). Therefore, 2-methyl-2-thiazoline exerts sufficient insect repellency even at lower concentrations than DEET. Furthermore, 2-methyl-2-thiazoline is a compound that does not activate human TRP channels, is used as a food additive, and is highly safe, making it a desirable compound.

[0023] Furthermore, when the insect repellent compound of this embodiment contains a compound represented by formula (2), it preferably contains 2-ethyl-4-methylthiazole (2E4MT) in which R1 is an ethyl group and R2 is a methyl group, because this compound exhibits a strong repellent effect against insects. Furthermore, when the insect repellent compound of this embodiment contains a compound represented by formula (3), it preferably contains thiomorpholine (TMO), in which R1 and R2 are both hydrogen atoms, because this compound exhibits a strong repellent effect against insects.

[0024] The insect repellent of this embodiment may contain one or more insect repellent compounds selected from the compounds represented by general formulas (1) to (3), and may contain other compounds in addition to the insect repellent compound of this embodiment, as necessary, within a range that does not impair the properties of the compounds represented by general formulas (1) to (3). Examples of other compounds include known insect repellent compounds and solvents other than the compounds represented by general formulas (1) to (3).

[0025] As known insect repellent compounds, for example, one or more selected from DEET, icaridin, IR3535, wasabi components, and essential oils such as eucalyptus and citronella can be used. Examples of the solvent include water, dimethyl sulfoxide (DMSO), ethanol, etc., and the solvent is appropriately selected depending on the type of insect repellent compound.

[0026] The insect repellent of this embodiment contains one or more insect repellent compounds selected from the compounds represented by general formulas (1) to (3). These insect repellent compounds induce strong repellent behavior as a physiological response of insects. Therefore, the insect repellent of this embodiment exhibits strong repellent action against insects.

[0027] (Insect repellent method) The insect repelling method of this embodiment includes the step of diffusing the insect repellent compound contained in the insect repellent of this embodiment into a space from which insects are desired to be repelled. The space from which it is desired to repel insects is a space from which it is desired to prevent the intrusion of insects, and may be either indoors or outdoors, and is not particularly limited.

[0028] As a method for dispersing the insect repellent compound into a space where insects are desired to be repelled, a known method can be used, and can be appropriately selected depending on the shape and size of the space where insects are desired to be repelled, the temperature, the type and amount of the insect repellent compound to be used, etc. Specific methods for dispersing insects into a space where it is desired to repel insects include a method in which the insect repellent compound is supported on a known substrate such as a resin and / or nonwoven fabric, and the substrate is placed in the space where it is desired to repel insects, thereby gradually releasing the insect repellent compound from the substrate into the space; and a method in which the insect repellent compound is placed in a known container with an openable opening, the opening of the container is opened, and the container is placed in the space where it is desired to repel insects, thereby gradually releasing the insect repellent compound from the container into the space.

[0029] The insect repellent method of the present embodiment includes a step of diffusing the insect repellent compound contained in the insect repellent into a space where insects are desired to be repelled, and therefore, according to the insect repellent method of the present embodiment, insects can be effectively repelled from the space where insects are desired to be repelled. [Example]

[0030] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0031] Example 1 The repellent effect of insect repellents on Drosophila melanogaster was investigated using the experimental setup shown in Figure 1(a) and Figure 1(b). Figure 1(a) is a schematic cross-sectional view of the experimental setup. Figure 1(b) is a photograph of an assay plate removed from the experimental setup shown in Figure 1(a).

[0032] As shown in Figure 1(b), a 100 mm diameter, four-section Petri dish 11 (product name: Divided Petri Dish, Four Sections; 25384-308, manufactured by VWR) was fitted with a quadrant-shaped acrylic block with a radius of 42 mm and a thickness of 6 mm, creating four quadrant sections 1a, 1b, 1c, and 1d. Next, 1.2 mL of a 0.5% by mass agarose solution containing 2 mmol / L sucrose was poured into each of the diagonally opposite sections 1a and 1b of the four sections 1a, 1b, 1c, and 1d, coating the quadrants and forming an agarose layer 12. This resulted in a two-choice position selection assay plate 10.

[0033] In two diagonal sections of the assay plate 10 (sections 1c and 1d in Figure 1(b)) that did not form the agarose layer 12, 1.2 mL of a 0.5% by mass agarose solution containing 2-methyl-2-thiazoline (2MT) at a concentration of 1 mmol / L as an insect repellent and sucrose at a concentration of 2 mmol / L was placed in each quadrant to coat the quadrants and form agarose layers 12 containing the insect repellent.

[0034] As shown in Figure 1(a), a tracing table 20 (SV531A, manufactured by Shinkosha) equipped with a light-emitting diode (LED) light 21 was prepared, and the LED light 21 (6800 lux) was turned on while covered with a red film 22 (#105, manufactured by LEE-Filters). A stage 23 made of an acrylic plate was then placed 5 cm above the top surface of the tracing table 20.

[0035] A total of 50 adult male Drosophila melanogaster 3 that had been starved for 24 hours were released onto the assay plate 10, and a transparent lid was placed on top. The assay plate 10 was then inverted and placed on an acrylic stage 23. The assay plate 10 was then maintained at a temperature of 25±0.5°C and a humidity of 40-60%, and the preference index (PI) was measured using the method described below. The results are shown in Figure 2(a) and Figure 2(b).

[0036] (Method of measuring Preference Index (PI)) As shown in Figure 1(a), the assay plate 10 on which Drosophila melanogaster 3 was released was photographed every minute using a digital camera 25 (FDR-AX60, Sony) from the side opposite the stage 23 of the assay plate 10. Using the still images obtained, the number of Drosophila melanogaster 3 present in two sections of the assay plate 10 containing an insect repellent (a solution containing either 2MT, 4E2MT, TMO, or 2MO) (sections containing the insect repellent (1c and 1d in Figure 1(b))) and the other two sections (sections not containing the insect repellent (1a and 1b in Figure 1(b))) was counted.

[0037] Then, a preference index (PI) was calculated for each elapsed time after the Drosophila melanogaster 3 was released onto the assay plate 10 using the following formula. Preference index (PI) (%) = {("Number of individuals in the section containing insect repellent" - "Number of individuals in the section not containing insect repellent") / "Total number of individuals in all sections"} × 100

[0038] Example 2 Except for using non-starved (sated) Drosophila melanogaster individuals, the preference index (PI) was measured under the same conditions as in Example 1 and by the same method as in Example 1. The results are shown in Figure 2(a) and Figure 2(b).

[0039] Example 3 The assay plate was prepared under the same conditions as in Example 1, except that the inside of each quadrant of the four sections 1a, 1b, 1c, and 1d was coated with a sucrose-free agarose solution. The preference index (PI) was measured in the same manner as in Example 1. The results are shown in Figure 2(a) and Figure 2(b).

[0040] Example 4 The preference index (PI) was measured under the same conditions and in the same manner as in Example 1, except that a 0.5% by mass agarose solution containing 2-methyl-2-thiazoline (2MT) as an insect repellent at a concentration of 1 mmol / L and sucrose at a concentration of 2 mmol / L was used instead of the 0.5% by mass agarose solution containing 2-methyl-4-ethylthiazoline (4E2MT) as an insect repellent at a concentration of 300 μmol / L and sucrose at a concentration of 2 mmol / L. The results are shown in Figure 2(a) and Figure 2(b).

[0041] Example 5 The preference index (PI) was measured under the same conditions and in the same manner as in Example 1, except that a 0.5% by mass agarose solution containing thiomorpholine (TMO) as an insect repellent at a concentration of 100 μmol / L and sucrose at a concentration of 2 mmol / L was used instead of the 0.5% by mass agarose solution containing 2-methyl-2-thiazoline (2MT) as an insect repellent at a concentration of 1 mmol / L and sucrose at a concentration of 2 mmol / L. The results are shown in Figure 2(a) and Figure 2(b).

[0042] (Comparative Example 1) The preference index (PI) was measured under the same conditions and in the same manner as in Example 1, except that 1.2 mL of a 0.5% by mass agarose solution containing 2 mmol / L sucrose without any insect repellent was placed in each of the four sections 1a, 1b, 1c, and 1d. The results are shown in Figure 2(a) and Figure 2(b).

[0043] (Comparative Example 2) The preference index (PI) was measured under the same conditions and in the same manner as in Example 1, except that a 0.5% by mass agarose solution containing 2-methyl-2-thiazoline (2MT) as an insect repellent at a concentration of 1 mmol / L and containing sucrose at a concentration of 2 mmol / L was used instead of the 0.5% by mass agarose solution containing 2-methyl-2-oxazoline (2MO) as an insect repellent at a concentration of 1 mmol / L and containing sucrose at a concentration of 2 mmol / L. The results are shown in Figure 2(a) and Figure 2(b).

[0044] Figure 2(a) is a graph showing the relationship between the time elapsed after releasing Drosophila melanogaster onto the assay plate and the preference index (PI). Figure 2(b) is a graph showing the preference index (PI) of Drosophila melanogaster at 60 minutes after releasing Drosophila melanogaster onto the assay plate.

[0045] As shown in Figures 2(a) and 2(b), the preference index (PI) values ​​for Examples 1 to 5 were lower than those for Comparative Examples 1 and 2. This confirmed that 2-methyl-2-thiazoline, 2-methyl-4-ethylthiazoline, and thiomorpholine all exhibit a repellent effect against Drosophila melanogaster.

[0046] Furthermore, from the results of Examples 1 and 2, it was confirmed that 2-methyl-2-thiazoline exhibits a stronger repellent effect on starved Drosophila melanogaster. Furthermore, the results of Examples 1 and 3 confirmed that 2-methyl-2-thiazoline exhibits a stronger repellent effect on starved Drosophila melanogaster, whether in the presence or absence of sucrose.

[0047] Example 6 The preference index (PI) was measured under the same conditions and by the same method as in Example 1, except that instead of a 0.5% by mass agarose solution containing 2-methyl-2-thiazoline (2MT) at a concentration of 1 mmol / L as an insect repellent and sucrose at a concentration of 2 mmol / L, a 0.5% by mass agarose solution containing 2-methyl-2-thiazoline (2MT) at concentrations of 10 μmol / L, 30 μmol / L, 100 μmol / L, 300 μmol / L, 600 μmol / L, 1 mmol / L, 3 mmol / L, and 10 mmol / L was used. The results are shown in Figure 2(c).

[0048] Figure 2(c) is a graph showing the relationship between the concentration of 2-methyl-2-thiazoline (2MT) and the preference index (PI). As shown in Figure 2(c), it was confirmed that a 2-methyl-2-thiazoline concentration of 1 mmol / L or higher exhibited a stronger repellent effect on Drosophila melanogaster.

[0049] Example 7 An agarose layer 12 was formed in the same manner as in Example 1, except that in each of the two diagonally opposite sections 1a and 1b of the four sections 1a, 1b, 1c, and 1d, a 0.25% by mass agarose solution containing sucrose at a concentration of 10 mmol / L was used instead of the 0.5% by mass agarose solution containing sucrose at a concentration of 2 mmol / L, and a two-choice position selection assay plate 10 was obtained.

[0050] In two diagonal sections of the assay plate 10 (sections 1c and 1d in Figure 1(b)) that did not form the agarose layer 12, 1.2 mL of a 0.25% by mass agarose solution containing 2-methyl-2-thiazoline (2MT) at a concentration of 1 mmol / L as an insect repellent and sucrose at a concentration of 10 mmol / L was placed in each quadrant to coat the quadrants and form agarose layers 12 containing the insect repellent. Then, 5 to 8 adult female Drosophila melanogaster at the time of egg laying were released onto the assay plate 10, and the plate was covered with a transparent lid.

[0051] The covered assay plate was then placed in a dark box lined with water-soaked paper and kept at a temperature of 25±0.5°C and a humidity of 80%. The oviposition index (OI) was measured 6 hours after the Drosophila melanogaster were released onto the assay plate using the method described below. The result was -93%. This confirmed that 2-methyl-2-thiazoline has a repellent effect not only on adult males but also on adult female Drosophila melanogaster during egg-laying.

[0052] (Method of measuring egg production index (OI)) Six hours after releasing the Drosophila melanogaster, the assay plates were visually observed, and the number of eggs laid was counted in two sections of the assay plate containing the insect repellent (aqueous solution containing 2MT) (sections containing the insect repellent) and the other two sections (sections not containing the insect repellent). Then, the egg-laying index (OI) 6 hours after releasing the Drosophila melanogaster onto the assay plate was calculated using the following formula. Oviposition index (OI) (%) = {("Number of eggs laid in sections with insect repellent" - "Number of eggs laid in sections without insect repellent") / "Total number of eggs laid in all sections"} x 100

[0053] [title] Avoidance of thiazoline compounds in Drosophila depends on multiple sensory pathways mediated by TrpA1 and olfactory receptors

[0054] [Abstract] Transient receptor potential (TRP) channels are major sensory molecules in animals and are involved in detecting diverse physical and chemical cues in the environment. Given the crucial role of TRPA1 channels in nociceptive and aversive behaviors in various insect species, TRPA1 activators are promising candidates for pest control. In this study, we demonstrated that 2-methylthiazoline (2MT), an artificial volatile thiazoline compound identified as a mouse TRPA1 stimulator, can be used as a novel repellent against Drosophila melanogaster. We observed that 2MT induced strong avoidance behavior in adult males, regardless of feeding status, in a dose-dependent manner, and also induced avoidance in females during oviposition behavior. These avoidance behaviors were mediated by TrpA1-mediated tactile chemosensation and olfactory perception via olfactory receptors. Silencing of TrpA1 revealed the important role of bitter taste neurons and nociceptive neurons in the legs and proboscis. Furthermore, we found that of the five isoforms, only TrpA1-C and TrpA1-D contribute to the avoidance of 2MT. We also found that these isoforms are directly activated by 2MT through covalent modification of evolutionarily conserved cysteine ​​residues. In conclusion, we identified that 2MT stimulates multiple sensory pathways and induces avoidance behavior in flies. We propose that 2MT and related chemicals may be useful for the development of novel insect repellents.

[0055] [preface] Pests, causing crop damage and vector-borne disease transmission, are now a global threat. Insecticides and repellents have been used for centuries as chemical treatments against pests (Debboun and Moore, 2006; Umetsu and Shirai, 2020). While insecticides are effective in controlling pests, most of them disrupt ecosystems, pose risks to humans, and may promote the emergence of pesticide-resistant species. The introduction of insect repellents is an alternative approach to overcome these problems, with DEET (N,N-Diethyl-meta-toluamide) being the most widely used since the 20th century (Leal, 2014; DeGennaro, 2015). However, due to limitations in their use and effectiveness, there is a strong need to develop novel repellents. To discover compounds that effectively repel pests, it is important to focus on key molecules related to sensory perception, particularly aversion responses.

[0056] Insects sense volatile chemicals through olfactory receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), and transient receptor potential (TRP) channels (Montell, 2021). They also sense nonvolatile chemicals through IRs, GRs, TRP channels, Pickpocket (Ppk) channels, and opsins (Montell, 2021). Among these, TRP channels play an important role in nociceptive defense behaviors and aversive responses to various chemical stimuli in many insect species. For example, TRPC channels are activated by carbon dioxide and camphor, citronellal, and citronellol, which are natural repellents for Drosophila melanogaster (Badsha et al., 2012; Zhang et al., 2013; Tian et al., 2022). TRPM channels are activated by menthol in the red flour beetle (Tribolium castaneum) (Shimomura et al., 2021). Hymenoptera-specific TRPA (HsTRPA) channels in the fire ant (Solenopsis invicta) are activated by citronellal, caryophyllene, octanoic acid, and decanoic acid (Wang X. et al., 2018). In honeybees (Apis mellifera), HsTRPA is activated by allyl isothiocyanate (AITC), cinnamaldehyde, and camphor (Kohno et al., 2010). TRPA1 channels have been extensively studied across species and are activated by a variety of natural stimulants, including citronellal, cinnamaldehyde, AITC, aristolochic acid, menthol, cinnamodial, and nepetalactone.These chemicals induce avoidance behaviors in Drosophila melanogaster, Tribolium castaneum, Helicoverpa armigera, and several mosquito species (Aedes aegypti, Anopheles gambiae, Anopheles stephensi, and Culex pipiens pallens) (Kang et al., 2010; Kim et al., 2010; Kwon et al., 2010; Du et al., 2015; Wei et al., 2015; Inocente et al., 2018; Li et al., 2019; Boonen et al., 2021; Melo et al., 2021; Shimomura et al., 2022). Therefore, insect TRPA1 activators represent promising leads for novel broad-spectrum repellents.

[0057] Trimethylthiazoline (TMT), a volatile compound found in fox urine and feces, is known to induce innate fear responses in rodents (Rosen et al., 2015). 2-Methylthiazoline (2MT), an artificial volatile thiazoline compound, can induce stronger fear responses in mice than TMT (Isosaka et al., 2015). 2-Sec-butyl-4,5-dihydrothiazole (SBT), a volatile mouse alarm pheromone, also has a similar thiazoline structure to TMT and induces fear responses in mice (Brechbuhl et al., 2013). The physiological responses induced by these thiazoline-related compounds (TMT, SBT, 2MT) and other artificial thiazoline analogs (Matsuo et al., 2021a) are mediated by TRPA1 (Wang Y. et al., 2018). TMT and 2MT are electrophilic chemicals that activate mouse TRPA1 by directly binding to cysteine ​​residues in the N-terminal cytoplasmic domain (Wang Y. et al., 2018). This mechanism of TRPA1 activation through covalent modification of cysteines is evolutionarily conserved across species (Kang et al., 2010). Notably, some of the cysteines in mouse TRPA1 that react with 2MT are conserved in other species, including pest insects (Kang et al., 2010; Kwon et al., 2010; Wei et al., 2015; Wang XD et al., 2021). Furthermore, 2MT effectively deters mice without direct contact, likely due to its volatility (Wang Y. et al., 2018). Based on these findings, we investigated the efficacy of thiazoline-related compounds as insect repellents and their molecular targets in Drosophila melanogaster. In this study, we found that 2MT induces a strong repellent response in Drosophila via multiple sensory pathways.This requires specific TrpA1 isoforms expressed in taste neurons and neurons that perceive noxious stimuli, and 2MT directly activates these TrpA1 isoforms by binding to an evolutionarily conserved cysteine ​​residue. Furthermore, OR-expressing olfactory sensory neurons (OSNs) were found to be able to recognize 2MT, especially at low concentrations, allowing insects to escape from 2MT over a wide range of concentrations. These findings suggest that 2MT functions as a novel insect repellent that stimulates multiple sensory pathways.

[0058] [result] [Figures 3-6: 2MT induces avoidance behavior in adult flies] Control flies (w 1118 The time course of the preference index (PI) for the chemical stimulus in mice (Fig. 3 and Fig. 5) and the PI after 60 min (Fig. 4 and Fig. 6) were shown. The dotted lines in Fig. 3 and Fig. 5 indicate the level at which mice showed no preference or avoidance behavior. Data are shown as moving means ± SEM.

[0059] Figures 3 and 4 show the PI of flies starved or non-starved for 24 hours in response to 100 μM quinine. PI of flies starved for 24 hours in response to 100 μM quinine in the absence of sucrose (non-sucrose). PI of flies starved for 24 hours in the absence of quinine (non-chemical). The same letters in Figure 4a and b indicate no significant difference based on one-way ANOVA with Tukey's multiple comparisons (N = 8).

[0060] Figures 5 and 6 show the PI of flies starved or non-starved for 24 hours in response to 1 mM 2MT. PI of flies starved for 24 hours in response to 1 mM 2MT in the absence of sucrose (non-sucrose). NS in Figure 6 is not significant; *P < 0.05; **P < 0.01 (Kruskal-Wallis test with Steel-Dwass multiple comparisons) (N = 9).

[0061] [Drosophila showed an avoidance response to 2MT] To evaluate the avoidance behavior of flies toward chemicals, we employed a two-choice place preference assay (Supplementary Figure 1A) (Sanchez-Alcaniz et al., 2017). Starved adult male flies were released onto an assay plate coated with sucrose-containing agarose. The plate was divided into four sections: two diagonal quadrants contained the chemical to be evaluated, while the remaining two quadrants were drug-free. Groups of approximately 50 flies were allowed to explore and select a place using their chemosensory abilities. The number of flies in each area reflected their preference or avoidance toward the chemical. Although the preference index (PI) showed considerable minute-by-minute fluctuations during the experiment (Supplementary Figure 1B), a consistent trend of decreasing PI was observed when a 21-minute running average was calculated (see Methods).

[0062] When quinine, which is known to induce aversive responses in flies via bitter taste neurons (Wang et al., 2004), was tested, flies showed aversion to quinine over time (Figures 3 and 4, Supplementary Figure 2A). On the other hand, when quinine was removed from the agarose, flies distributed evenly across all areas. This result demonstrated that this assay can detect chemical avoidance in a population of flies. In contrast to starved flies, sated flies did not show aversion to the same concentration of quinine (Figures 3 and 4, Supplementary Figure 2A). Furthermore, removing sucrose from the agarose did not cause flies to show aversion to quinine (Figures 3 and 4, Supplementary Figure 2A). These results suggest that the quinine aversion detected by this position preference assay is primarily dependent on feeding behavior.

[0063] Next, we investigated 2MT, a volatile compound that induces innate fear responses in mice (Isosaka et al., 2015). Its derivatives are also found in several fruits and vegetables (Servili et al., 2000; Fernandez et al., 2001; Takahashi and Shibamoto, 2008). Flies consistently and immediately avoided 1 mM 2MT (Figures 5 and 6; Supplementary Figure 2B). Unlike quinine, 2MT aversion was observed regardless of the presence or absence of sucrose in the agarose gel and was even more pronounced in sated flies (Figures 5 and 6; Supplementary Figure 2B). In subsequent experiments, we performed place preference assays using flies starved for 24 h in the presence of 2 mM sucrose. Taken together, these results suggest that 2MT avoidance is regulated not only by taste but also by other sensory pathways.

[0064] [Figures 7-16; 2MT escape is reduced in TrpA1 and Orco mutants] Figures 7-12 show the time course of PI for 1 mM 2MT (Figures 7, 9, and 11) and the PI after 60 minutes (Figures 8, 10, and 12). The dotted lines in Figures 7, 9, and 11 indicate the absence of preference or avoidance. Data are shown as moving means ± SEM.

[0065] Figures 7 and 8 show the control (w 1118 ), TrpA1 1 , Orco 2 , PI relative to 1 mM 2MT in flies. In Figure 8, **P<0.01; ***P<0.001 (Kruskal-Wallis test with Steel-Dwass multiple comparisons) (N=9-12). Figures 9 and 10 show the control (w 1118 ), pain 4 , wtrw 2 , PI against 1 mM 2MT in flies. In Figure 10, NS, not significant; **P < 0.01 (Kruskal-Wallis test with Steel-Dwass multiple comparisons) (N = 8). Figures 11 and 12 show the control (w1118 ), Trp MB03672 (Trp MB ), Trpl MB10553 (Trpl MB ), Trpγ G4 , PI against 1 mM 2MT in flies. In Figure 12, NS, not significant by Kruskal-Wallis test with Steel's multiple comparisons (N = 7-8).

[0066] Figures 13 and 14 show the control (w 1118 ), TrpA1 1 , Orco 2 , PI of 100 μM 2MT in flies. In FIG. 14, NS, not significant; **P < 0.01; ***P < 0.001; (one-way ANOVA with Tukey's multiple comparisons) (N = 9). Figure 15 shows the control (w 1118 ), TrpA1 1 , Orco 2 , Dose dependence of PI on 2MT (10 μM–10 mM) after 60 min in flies ( N = 6–17). Figure 16 shows the control (w 1118 ), TrpA1 1 , Orco 2 , Oviposition index (OI) in flies at 1 mM 2MT. Data are shown as mean ± SEM. NS, not significant; ***P < 0.001 (Kruskal-Wallis test with Steel-Dwass multiple comparisons) (N = 11-12).

[0067] [TrpA1 and OR play important roles in bypassing 2MT] To clarify which sensory molecules are involved in 2MT avoidance, we examined mutants of TRP channels known to be involved in chemical avoidance in adult flies (Al-Anzi et al., 2006; Kang et al., 2010; Kim et al., 2010; Kwon et al., 2010; Badsha et al., 2012; Zhang et al., 2013; Mandel et al., 2018; Li et al., 2020; Boonen et al., 2021; Melo et al., 2021). A TrpA1 null mutation nearly abolished the avoidance response to 1 mM 2MT (Figures 7 and 8; Supplementary Figure 3A). In contrast, other TRP channel mutants, including those from the TRPA subfamily (painless; pain and water witch; wtrw) and TRPC subfamily (Trp, Trp-like; Trpl, Trpγ), showed little or no reduction in avoidance behavior to the same concentration of 2MT (Figures 9-12, Supplementary Figures 3B and C). These results indicate that TrpA1 plays an essential role in the detection of 2MT in flies.

[0068] Because 2MT is a volatile compound, we investigated whether olfaction is required for 2MT avoidance. Flies carrying a null mutation in the odorant receptor co-receptor (Orco), which causes olfactory dysfunction (Larsson et al., 2004), showed a partial but significant impairment in avoidance responses to 1 mM 2MT at all test time points (30, 60, and 90 min) compared with controls (Figures 7 and 8, Supplementary Figure 3A). Furthermore, Orco mutant flies failed to avoid low concentrations of 2MT (30, 100, and 300 μM). Notably, TrpA1 mutant flies exhibited avoidance behavior indistinguishable from controls at these concentrations (Figures 13–15, Supplementary Figures 3D and 4). Therefore, we conclude that TrpA1 and olfactory receptors are involved in tactile chemosensory and olfactory-dependent detection of 2MT, respectively, and that their contributions depend on the chemical concentration.

[0069] [2MT influences female reproductive behavior through TrpA1] We investigated the effect of 2MT on another behavioral response, female oviposition behavior. Female flies utilize chemosensory abilities to avoid oviposition in the presence of noxious or aversive chemicals (Stensmyr et al., 2012; Melo et al., 2021). An oviposition assay, similar to the two-choice positional preference assay, was performed by releasing adult female flies onto agarose-coated plates (divided into four compartments). After 6 h, the number of eggs in each compartment was counted and the oviposition index (OI) was calculated. Control and Orco mutant flies strongly avoided oviposition on agarose containing 1 mM 2MT (Figure 16). In contrast, TrpA1 mutant flies did not exhibit this significant avoidance response (Figure 16). These results indicate that 2MT inhibits female oviposition via TrpA1. As observed in both males (two-alternative position choice assay) and females (oviposition assay), the action of 2MT through TrpA1 induces a general avoidance response in adult flies.

[0070] In mice, exposure to 2MT induces freezing behavior and suppressed locomotor activity (Isosaka et al., 2015; Matsuo et al., 2021b). To assess the locomotor activity of flies exposed to 2MT, we videotaped their spontaneous walking and measured the total distance traveled over a 10-minute test period using a video tracking system. Flies were exposed to 2MT at concentrations ranging from 100 μM to 10 mM, and their locomotor activity was compared with that of flies not exposed to the chemical. Control flies showed no difference in locomotor activity at any test period, regardless of the concentration of 2MT (Supplementary Figure 5A). TrpA1 mutant flies showed increased locomotion compared to control flies in the presence or absence of 2MT up to 1 mM. However, in the presence of 10 mM 2MT, locomotor activity decreased for unknown reasons (Supplementary Figure 5B). These results suggest that the physiological effects of 2MT differ between mice and flies.

[0071] [Figures 17-22; Thiazoline-related compounds elicited different behavioral responses] Figures 17 to 22 show the time course of PI for thiazoline-related compounds (Figures 17, 19, and 21) and the PI after 60 minutes (Figures 18, 20, and 22). The dotted lines in Figures 17, 19, and 21 indicate levels indicating no preference or avoidance. Data are shown as moving means ± SEM.

[0072] Figures 17 and 18 show the control (w 1118 ), TrpA1 1 , Orco 2 , PI against 300 μM 4E2MT in flies. In FIG. 18, NS, not significant; ***P < 0.001 (one-way ANOVA with Tukey's multiple comparisons) (N = 9). Figures 19 and 20 show the control (w 1118 ) and TrpA1 1 , PI against 100 μM TMO in flies. In FIG. 20, NS, not significant by Student's t-test (N=10). Figures 21 and 22 show the control (w 1118 ) and TrpA1 1 , PI to 1 mM 2MO in flies. In Figure 22, NS, not significant by Student's t-test (N = 8).

[0073] [The thiazoline-related compound 4E2MT induces avoidance responses via TrpA1] Because several thiazoline-related compounds activate mouse TRPA1 (Matsuo et al., 2021a), we also examined the avoidance responses induced by other thiazoline-related compounds. 2-Methyl-4-ethylthiazoline (4E2MT) induced avoidance behavior in control flies, but partially reduced avoidance behavior in TrpA1 mutant flies (Figures 17 and 18, Supplementary Figure 6A). 4E2MT is volatile, but unlike 2MT, it had no effect on the avoidance response in Orco mutant flies. Thiomorpholine (TMO) induced avoidance behavior in both control and TrpA1 mutant flies (Figures 19 and 20, Supplementary Figure 6B), suggesting that TMO acts on an unknown sensory molecule. 2-Methyl-2-oxazoline (2MO), which does not activate mouse TRPA1 (Wang Y. et al., 2018; Matsuo et al., 2021a), did not induce any clear behavioral responses in either control or TrpA1 mutant flies (Figures S1 and S2, Supplementary Figure 6C). In summary, of the thiazoline-related compounds tested, 2MT and 4E2MT exerted their avoidance effects primarily via TrpA1, but only 2MT induced avoidance behaviors that were also mediated by olfaction.

[0074] [Figures 23-32; TrpA1 expression in taste and nociceptive neurons was required for 2MT avoidance] Figures 23 to 32 show the time course of PI for 2MT (Figures 23, 25, 27, 29, and 31) and the PI after 60 minutes (Figures 24, 26, 28, 30, and 32). The dotted lines in Figures 23, 25, 27, 29, and 31 indicate levels that show no preference or avoidance. Data are shown as moving means ± SEM.

[0075] Figures 23 and 24 show PI to 1 mM 2MT in Gr66a-GAL4 / + (Gr66a-G4 / +), + / UAS-dicer2; + / UAS-TrpA1 RNAi (TrpA1 RNAi / +), and Gr66a-GAL4 / UAS-dicer2; + / UAS-TrpA1 RNAi (Gr66a>TrpA1 RNAi) flies. Figures 25 and 26 show PI to 1 mM 2MT in ppk-GAL4 / + (ppk-G4 / +), TrpA1 RNAi / +, + / UAS-dicer2; ppk-GAL4 / UAS-TrpA1 RNAi (ppk>TrpA1 RNAi) flies. Figures 27 and 28 show PI to 1 mM 2MT in Orco-GAL4 / + (Orco-G4 / +), TrpA1 RNAi / +, and Orco-GAL4 / UAS-dicer2; + / UAS-TrpA1 RNAi (Orco>TrpA1 RNAi) flies. In Figures 24, 26, and 28, NS, not significant; *P < 0.05; **P < 0.01 (Kruskal-Wallis test with Steel-Dwass multiple comparisons) (N = 8; note: TrpA1 RNAi / + data are shared between Figures 24 and 26).

[0076] Figures 29-32 show the PI response to 100 μM 2MT (Figures 29 and 30) and 1 mM 2MT (Figures 31 and 32) in Orco-GAL4 / + (Orco-G4 / +), + / UAS-Kir2.1::GFP (Kir2.1 / +), and Orco-GAL4 / UAS-Kir2.1::GFP (Orco>Kir2.1) flies. In Figure 30, NS, not significant; *P < 0.05; **P < 0.01 (Kruskal-Wallis test with Steel-Dwass multiple comparisons) (N = 9-10).

[0077] [Multiple sensory neurons are involved in 2MT avoidance] TrpA1 is expressed in adult fly bitter taste neurons (Kang et al., 2010, 2011; Kim et al., 2010; Leung et al., 2020), olfactory neurons (Kwon et al., 2010), and nociceptor neurons (Khuong et al., 2019). To clarify which sensory neurons are involved in 2MT avoidance, we performed targeted gene knockdown experiments of TrpA1 in specific sensory neurons using a GAL4 / UAS binary expression system and RNA interference. Knockdown of TrpA1 in bitter taste neurons with Gr66a-GAL4 or in nociceptor neurons with ppk-GAL4 significantly reduced 2MT avoidance (Figures 23-26, Supplementary Figure 7A), whereas knockdown in olfactory neurons with Orco-GAL4 had no significant effect (Figures 27 and 28, Supplementary Figure 7B).

[0078] Because Orco mutant flies exhibited altered 2MT avoidance, especially at low concentrations, we next expressed Kir2.1 and inhibited neural activity in olfactory neurons (Figures 29-32). Inhibition of electrical activity in olfactory neurons with Orco-GAL4 abolished 100 μM 2MT avoidance and partially impaired 1 mM 2MT avoidance (Figures 29-32; Supplementary Figure 7B), consistent with the results obtained with Orco mutant flies (Figures 7, 8, 13, and 14). These results suggest that adult flies detect 2MT through TrpA1, which is expressed in bitter and nociceptive neurons, in conjunction with olfactory receptors expressed in olfactory neurons.

[0079] [Figures 33-36; TrpA1-C and TrpA1-D isoforms were involved in 2MT escape] Figures 33 to 36 show the time course of PI for 1 mM 2MT (Figures 33 and 35) and the PI after 60 minutes (Figures 34 and 36). The dotted lines in Figures 33 and 35 indicate the level of no preference or avoidance. Data are shown as moving means ± SEM.

[0080] Figures 33 and 34 show TrpA1 1 / TrpA1-T2A-GAL4(TrpA1-KI), TrpA1 1 / TrpA1-KO (TrpA1-KO), TrpA1 1 / TrpA1-CKI-T2A-GAL4 (TrpA1-CKI), and TrpA1 1 PI in response to 1 mM 2MT in / TrpA1-DKI-T2A-GAL4 (TrpA1-DKI) flies. The same letter in Figure 34a and b indicates no significant difference based on one-way ANOVA with Tukey's multiple comparisons (N = 12-16).

[0081] Figures 35 and 36 show the TrpA1-KI, TrpA1-KO, and TrpA1 1 / TrpA1-AKI-T2A-GAL4(TrpA1-AKI), TrpA1 1 / TrpA1-BKI-T2A-GAL4 (TrpA1-BKI), and TrpA1 1 PI in response to 1 mM 2MT in / TrpA1-EKI-T2A-GAL4 (TrpA1-EKI) flies. The same letter in Figure 36(a) and (b) indicates no significant difference based on the Kruskal-Wallis test with Steel-Dwass multiple comparisons (N = 12-21).

[0082] [TrpA1-C and TrpA1-D isoforms play a key role in the avoidance response to 2MT] Drosophila TrpA1 has five splicing variants: TrpA1-A, TrpA1-B, TrpA1-C, TrpA1-D, and TrpA1-E (Zhong et al., 2012; Gu et al., 2019). When expressed in cultured cells, all isoforms except TrpA1-E respond to the wasabi component AITC. However, the contribution of each isoform to thermosensation and chemosensation differs between larvae (Zhong et al., 2012; Gu et al., 2019) and adults (Kang et al., 2011; Du et al., 2015; Leung et al., 2020). To determine which TrpA1 isoform is essential for 2MT bypass, we used TrpA1 isoform-specific knock-in (KI) lines (Gu et al., 2019). This KI line allows expression of only a single TrpA1 isoform by mutating exons specific to other isoforms. Because homozygous KI lines have poor viability, heterozygotes for KI or knockout (KO) lines are used to express only TrpA1. 1 TrpA1-CKI and TrpA1-DKI heterozygotes exhibited strong avoidance behavior toward 2MT, similar to TrpA1-KI heterozygotes expressing all isoforms (Figures 33 and 34, Supplementary Figure 8A). Conversely, TrpA1-AKI, TrpA1-BKI, and TrpA1-EKI heterozygote flies did not exhibit avoidance behavior toward the same concentration of 2MT, indicating that TrpA1-KO / TrpA1 1 The responses of transheterozygotes were similar to those of transheterozygotes (Figures 35 and 36, Supplementary Figure 8B). These results indicate that expression of either the TrpA1-C or TrpA1-D isoform is sufficient for adult flies to respond to 2MT.

[0083] [Figures 37-42; TrpA1-C and TrpA1-D isoforms were expressed in leg sensory neurons] The vertical direction in Figures 37 to 41 is the length direction of the scale bar. Figures 37 to 41 show GFP expression in the forelimb (left), middle leg (center), and hind leg (right), respectively. The arrow indicates the cell body of the ppk neuron in the tarsal segments 1-3. The triangle indicates the cell body of the ppk neuron and Gr66a neuron in the forepaw segment 5 of the forelimb. In Figures 37 to 41, the photographs placed at the top of the vertically long photographs are enlarged images of the cell body near the arrow or triangle within the vertically long photograph.

[0084] Figure 37 shows TrpA1-T2A-GAL4 / UAS-mCD8::GFP. Figure 38 shows TrpA1-CKI-T2A-GAL4 / UAS-mCD8::GFP. Figure 39 shows TrpA1-DKI-T2A-GAL4 / UAS-mCD8::GFP. Figure 40 shows Gr66a-GAL4 / UAS-mCD8::GFP. Figure 41 shows ppk-GAL4 / UAS-mCD8::GFP. The scale bars in Figures 37 to 41 represent 100 μm (vertical photographs) or 10 μm (photographs placed on top of vertical photographs).

[0085] Figure 42 shows mCherry (left), GFP (middle), and overlay images (right) of the legs. A magnified image of Gr66a neurons in the forelimbs (top) in lexAop2-mCherry / Gr66a-GAL4; TrpA1-T2A-LexA / UAS-mCD8::GFP. A magnified image of ppk neurons in the forelimbs, middle, and hindlimbs (bottom) in lexAop2-mCherry / UAS-mCD8::GFP; TrpA1-T2A-LexA / ppk-GAL4. Scale bars represent 10 μm. Expression patterns were confirmed in triplicate for all samples.

[0086] [TrpA1-C and TrpA1-D isoforms are expressed in leg sensory neurons] To determine the expression patterns of TrpA1-C and TrpA1-D isoforms in chemosensory and nociceptive neurons, we examined the expression of membrane-bound green fluorescent protein (mCD8::GFP) induced in TrpA1 isoform-specific KI lines. These KI lines contain a T2A-GAL4 sequence immediately upstream of the stop codon, allowing GAL4 expression to be specific to the cells expressing each isoform (Gu et al., 2019). Since their expression patterns in the proboscis and maxillary palp have been previously described (Gu et al., 2019), we examined the expression patterns of TrpA1-C and TrpA1-D in sensory neurons in the legs and proboscis tip.

[0087] First, we observed TrpA1-T2A-GAL4 (TrpA1-KI) flies expressing all isoforms to determine the overall TrpA1 expression pattern. In the forelimbs, two large cell bodies located in the fifth segment of the forelimb were clearly labeled, similar to the pattern of Gr66a- and ppk-positive neurons (Figure 37, Figure 40, Figure 41, Supplementary Figure 9A). This expression was also observed in TrpA1-CKI-T2A-GAL4 and TrpA1-BKI-T2A-GAL4 flies, but not in TrpA1-DKI-T2A-GAL4 and TrpA1-AKI-T2A-GAL4 flies (Figure 38 and Figure 39, Supplementary Figure 9B and C). Furthermore, TrpA1-KI flies showed weak GFP signals in neurons with small cell bodies located in the proximal tarsal segments of the forelimbs, midlegs, and hindlegs. Its location and shape resembled those of nociceptor neurons expressing ppk-GAL4 (Figures 37 and 41, Supplementary Figure 9A). This ppk-like expression pattern was observed in both TrpA1-CKI-T2A-GAL4 and TrpA1-DKI-T2A-GAL4 flies (Figures 38 and 39), but not in TrpA1-AKI-T2A-GAL4 or TrpA1-BKI-T2A-GAL4 flies (Supplementary Figures 9B and C). To confirm TrpA1 expression in taste and nociceptor neurons, we crossed flies expressing mCD8::GFP with TrpA1-T2A-LexA, which expresses red fluorescent protein (mCherry) in all isoform-expressing cells, to flies expressing mCD8::GFP with Gr66a-GAL4 or ppk-GAL4 (Figure 42). As expected from the individual labeling (Figures 37, 40, and 41), TrpA1 colocalized with Gr66a and Ppk in all legs (Figure 42). We also examined the expression patterns of Gr66a- and ppk-GAL4 in the proboscis tip, along with TrpA1-T2A-LexA. TrpA1 was widely expressed in taste neurons and showed colocalization with a subset of Gr66a neurons (Supplementary Figure 10A). However, coexpression of Ppk and TrpA1 was not observed in the proboscis tip (Supplementary Figure 10B).These results and the behavioral analysis in Figure 4 suggest that bitter taste neurons expressing TrpA1-C and nociceptive neurons expressing TrpA1-C and TrpA1-D contribute to the aversive response to 2MT.

[0088] [Figures 43-50; 2MT activates TrpA1-C and TrpA1-D isoforms via cysteine ​​modification] Figures 43, 44, 46, and 47 show representative Fura-2 responses to 2MT (Figures 43 and 44) ​​or 4E2MT (Figures 46 and 47) in cells expressing TrpA1-C (Figures 43 and 46) or TrpA1-D (Figures 44 and 47). S2R+ cells were stimulated with 10 mM 2MT or 10 mM 4E2MT. To assess channel function, 100 μM NMM was added. Cell viability was confirmed by the addition of 2.5 μM ionomycin (Iono).

[0089] Figures 45 and 48 show Fura-2 dose-response curves for TrpA1-C, TrpA1-D, and mock control (cells not expressing anything, Mock) to 2MT (Figure 45) or 4E2MT (Figure 48). Data are shown as mean ± SEM.

[0090] Figure 49 shows the genomic structure of the TrpA1-C and TrpA1-D isoforms. Coding and non-coding exons are shown as white and black boxes, respectively, and alternatively spliced ​​exons are shown as checkered and hatched boxes.

[0091] Figure 50 shows the maximum Ca response to 10 mM 2MT in cells expressing TrpA1-C (left, WT), TrpA1-C C480S (left), TrpA1-D (middle, WT), TrpA1-D C480S (middle), TrpA1-D C729S (middle), TrpA1-D C480S / C729S (middle), and mock control (right, Mock). 2+ i Increased (Ca 2+ iData are shown as mean ± SEM (N = 6-13). *P < 0.05; ***P < 0.001 in Figure 50 (Student's t-test or Kruskal-Wallis test with Steel's multiple comparisons).

[0092] [TrpA1-C and TrpA1-D isoforms are activated by 2MT via an evolutionarily conserved cysteine ​​residue] To examine the responsiveness of Drosophila TrpA1-C and TrpA1-D isoforms to 2MT, in vitro calcium imaging was performed using S2R+ cells expressing TrpA1-C or TrpA1-D. 2MT treatment significantly increased the intracellular calcium ion concentration ([Ca 2+ ] i ) levels in a dose-dependent manner, whereas no such response was observed in control cells (Fig. 43-45). Notably, TrpA1-D significantly increased [Ca] levels compared with TrpA1-C. 2+ ] i The addition of 4E2MT showed a strong increase in [Ca 2+ ] i Increases in [Ca] were also observed in TrpA1-C and TrpA1-D expressing cells, but the nonspecific effects of the compounds became more pronounced at higher concentrations. 2+ ] i The increase in TrpA1 expression was indistinguishable from that in control cells (Figs. 46 to 48).

[0093] Previous studies have shown that 2MT activates mouse TRPA1 by covalently modifying six cysteine ​​residues (Wang Y. et al., 2018). Two cysteine ​​residues are conserved in Drosophila TrpA1: C480 and C729 in TrpA1-D (corresponding to mouse C415 and C666, respectively), and C480 in TrpA1-C (corresponding to mouse C415) (Figure 49, Supplementary Figure 11). To assess the role of these conserved cysteine ​​residues in Drosophila TrpA1, we substituted each residue with serine in TrpA1-C (C480S) and TrpA1-D (C480S, C729S, C480S / C729S). Mutation of single or multiple cysteine ​​residues in these isoforms significantly increased the [Ca] response to 2MT. 2+ ] i The response was significantly reduced, almost reaching background levels (Figure 50). Importantly, we confirmed that the TrpA1-D double mutant retained its responsiveness to citronellal, which activates the channel through a noncovalent mechanism (Du et al., 2015; Boonen et al., 2021). This indicates that the mutant channel is still functional (Supplementary Figure 12). These results strongly suggest that 2MT activates Drosophila TrpA1 by specifically interacting with these conserved cysteine ​​residues.

[0094] [Consideration] In this study, we investigated the effects of the thiazoline-related compound 2MT on behavioral responses in both males and females of Drosophila melanogaster and investigated its underlying mechanisms. Avoidance responses to high concentrations (300 μM–3 mM) of 2MT were primarily mediated by the gustatory and nociceptive pathways via TrpA1, whereas avoidance responses to low concentrations (<300 μM) of 2MT were mediated by the olfactory pathway involving olfactory receptors. Unlike bitter substances that typically exert their effects through the proboscis's taste pathway, 2MT exerts its repellent effect even when its involvement in feeding is minimal (Figures 3–6). This indicates its potential as a promising lead compound for the development of insect repellents. Furthermore, a clear avoidance response was observed in TrpA1 mutants at higher concentrations of 2MT (≥3 mM) (Figures 7–16), suggesting the existence of additional receptors and detection mechanisms for 2MT.

[0095] TrpA1 is expressed in Drosophila gustatory, nociceptive, and olfactory neurons (Kang et al., 2010, 2011; Kim et al., 2010; Kwon et al., 2010; Khuong et al., 2019; Leung et al., 2020). Knockdown of TrpA1 in gustatory and nociceptive neurons, but not in olfactory neurons, reduced the avoidance response to 2MT (Figures 23–32). This indicates a functional role of TrpA1 in the tactile chemosensory pathway. Furthermore, Orco mutation and inhibition of olfactory neuron activity using Kir2.1 resulted in a reduction in 2MT avoidance, indicating the contribution of the olfactory pathway to 2MT avoidance (Figures 7–16, 23–32). Notably, similar 2MT characteristics have been reported in mouse studies: TRPA1 expressed in the trigeminal and vagus nerves mediates 2MT responses, but TRPA1 expressed in olfactory neurons does not (Matsuo et al., 2021a). Nevertheless, olfactory neurons themselves are required for 2MT responses (Matsuo et al., 2021a). Similar to 2MT, other TRP channel activators also drive multiple sensory pathways in insects. Flies detect the naturally occurring insect repellent citronellal via olfactory receptors and TrpA1 expressed in olfactory and gustatory neurons, and Trpγ expressed in olfactory neurons (Kwon et al., 2010; Du et al., 2015; Tian et al., 2022). The repellent effect of camphor is mediated by Trpl, which is expressed in gustatory and olfactory neurons (Zhang et al., 2013; Wang Q. et al., 2021), and menthol-induced repellency involves TrpA1 and olfactory receptors (Boonen et al., 2021; Wang Q. et al., 2021). Taken together, the detection of 2MT and the subsequent repellent response involve multiple sensory processes, and such mechanisms are considered to be important characteristics of TRP channel activators as insect repellents.

[0096] Interestingly, Orco mutant flies showed attractive, but not aversive, responses to low concentrations (30, 100, and 300 μM) of 2MT (Figures 7–16; Supplementary Figure 4). This paradoxical effect of 2MT in mutant flies suggests that 2MT is detected not only as an aversive cue but also as an attractive cue by an unidentified chemosensory receptor in flies. Structurally similar to 2MT, 2-methylthiazolidine is a component of the male sex pheromone of cockroaches (Nauphoeta cinerea) and is highly attractive to females at very low concentrations (Sirugue et al., 1992). 2MT also exhibits attractive effects on female cockroaches, but the amount detected in male glands is too small to exert an attractive effect. These findings may support the possibility that an unknown chemosensory receptor is involved in the attractive behavior of 2MT at low concentrations in flies. At the tip of the proboscis, TrpA1 is present in both bitter and other taste neurons (Supplementary Figure 10). Neurons expressing only TrpA1, distinct from Gr66a, may contribute to the mild attraction of Drosophila to 2MT.

[0097] We also found that other thiazoline-related chemicals, 4E2MT and TMO, induced avoidance responses (Figures 17–22). These chemicals activate mouse TRPA1 and induce hypoxia resistance in mice similar to that induced by 2MT (Matsuo et al., 2021a). However, unlike 2MT, TMO-induced avoidance responses were not reduced in TrpA1 mutant flies (Figures 17–22), suggesting that flies detect TMO via a TrpA1-independent pathway. On the other hand, 4E2MT-induced avoidance was unchanged in Orco mutants and partially reduced in TrpA1 mutants (Figures 17–22). Furthermore, 4E2MT induced nonspecific calcium responses in cultured Drosophila cells lacking TrpA1 expression (Figures 43–50). These results suggest that 4E2MT acts on unknown receptors or nonspecifically in Drosophila cells.

[0098] Drosophila has five TrpA1 isoforms. All isoforms are expressed in esophageal taste neurons (Gu et al., 2019). While TrpA1-C and TrpA1-D have been reported to be expressed in taste neurons at the tip of the proboscis, the TrpA1-A, TrpA1-B, and TrpA1-E isoforms are either not expressed or only weakly expressed. Expression of TrpA1-C and TrpA1-D was observed in chemosensory and / or nociceptive neurons in the legs (Figures 37-42). 2MT avoidance was observed only in TrpA1-CKI and TrpA1-DKI flies expressing the TrpA1-C and TrpA1-D isoforms, respectively, but not in TrpA1-AKI, TrpA1-BKI, or TrpA1-EKI flies (Figures 33-36). This difference in expression patterns may reflect different functions of the isoforms in 2MT detection. The TrpA1-B isoform, like the TrpA1-C isoform, was weakly expressed in Gr66a-bitter neurons (Supplementary Figure 9). However, unlike TrpA1-C, TrpA1-B did not contribute to 2MT avoidance (Figures 33-36). This similar expression pattern may be due to the shared exon between these isoforms (Zhong et al., 2012; Gu et al., 2019). Consistent with this idea, the TrpA1-C and TrpA1-D isoforms are expressed in ppk nociceptor neurons in the forelimb, middle limb, and hind limb (Figures 37-42), and these isoforms share a common first exon (Zhong et al., 2012; Gu et al., 2019). The expression pattern of each isoform may depend on specific exons / introns.

[0099] Previous studies have demonstrated differences in chemical sensitivity among Drosophila TrpA1 isoforms. TrpA1-C and D are more sensitive to citronellal than TrpA1-A, and TrpA1-A is more sensitive to menthol than TrpA1-C and D (Du et al., 2015; Boonen et al., 2021). TrpA1-B is the least sensitive to both citronellal and menthol (Boonen et al., 2021). The plant chemical aristolochic acid activates TrpA1-D but not TrpA1-C (Leung et al., 2020). Nepetalactone, a natural repellent found in catnip, activates TrpA1-C but not TrpA1-A (Melo et al., 2021). Furthermore, chemical sensitivity of TRPA1 differs among isoforms in mosquitoes such as Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens pallens (Du et al., 2015; Li et al., 2019; Melo et al., 2021). Taken together, the physiological roles of each isoform may depend on variations in chemical sensitivity and expression patterns.

[0100] Mammalian TRPA1 is activated by electrophiles through covalent modification of specific cysteine ​​residues, and Drosophila TrpA1 also responds to electrophiles using evolutionarily conserved cysteine ​​residues (Hinman et al., 2006; Macpherson et al., 2007; Kang et al., 2010). Previous studies have identified six cysteine ​​residues involved in the activation of mouse TRPA1 by 2MT (Wang Y. et al., 2018). Two of these cysteine ​​residues (C480 and C729) are also conserved in fly TrpA1: C480 and C729 in TrpA1-D and C480 in TrpA1-C (Figures 43–50, Supplementary Figure 11). The calcium response of TrpA1-D was stronger than that of TrpA1-C, and a single mutation of C729 resulted in reduced activation by 2MT (Figures 43–50). This mutant retained an additional cysteine ​​(C480), and the calcium response was comparable to that of the TrpA1-C isoform bearing C480. The C480 substitution in TrpA1-C and the double cysteine ​​mutation in TrpA1-D reduced the calcium response to background levels, indicating that these cysteine ​​residues are essential for 2MT-dependent channel activation.C480 and C729 of TRPA1 are found in moths (Bombyx mori, Bombyx mandarina, Manduca sexta, Tuta absoluta, Galleria mellonella, Helicoverpa armigera), aphids (Acyrthosiphon pisum), and mosquitoes (Anopheles gambiae, Aedes aegypti, Culex pipiens) (Kang et al., 2010; Kwon et al., 2010; Wei et al., 2015; Wang The repellent effect of 2MT was highly conserved across a wide range of insect species, including agricultural pests such as the bug (Lygus hesperus) and disease vectors (Supplementary Figure 13). This suggests that the repellent effect of 2MT may be maintained across these species. Further research is needed on the effects of 2MT on these insects at the behavioral and molecular biological levels.

[0101] Our results suggest that insect TRP channels may be promising targets for the development of repellents and insecticides. Recent studies have identified commercially available insecticides, such as pymetrozine and pyrifluquinazone, as activators of insect TRPV channels (Inactive, Nanchung) expressed in stretch receptor cells (Nesterov et al., 2015). These synthetic chemicals induce insecticidal effects by disrupting normal locomotor and feeding behaviors of pest insects. In addition, we propose that 2MT has important potential as an insect repellent for the following reasons: First, it functions through both olfactory and tactile chemosensation. Second, mere contact with 2MT effectively induces aversion without the need for feeding. Notably, a previous report failed to detect activation of human TrpA1 by 2MT, despite the conservation of four critical cysteines (Wang Y. et al., 2018). It is also worth noting that 2MT is commercially used as a food additive, but its concentration range is generally 0.1–10 ppm (Fernandez et al., 2002). Considering these findings and our results, 2MT and related chemicals may be valuable resources for the development of novel insect repellents and insecticides that target insect TRP channels as promising targets.

[0102] Materials and Methods [Fly strain] Flies were reared on glucose-yeast-cornmeal medium: 2500 ml reverse osmosis water, 180 g cornmeal (Oriental Yeast), 100 g dried brewer's yeast Ebios (#128-297405, Mitsubishi Tanabe Pharma), 19 g agar (#RSU-AL01, Rikaken), 250 g glucose (#TDH, Sanei Saccharification), 24 ml methyl 4-hydroxybenzoate (10% in 70% ethanol; #H5501, Sigma-Aldrich), and 8 ml propionic acid (#81910, Sigma-Aldrich). Flies were reared in vials or bottles at 25°C under a 12-h light-dark cycle. 1118were backcrossed for 10 generations onto the wild-type Canton-S (CS) genetic background and used as a control. The fly strains used in this study are as follows: TrpA1 1 [Bloomington stock center (BL) #26504], Orco 2 (BL #23130), wtrw 2 (BL #59038), Trp MB03672 (BL #23636), Trpl MB10553 (BL #29134), Trpγ G4 (BL # 64313), Orco-GAL4 (BL #26818), UAS-dicer2 (BL #24650), UAS-TrpA1 RNAi (BL #36780), UAS-Kir2.1::GFP (Drosophila Genetic Resource Center, Kyoto Institute of Technology #108846), UAS-mCD8::GFP (BL #5137 and #32195). The following stocks were provided by the indicated researchers: TrpA1-T2A-GAL4, TrpA1-KO, TrpA1-AKI-T2A-GAL4, TrpA1-BKI-T2A-GAL4, TrpA1-CKI-T2A-GAL4, TrpA1-DKI-T2A-GAL4, TrpA1-EKI-T2A-GAL4, and lexAop2-mCherry; TrpA1-T2A-LexA (Dr. Y. Xiang) (Gu et al., 2019), Gr66a-GAL4 (Dr. H. Amlein), ppk-GAL4 B-3207d (Dr. D.N. Cox), and pain 4 (Dr. C. Montell) (Liu et al., 2023). UAS-dicer2, UAS-TrpA1 RNAi, UAS-mCD8::GFP, TrpA1-T2A-GAL4, TrpA1-KO, TrpA1-KI-T2A-GAL4, and lexAop2-mCherry; all lines except the TrpA1-T2A-LexA line have a CS genetic background. 1118 The flies were backcrossed to the wild-type flies for five generations.

[0103] [Chemical substances] The following chemicals were purchased from Tokyo Chemical Industry Co., Ltd.: quinine hydrochloride dihydrate (#Q0030), 2-methylthiazoline (2MT; #M0285), 2-methyl-4-ethylthiazoline (4E2MT; #M0689), thiomorpholine (TMO; #T1007), and 2-methyl-2-oxazoline (2MO; #M0857). N-methylmaleimide (NMM; #389412) and citronellal (#373753) were purchased from Sigma-Aldrich. Quinine was dissolved in water and stored at -20 °C. 2MT, TMO, and 2MO were dissolved in water immediately before use in behavioral assays. 4E2MT was dissolved in dimethyl sulfoxide (DMSO) immediately before use in behavioral assays. 2MT and 4E2MT were dissolved in the bath solution and sonicated for 1 minute in an ultrasonic cleaner (#MCS-2P, AS ONE) immediately before use (see below for the composition of the bath solution). NMM was dissolved in DMSO and stored at -20°C.

[0104] [Two-choice positional preference assay] Assays were performed following a similar protocol as previously described (Sanchez-Alcaniz et al., 2017). Male flies (3–8 days old) were used for all behavioral assays. A total of 50 adult males were collected and maintained in vials containing regular food until the starvation treatment began. To induce starvation, flies were fasted in vials containing 1 ml of 1% agarose for 24 h prior to the assay. Non-starved flies were transferred to vials containing food 24 h prior to the experiment.

[0105] The assay plate consisted of a 100 mm diameter, quadrant-sectioned Petri dish (#25384-308, VWR) and a custom-made, quadrant-sectioned acrylic block. An acrylic block was fitted into each quadrant of the Petri dish, and a small amount of 1% agarose was added to fill the gaps. After the agarose solidified, the surface of the acrylic block in each quadrant was covered with 0.5% agarose containing the drug or vehicle (1.2 ml per quadrant). Unless otherwise noted, all areas contained 2 mM sucrose. The test drug was alternately present or absent within each quadrant. When chemicals were dissolved in DMSO, the final DMSO concentration was 0.1%. Flies in vials were gently anesthetized on ice for up to 70 seconds and immediately transferred to the assay plate. The assay plate was then capped and placed upside down on the acrylic stage, allowing flies to contact the agarose by negative taxis. The acrylic stage was placed 5 cm above the LED tracing platform (6800 lux; SV531A, Shinkosha), and the LED light was covered with red film (#105, LEE Filters). The assay chamber was covered with a light-blocking curtain. Still images of the assay plate were taken from above every minute using a digital camera (FDR-AX60, Sony) for up to 120 minutes. All behavioral experiments began in the morning [zeitgeber time (ZT) 1.5-2]. A schematic diagram of the setup is shown in Supplementary Figure 1.

[0106] The number of flies in each area was quantified using a custom ImageJ macro (Sanchez-Alcaniz et al., 2017). The preference index (PI) was calculated using the following formula: PI (%) = (N chemical -N control ) / (N chemical +N control ) × 100, where N chemical and N control represents the number of flies in the chemical-containing and control areas, respectively. PI was calculated over time using a 21-minute moving average.

[0107] [Egg-laying assay] Four- to eight-day-old female flies were used. A total of 30 to 40 adult females and 10 adult males were collected and reared in vials containing standard diet. Four days before the assay, flies were transferred to food vials containing yeast paste. Assay plates were prepared using the same procedure as for the two-alternative positional preference assay described above. To increase egg production, the surface of an acrylic block was covered with soft agarose (0.25%) containing 10 mM sucrose. Five to eight female flies were gently transferred to each assay plate using an aspirator. The assay plates were then placed in a dark box lined with water-soaked paper. Females were allowed to lay eggs from morning (ZT 2-2.5) to evening (ZT 8-8.5) at 25°C.

[0108] The number of eggs in each area was counted manually. Oviposition index (OI) was calculated using the following formula: OI (%) = (E chemical -E control ) / (E chemical + E control ) × 100, where E chemical and E control represents the number of eggs in the chemical-containing area and the control area, respectively. Trials with a total egg count of less than 20 were excluded from the analysis.

[0109] [Measurement of exercise volume] Assay plates were prepared using a 96-well microplate (#1-1601-02, Violamo). Each well was filled with 180 μl of 1% agarose (with or without drug). Once the agarose solidified, the plate was sealed with a plate seal (#547-SBS-PET, Watson), and a cross was cut into each well. Male flies (3–8 days old) were individually transferred into each well using an aspirator. Flies were allowed to behave freely within the wells and recorded in the assay chamber using a digital camera (FDR-AX60, Sony) in the same manner as in the two-choice place choice assay described above. After placing the assay plate in the chamber, recordings were made for 10 minutes at a frame rate of 30 frames per second. All behavioral experiments were performed in the afternoon (ZT 5.5–6.5). The total distance traveled (mm) as an index of locomotor activity ( Sakai et al., 2009 ) was calculated using the video tracking software Move-tr / 2D (version 8.4, Library).

[0110] [Confocal microscope] To observe the expression patterns of TrpA1 isoforms, UAS-mCD8::GFP was crossed with TrpA1-T2A-GAL4, TrpA1-AKI-T2A-GAL4, TrpA1-BKI-T2A-GAL4, TrpA1-CKI-T2A-GAL4, TrpA1-DKI-T2A-GAL4, Gr66a-GAL4, and ppk-GAL4 B-3207d. To observe the coexpression of TrpA1 with Gr66a-GAL4 and ppk-GAL4, lexAop2-mCherry;TrpA1-T2A-LexA was crossed with Gr66a-GAL4;UAS-mCD8::GFP and UAS-mCD8::GFP;ppk-GAL4. Forelegs, middle legs, hind legs, and proboscises were dissected from male flies (4-10 days old) in phosphate-buffered saline (PBS) and mounted in 50% glycerol / PBS. Fluorescent and bright-field images of GFP and mCherry were acquired using a confocal laser scanning microscope (FV1200, Olympus) with a 30x / 1.05 UPLSAPO30X SIR dry objective. Z-sections were taken at 1 μm intervals. Images were analyzed using FLUOVIEW (version 4.2c, Olympus) and Fiji software (Schindelin et al., 2012).

[0111] [Cloning] Cloning of the TrpA1-C and TrpA1-D isoforms required the use of w 1118 RT-PCR was performed using total RNA extracted from third-instar larvae of the genus Pseudomonas aeruginosa according to the manufacturer's protocol. 20A 13 μl reaction premix containing 10 mM dNTP mix, 5 mg of total RNA in DEPC-treated water was prepared in a 0.2 ml PCR tube. This premix was incubated at 65°C for 5 minutes and then cooled on ice for at least 1 minute. The incubated reaction premix was then combined with 7 μl of a mix containing 4 μl of 5X SuperScript IV RT buffer, 1 μl of 100 mM DTT, 1 μl of SuperScript IV Reverse Transcriptase (#18090010, Invitrogen), and 1 μl of RNaseOUT Recombinant Ribonuclease Inhibitor (#10777019, Invitrogen). The reaction was incubated at 55°C for 10 minutes, followed by 80°C for 10 minutes. The reverse transcription product was stored at -20°C until use.

[0112] PCR was performed to amplify TrpA1-C and TrpA1-D cDNAs using a common forward primer containing a KOZAC sequence upstream of the start codon and a common reverse primer (Supplementary Table 1). Phusion High-Fidelity DNA Polymerase (#M0530S, Invitrogen) was mixed with 2 μl (~500 ng) of undiluted reverse-transcribed product in a 50 μl reaction. The PCR protocol consisted of an initial denaturation at 98°C for 30 s, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 66°C for 30 s, and extension at 72°C for 2 min 40 s. A final extension was performed at 72°C for 2 min. The PCR product and pAc5.1-V5-His A vector (#V411020, Invitrogen) were digested with NotI-HF (#R3189S, New England Biolabs (NEB)) and XbaI (#R0145S, NEB). The digested PCR product and vector were ligated using Ligation High Ver. 2 (#LGK-201, Toyobo) at 16°C for 30 minutes and transformed into DH5α competent cells. The E. coli culture was placed in a 10 cm Petri dish (#SH90-15, IWAKI) and plated on LB agar medium (#20067-85, Nacalai Tesque) containing 100 μg / mL ampicillin (#016-23301, Wako) and cultured overnight at 37°C. Colonies were inoculated into Plasgrow II (#08202-75, Nacalai Tesque) containing 100 μg / mL ampicillin and grown overnight in a shaker at 37°C. Plasmids were extracted using NucleoSpin Plasmid EasyPure (#U0727C, Takara Bio). The presence of dTrpA1-C or dTrpA1-D in the constructs was confirmed by digestion with Ssp I-HF (#R3132S, NEB). The following fragments were obtained: TrpA1-C / pAc5.1-V5-His A: 4063, 2751, 992, 637, 321, 284 bp; TrpA1-D / pAc5.1-V5-His A: 6817, 992, 637, 321, 284 bp. All sequences were confirmed (Supplementary Figure 14).Plasmids were extracted using NucleoBond Xtra Midi (#U0410B, Macherey-Nagel) and stored at -20°C.

[0113] [Site-directed mutagenesis] PCR was performed to replace cysteine ​​at positions 480 and / or 729 of wild-type TrpA1-C / pAc5.1-V5-His A or TrpA1-D / pAc5.1-V5-His A with serine. The cysteine ​​codons in the target regions were replaced with serine using a pair of overlapping oligonucleotides containing the specified mutations. The synthetic oligonucleotide primers used to introduce the mutations are listed in Supplementary Table 2.

[0114] A two-step PCR reaction was performed using Phusion High-Fidelity DNA Polymerase (#M0530S, NEB). The reaction premix contained 200 μM dNTPs, 10 μM forward and reverse oligonucleotides, 200 pg template DNA, 5X Phusion buffer, and 0.2 units of Phusion High-Fidelity DNA Polymerase dissolved in MilliQ water. The PCR protocol consisted of an initial denaturation step at 98°C for 30 seconds, followed by 25 cycles of denaturation at 98°C for 10 seconds and annealing / extension at 72°C for 3 minutes. A final extension was performed at 72°C for 5 minutes. The amplified plasmid was demethylated with DpnI at 37°C for 1 hour, transformed into DH5α E. coli, and grown overnight. The resulting colonies were purified using Nucleobond Xtra Midi (740410.50, Macharey-Nagel) and used for subsequent experiments.

[0115] [Calcium imaging] Schneider 2 R+ (S2R+) cells were cultured in 60-mm dishes (#353002, Falcon) at 25°C in 4 ml of Schneider's medium (#21720-024, Gibco) containing 10% inactivated fetal bovine serum (#10437-028, Gibco) and 50 mg / mL penicillin / 50 units / mL streptomycin (#15140-122, Gibco). For transient transfection, a mixture of 1 μg of pAc5.1-V5-His expression vector containing TrpA1-C, TrpA1-D, or each isoform mutant and 0.1 μg of DsRed / pAc5.1-V5-His was prepared at a 10:1 ratio using X-tremeGENE9 Transfection Reagent (#06365787001, Roche) in 1X OPTI-MEM medium (#31985-070, Thermo Fisher Scientific) according to the manufacturer's protocol. The transfection mix was incubated at room temperature for 15 minutes. S2R+ cells were replated onto 12-mm coverslips (Matsunami Glass) in a 35-mm dish (#1000-035, IWAKI) containing 2 ml of medium, and the transfection mix was added to the medium. The cells were cultured for 48 hours in a 25°C incubator. To transfect cells with Fura-2 AM, 1 mL of the following mixture was added to the culture medium and incubated for 1–3 hours in a 25°C incubator: 5 μM Fura-2 AM (#F-1201, Life Technologies), 1 mM probenecid (#162-26112, Wako), and 0.02% pluronic F-127 surfactant (#P2443, Sigma-Aldrich). Coverslips were placed in a chamber (#RC-26G; Warner Instruments) filled with bath solution [130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 30 mM sucrose, and 10 mM N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)], pH adjusted to 7.2 with NaOH. 500 μM probenecid was dissolved in the bath solution to retain Fura-2 intracellularly.The chamber was connected to a gravity flow system to administer various drug stimuli. A xenon lamp was used as the illumination source. To obtain the fluorescence intensity of calcium-bound and calcium-free Fura-2, cells were excited at 340 and 380 nm, respectively, and 510 nm fluorescence was monitored using a CCD camera, CoolSNAP DYNO (Photometrics), connected to a fluorescence microscope (#TE-300, Nikon). Data were analyzed using a NIS element AR (Nikon).

[0116] DsRed-positive cells were analyzed, and traces of each cell were obtained using the NIS element AR. 2+ i The change in Ca2 + i response = (F res -F min ) / (F max -F min ) To standardize the responses, responses were recorded every 3 seconds (F res ) and the maximum value (F max ), to the minimum value of the basal phase (F min After normalization, the Ca concentration during the stimulation period was subtracted for further analysis. 2+ i The maximum increase in Ca 2+ i max) was extracted.

[0117] [statistics] Data are presented as moving means ± SEM or means ± SEM. The number of experiments performed (N) is indicated in the figure legends. Data normality was assessed using the Shapiro-Wilk test. If data were normally distributed, pairwise comparisons were performed using Student's t test or Welch's t test, and one-way analysis of variance (ANOVA) with Dunnett's or Tukey's post hoc analysis was performed for multiple pairwise comparisons. For non-normally distributed data, pairwise comparisons were performed using the Mann-Whitney U test, and multiple pairwise comparisons were performed using nonparametric ANOVA (Kruskal-Wallis test) with post hoc analysis using the Steel or Steel-Dwass test. EZR (version 1.61; Saitama Medical Center, Jichi Medical University), a graphical user interface for R (The R Foundation for Statistical Computing) (Kanda, 2013), was used for all statistical analyses. Statistical significance was indicated by asterisks, *P < 0.05, **P < 0.01, ***P < 0.001. NS indicates not significant. [Explanation of symbols]

[0118] 1a, 1b, 1c, 1d...section, 3...Drosophila melanogaster, 10...assay plate, 11...four-section Petri dish, 12...agarose layer, 20...tracing table, 21...light-emitting diode (LED) light, 22...red film, 23...stage, 25...digital camera 25.

Claims

1. An insect repellent comprising one or more insect repellent compounds selected from the compounds represented by the following general formulas (1) to (3): 【Chemical 1】 (In formulas (1) to (3), R １ and R ２ are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 6 carbon atoms, or an alkylthio group having 1 to 5 carbon atoms.

2. R in the formulas (1) to (3) １ 2. The insect repellent of claim 1, wherein is a methyl group or an ethyl group.

3. 2. The insect repellent according to claim 1, wherein the insect repellent compound is a compound represented by formula (1).

4. A method for repelling insects, comprising diffusing the insect repellent compound contained in the insect repellent according to any one of claims 1 to 3 into a space where insects are desired to be repelled.

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