Application of cis-9-tricosene as murine repellent or in preparation of murine repellent

Abstract

The invention provides an application of cis-9-tricosene as a mouse repellent or in preparation of the mouse repellent. According to the present invention, the dopaminergic pathway of a ventral capped area (VTA)-medial olfactory tuberous nodule (mOT) in a mouse can coordinate the odor guiding behavior by adjusting the dopamines D1 and D2 spinous projective neurons (D1and D2 spinous projective neurons, SPNs) in the mOT, and the D2SPNs is mainly used for adjusting the avoidance of (Z)-9-TE), such that the dopaminergic pathway can coordinate the odor guiding behavior by adjusting the dopamines D1 and D2 spinous projective neurons (D1and D2 spinous projective neurons (SPNs) in the mOT, and the D2SPNs can coordinate the odor guiding behavior by adjusting the dopes D1 and D2 spinous projective neurons (SPNs) in the mOT. Therefore, the cis-9-tricosene can be used as the mouse repellent or used for preparing the mouse repellent. The cis-9-tricosene is prepared into the rodent repellent, so that rodents can be efficiently and accurately prevented and treated, adverse influence on human beings or the environment is avoided, and the method is a novel efficient, safe, green and environment-friendly rodent pest prevention and treatment way.

Description

[0001] This application claims to have access to the manuscript submitted by the Institute of Zoology, Chinese Academy of Sciences on May 20, 2025, entitled "..." The Chinese patent application number for the application of linoleic acid / cis-9-tetratriene in influencing the orientation behavior of rodents is [file number missing]. Priority is given to 202510649376.3, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of rodent control, specifically involving cis-9-tetratriene as a rodent repellent or its application in the preparation of rodent repellents. Background Technology

[0003] Rodents are considered a major global pest, primarily due to their disruptive impact on agriculture, disease transmission, and negative influence on ecosystems. Rodents not only steal crops but also gnaw on various items, including electrical wires, pipes, and furniture, causing economic losses. Furthermore, their feces and urine can transmit diseases such as plague, hemorrhagic fever, and Lyme disease, posing a serious threat to human health.

[0004] These adverse effects have prompted continuous efforts to trap, repel, or poison rodents. Current rodent control measures mainly include three aspects: physical control, chemical control, and environmental management. Physical control involves using rodent traps such as mousetraps, glue boards, and cages to capture rodents. These tools are simple and easy to implement, suitable for small-scale rodent control. Alternatively, electronic rodent repellents can be used, which utilize high-frequency sound to interfere with the rodent's auditory system, causing them to stay away from specific areas. This method is non-toxic and harmless, suitable for use in homes and offices. Chemical control also includes rodenticides and poison baits, typically using anticoagulant, slow-acting rodenticides such as bromadiolone and difenoconazole. Poison baits can also be placed in areas where rodents frequently appear, such as near rodent burrows or in kitchen corners.

[0005] However, existing methods generally suffer from low efficiency, environmental pollution, or insufficient safety, thus necessitating the development of a highly efficient, safe, green, environmentally friendly, and non-target organism-safe rodent control technology. It is noteworthy that insects, as natural prey for rodents, especially wild rodents, possess surface compounds such as linoleic acid and (Z)-9-tricosene, which have potential roles in wild rodent control. Currently, there is no relevant research or practical experience. Although these two substances have been confirmed as pheromone components in insects, their potential attraction (trapping) or repellency effects on rodents have not been reported, and there are no precedents for their application in eco-friendly rodent control. Summary of the Invention

[0006] One of the objectives of this invention is to provide a new use for cis-9-tetratriene.

[0007] The novel use of cis-9-tetratriene provided by this invention is the application of cis-9-tetratriene or a mixture with cis-9-tetratriene as the main component in influencing the homing behavior of rodents.

[0008] Specifically, cis-9-tetratriene ((Z)-9-TE) or mixtures with cis-9-tetratriene as the main component are used as rodent repellents or in the preparation of rodent repellents.

[0009] Animal experiments have shown that mice exhibit avoidance behavior toward cis-9-tetratriene ((Z)-9-TE).

[0010] More specifically, the VTA-mOT dopaminergic pathway in mice coordinates odor-guided behavior by regulating D1 and D2 SPNs in mOT. D1 SPNs mainly mediate preference for LA, while D2 SPNs mainly regulate avoidance of (Z)-9-TE.

[0011] The present invention also provides a rodent repellent.

[0012] The rodent repellent provided by the present invention comprises cis-9-tetratriene ((Z)-9-TE) and excipients.

[0013] The present invention also provides a rodent attracting device, the rodent attracting device comprising an attracting device body, wherein the rodent attractant is placed in the attracting device body.

[0014] The present invention also provides a rodent repelling device, the rodent repelling device comprising a repelling device body, wherein the rodent repellent is placed in the repelling device body.

[0015] Animal experiments of this invention show that mice prefer satiated larvae to starved larvae because satiated larvae contain relatively high levels of linoleic acid (LA) and low levels of cis-9-tetratriene ((Z)-9-TE). Under physiological conditions, the VTA-mOT dopaminergic pathway responds to dopamine released from the ventral tegmental area by regulating D1 and D2 SPNs in the mOT. The difference in the degree of dopamine response between D1 and D2 SPNs creates an unbalanced "seesaw" state, leading to a preference for linoleic acid and an avoidance behavior towards cis-9-tetratriene in mice. Therefore, using linoleic acid as a rodent attractant or cis-9-tetratriene as a rodent repellent can effectively control rodents without causing adverse effects on humans or the environment, representing a novel, efficient, safe, and environmentally friendly rodent control strategy. Attached Figure Description

[0016] Figure 1Mice's preference for satiated bollworm larvae is not affected by their feeding status. (A), (B), (C) Behavioral diagrams and trajectory diagrams; (D) Comparison of the amount of satiated and starved bollworm larvae consumed by non-fasting mice; (E) Percentage of non-fasting mice consuming satiated or starved bollworm larvae; (F) Number of non-fasting mice selectively consuming satiated or starved bollworm larvae; (G) Percentage of non-fasting mice selectively consuming satiated or starved bollworm larvae; (H) Time spent by non-fasting mice around satiated or starved bollworm larvae; (I) Time spent by non-fasting mice around satiated or starved bollworm larvae. (J) Distance traveled by fasted or starved mice around bollworm larvae; (K) Percentage of fasted mice that consumed saturated or starved bollworm larvae; (L) Number of fasted mice that selectively consumed saturated or starved bollworm larvae; (M) Percentage of fasted mice that selectively consumed saturated or starved bollworm larvae; (N) Time spent by fasted mice around saturated or starved bollworm larvae; (O) Distance traveled by fasted mice around saturated or starved bollworm larvae. Data are presented as mean ± standard error. *P<0.05, ***P<0.001, ****P<0.0001.

[0017] Figure 2 The effect of visual information on mouse preference for bollworm larvae: (A), (B), (G) Behavioral diagrams and trajectory maps; (C) Time spent by non-fasting mice around satiated or starved bollworm larvae; (D) Total time spent by non-fasting mice on one side of satiated or starved bollworm larvae; (E) Distance moved by non-fasting mice around satiated or starved bollworm larvae; (F) Total distance moved by non-fasting mice on one side of satiated or starved bollworm larvae; (H) Time spent by non-fasting mice around satiated or starved bollworm larvae; (I) Total time spent by non-fasting mice on one side of satiated or starved bollworm larvae; (J) Distance moved by non-fasting mice around satiated or starved bollworm larvae; (K) Total distance moved by non-fasting mice on one side of satiated or starved bollworm larvae. *P<0.05, **P<0.001.

[0018] Figure 3The effect of the dominant olfactory system of mice on the preference for satiated or starved bollworm larvae. (A), (B), (C) Behavioral diagrams and trajectory maps; (D) Time spent by non-fasted mice treated with saline around satiated or starved bollworm larvae; (E) Distance moved by non-fasted mice treated with saline around satiated or starved bollworm larvae; (F) Time spent by non-fasted mice treated with methimazole around satiated or starved bollworm larvae; (G) Distance moved by non-fasted mice treated with methimazole around satiated or starved bollworm larvae; (H) Time spent by fasted mice treated with saline around satiated or starved bollworm larvae; (I) Distance moved by fasted mice treated with saline around satiated or starved bollworm larvae; (J) Time spent by fasted mice treated with methimazole around satiated or starved bollworm larvae; (K) Distance moved by fasted mice treated with methimazole around satiated or starved bollworm larvae. *P<0.05, **P<0.01, ns, no significant difference.

[0019] Figure 4 The effect of feed residue odor on the preference of mice for feeding on bollworm larvae. (A), (B), (E) Behavioral diagrams and trajectory diagrams; (C) Time spent by non-fasting mice around the experimental or control groups; (D) Distance moved by non-fasting mice around the experimental or control groups; (F) Time spent by fasting mice around the experimental or control groups; (G) Distance moved by fasting mice around the experimental or control groups. No significant difference was found in the values ​​of ns.

[0020] Figure 5 The effect of bollworm larvae excrement on mouse preference for satiated bollworm larvae. (A), (B), (E) Behavioral diagrams and trajectory diagrams; (C) Time spent by non-fasting mice around satiated or starved bollworm larvae excrement; (D) Distance traveled by non-fasting mice around satiated or starved bollworm larvae excrement; (F) Time spent by fasting mice around satiated or starved bollworm larvae excrement; (G) Distance traveled by fasting mice around satiated or starved bollworm larvae excrement. No significant difference was observed in ns.

[0021] Figure 6Analysis of compounds on the surface of cotton bollworm larvae under saturation or starvation treatment using gas chromatography-mass spectrometry (GC-MS). (A) Representative chromatograms showing significant differences in compounds. Arrows indicate compounds that showed significant differences between saturated and starved cotton bollworm larvae at the corresponding retention time points. (B) Comparison of the relative abundance of compounds on the surface of saturated and starved cotton bollworm larvae. The experiment was repeated six times, with twenty larvae in each of the fed and unfed groups. Data are presented as mean ± standard error. *P<0.05, **P<0.01. Bar charts without statistical symbols indicate no significant difference between the two groups.

[0022] Figure 7 Comparison of compounds on the surface of cotton bollworm larvae that showed significant differences after being fed or starved. (A) Relative content of linoleic acid on the surface of cotton bollworm larvae after being fed or starved; (B) Relative content of (Z)-9-tricosene on the surface of cotton bollworm larvae after being fed or starved; (C) Relative content of 3,5-di-tert-butylphenol on the surface of cotton bollworm larvae after being fed or starved; (D) Relative content of heptacosane on the surface of cotton bollworm larvae after being fed or starved; (E) Relative content of pentacosane on the surface of cotton bollworm larvae after being fed or starved; (F) Relative content of 3-ethyl-5-(2-ethylbutyl)octadecane on the surface of cotton bollworm larvae after being fed or starved. *P<0.05, **P<0.01.

[0023] Figure 8Mice exhibited a preference for linoleic acid and avoidance behavior towards cis-9-dococtitriene. (A), (B), (C) Behavioral diagrams and trajectory diagrams; (D) Time spent by unfasted mice around mineral oil and linoleic acid; (E) Distance traveled by unfasted mice around mineral oil and linoleic acid; (F) Time spent by fasted mice around mineral oil and linoleic acid; (G) Distance traveled by fasted mice around mineral oil and linoleic acid; (H) Time spent by unfasted mice around mineral oil and cis-9-dococtitriene; (I) Distance traveled by unfasted mice around mineral oil and cis-9-dococtitriene; (J) Time spent by fasted mice around mineral oil and cis-9-dococtitriene; (K) Distance traveled by fasted mice around mineral oil and cis-9-dococtitriene. (L) Time spent in the vicinity of linoleic acid and cis-9-dococtitriene in unfasted mice; (M) Distance traveled in the vicinity of linoleic acid and cis-9-dococtitriene in unfasted mice; (N) Time spent in the vicinity of linoleic acid and cis-9-dococtitriene in fasted mice; (O) Distance traveled in the vicinity of linoleic acid and cis-9-dococtitriene in fasted mice. *P<0.05, **P<0.01, ns not significant.

[0024] Figure 9 Unfasted mice did not exhibit significant preference or avoidance behaviors towards 3,5-di-tert-butylphenol, pentadecane, heptadecane, or oleic acid. (A) Time spent by unfasted mice around mineral oil and 3,5-di-tert-butylphenol; (B) Distance traveled by unfasted mice around mineral oil and 3,5-di-tert-butylphenol; (C) Time spent by unfasted mice around mineral oil and n-pentadecane; (D) Distance traveled by unfasted mice around mineral oil and n-pentadecane; (E) Time spent by unfasted mice around mineral oil and n-heptadecane; (F) Distance traveled by unfasted mice around mineral oil and n-heptadecane; (G) Time spent by fasted mice around mineral oil and oleic acid; (H) Distance traveled by unfasted mice around mineral oil and oleic acid. No significant differences were observed in the data (ns).

[0025] Figure 10The preferences of fasted and non-fasted mice for different proportions of linoleic acid and cis-9-docosahexaene mixtures. (A), (B), (C) Behavioral diagrams; (D) Time spent by mice around a mixture of linoleic acid and cis-9-docosahexaene with linoleic acid as the predominant component and mineral oil; (E) Distance of mice around a mixture of linoleic acid and cis-9-docosahexaene with linoleic acid as the predominant component and mineral oil; (F) Time spent by mice around a mixture of linoleic acid and cis-9-docosahexaene with cis-9-docosahexaene as the predominant component and mineral oil; (G) Preferences of mice around a mixture of linoleic acid and cis-9-docosahexaene with cis-9-docosahexaene as the predominant component. (H) The distance a mouse moves around a mixture of linoleic acid and cis-9-docosahexaene with a linoleic acid majority and a mixture of linoleic acid and cis-9-docosahexaene with a cis-9-docosahexaene majority; (I) The distance a mouse moves around a mixture of linoleic acid and cis-9-docosahexaene with a linoleic acid majority and a mixture of linoleic acid and cis-9-docosahexaene with a cis-9-docosahexaene majority.

[0026] Figure 11 Brandt's voles and brown rats that were not fasted showed a preference for linoleic acid and an avoidance behavior towards cis-9-dococtitriene. (A), (B), (C) Behavioral diagrams; (D) Time Brandt's voles spent around mineral oil and linoleic acid; (E) Distance Brandt's voles moved around mineral oil and linoleic acid; (F) Time Brown rats spent around mineral oil and linoleic acid; (G) Distance Brown rats moved around mineral oil and linoleic acid; (H) Time Brandt's voles spent around mineral oil and cis-9-dococtitriene; (I) Distance Brandt's voles moved around mineral oil and cis-9-dococtitriene; (J) Time Brown rats spent around mineral oil and cis-9-dococtitriene; (K) Distance Brown rats moved around mineral oil and cis-9-dococtitriene. (L) Time spent by Brandt's voles around linoleic acid and cis-9-dococtitriene; (M) Distance traveled by Brandt's voles around linoleic acid and cis-9-dococtitriene; (N) Time spent by brown rats around linoleic acid and cis-9-dococtitriene; (O) Distance traveled by brown rats around linoleic acid and cis-9-dococtitriene. *P<0.05, **P<0.01, ***P<0.001.

[0027] Figure 12Chemogenetic manipulation of the VTA-mOT dopaminergic pathway altered the preference of non-fasted mice for linoleic acid (LAA) over cis-9-dococtiformin (CDR). (A) Schematic diagram of chemogenetic inhibitory virus injection, cannula implantation, and administration strategies; (B) Bidirectional odor preference test behavioral paradigm (top) and representative movement trajectories (middle and bottom); (C) Time spent around LAA and CDR in saline-treated hM4Di (inhibitory DREADD) mice; (D) Distance traversed around LAA and CDR in saline-treated hM4Di (inhibitory DREADD) mice; (E) Time spent around LAA and CDR in CNO-treated hM4Di (inhibitory DREADD) mice; (F) Distance traversed around LAA and CDR in CNO-treated hM4Di (inhibitory DREADD) mice; (G) (H) Schematic diagrams of viral injection, cannula implantation, and drug delivery strategies for chemogenetic activation; (I) Behavioral paradigms of the bidirectional odor preference test (top) and representative movement trajectories (middle and bottom); (II) Time spent by saline-treated hM3Dq-expressing mice around linoleic acid and cis-9-dococtitriene; (III) Distance moved by saline-treated hM3Dq-expressing mice around linoleic acid and cis-9-dococtitriene; (IV) Time spent by CNO-treated hM3Dq-expressing mice around linoleic acid and cis-9-dococtitriene; (V) Distance moved by CNO-treated hM3Dq-expressing mice around linoleic acid and cis-9-dococtitriene. Data are presented as mean ± standard error. *P < 0.05, **P < 0.01. No significant difference was observed within ns.

[0028] Figure 13Chemogenetic activation of the VTA-mOT dopaminergic pathway eliminated the preference for linoleic acid and avoidance behavior towards cis-9-docosahexaene in fasted mice. (A) Time spent around linoleic acid and mineral oil in mice expressing hM3Dq (activating DREADD) treated with saline or CNO; (B) Distance traversed around linoleic acid and mineral oil in mice expressing hM3Dq (activating DREADD) treated with saline or CNO; (C) Time spent around mineral oil and cis-9-docosahexaene in mice expressing hM3Dq (activating DREADD) treated with saline or CNO; (D) Distance traversed around mineral oil and cis-9-docosahexaene in mice expressing hM3Dq (activating DREADD) treated with saline. Data are expressed as mean ± standard error. *P < 0.05, **P < 0.01. No significant difference was observed within ns.

[0029] Figure 14 Chemogenetic manipulation of the VTA-mOT pathway did not affect the preference for linoleic acid or the avoidance behavior of cis-9-tetratriene in non-fasted mice expressing mCherry. (A) Schematic diagram of virus injection, cannula implantation, and drug administration strategy for control group mCherry mice; (B) Schematic diagram of odor preference test behavioral paradigm; (C) Time spent by mCherry-expressing mice treated with saline or CNO around mineral oil and linoleic acid; (D) Distance moved by mCherry-expressing mice treated with saline or CNO around mineral oil and linoleic acid; (E) Time spent by mCherry-expressing mice treated with saline or CNO around mineral oil and cis-9-dococtitriene; (F) Distance moved by mCherry-expressing mice treated with saline or CNO around mineral oil and cis-9-dococtitriene; (G) Time spent by mCherry-expressing mice treated with saline or CNO around linoleic acid and cis-9-dococtitriene; (H) Distance moved by mCherry-expressing mice (control group) treated with saline or CNO around linoleic acid and cis-9-dococtitriene. Data are expressed as mean ± standard error. *P < 0.05, **P < 0.01. There was no significant difference in ns.

[0030] Figure 15Chemically manipulate the VTA-mOT dopaminergic pathway to alter the initial preference and avoidance behavior of unfasted mice towards cotton bollworm larvae. (A) Time spent by mice expressing hM4Di after saline treatment around fed or starved bollworm larvae; (B) Distance moved by mice expressing hM4Di after saline treatment around fed or starved bollworm larvae; (C) Time spent by mice expressing hM3Dq after saline treatment around fed or starved bollworm larvae; (D) Distance moved by mice expressing hM3Dq after saline treatment around fed or starved bollworm larvae; (E) Time spent by mice expressing hM4Di after CNO treatment around fed or starved bollworm larvae; (F) Distance moved by mice expressing hM4Di after CNO treatment around fed or starved bollworm larvae; (G) Time spent by mice expressing hM3Dq after CNO treatment around fed or starved bollworm larvae; (H) Distance moved by mice expressing hM3Dq after CNO treatment around fed or starved bollworm larvae. Data are expressed as mean ± standard error. *P < 0.05, **P < 0.01. No significant difference was observed within ns.

[0031] Figure 16Chemogenetic manipulation of the VTA-mOT dopaminergic pathway did not affect the olfactory perception and motor abilities of unfasted mice. (A) Immunostaining of enhanced green fluorescent protein (EGFP) and tyrosine hydroxylase (TH, red) in VTA neurons of hM4Di-expressing mice (left), scale bar: 100 μm. Viral transfection efficiency was assessed by counting the proportion of neurons co-expressing hM4Di and TH to the total number of hM4Di-expressing neurons. Quantitative analysis was performed on 9 brain slices from three mice (3 brain slices from each mouse) (right); (B) Immunostaining of red fluorescent protein (mCherry) and tyrosine hydroxylase (TH, pink) in VTA neurons of hM3Dq-expressing mice (left), scale bar: 100 μm. Viral transfection efficiency was assessed by statistically analyzing the proportion of neurons co-expressing hM3Dq and TH to the total number of neurons expressing hM4Di. Nine brain slices from three mice (nine brain slices from each mouse) were quantitatively analyzed (right); (C) Representative electrophysiological recordings of spontaneous firing in acute brain slices containing VTA from hM4Di-expressing mice after CNO administration (top), and neuronal firing frequencies after administration of artificial cerebrospinal fluid or CNO (bottom). Eight cells from three mice were quantified in each group, with membrane clamp potentials of –50 mV; (D) Acute brain slices containing VTA from hM3Dq-expressing mice after CNO administration. Representative electrophysiological recordings of spontaneous discharge after CNO administration (top); neuronal firing frequency after administration of artificial cerebrospinal fluid or CNO (bottom); quantification of 8 cells from 3 mice in each group; membrane clamp potential of –50 mV; (E) Latency of first finding of food particles in mice expressing mCherry (left), hM3Dq (middle), and hM4Di (right) in the buried food search test; 8 mice in each group participated in the experiment; (F) Total distance traveled by mice expressing mCherry (left), hM3Dq (middle), and hM4Di (right) in the bidirectional odor preference test; 8 mice in each group participated in the experiment. Data are expressed as mean ± standard error. **P < 0.01, ns, no significant difference.

[0032] Figure 17In a bidirectional odor preference test, antagonism of D1 and D2 receptors in the medial olfactory tubercle (mOT) mediated the response characteristics of mice to linoleic acid and cis-9-tetratriene. (A) Schematic diagram of mOT local administration strategy; (B, C, D) Schematic diagrams showing behavioral paradigms (top) and representative movement trajectories of non-fasting mice treated with SCH23390 (B, D; bottom) and eticlopride (C; bottom); (E) Odor preference index of linoleic acid dwell time in non-fasting mice after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) into the mOT in a single-compound linoleic acid preference experiment (mineral oil as control); (F) Linoleic acid preference experiment (mineral oil as control). (G) Odor preference index of the distance mice traveled around linoleic acid after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) into the mOT; (H) Odor preference index of the time mice spent around linoleic acid after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) into the mOT; (F) Odor preference index of the distance mice traveled around linoleic acid after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) into the mOT; (F ... Odor preference index of mice after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) at mOT, measuring the distance they moved around linoleic acid; (I) Odor preference index of non-fasted mice after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) at mOT, measuring the distance they moved around cis-9-tetratriene; (J) Odor preference index of non-fasted mice after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) at mOT, measuring the distance they moved around cis-9-tetratriene; In the experiment (mineral oil as the control group), the odor preference index of non-fasted mice after local injection of physiological saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) at mOT was measured by the time spent around cis-9-tetratriene; (K) In the single compound cis-9-tetratriene preference experiment (mineral oil as the control group), the odor preference index of non-fasted mice after local injection of physiological saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) at mOT was measured by the time spent around cis-9-tetratriene.(L) Odor preference index of non-fasted mice after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) in a single compound cis-9-dococtitriene preference experiment (mineral oil as control group), based on their dwell time around cis-9-dococtitriene; (M) Odor preference index of non-fasted mice after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) in a linoleic acid and cis-9-dococtitriene preference experiment, based on their dwell time around the two compounds; (N) Odor preference index of non-fasted mice after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) in a linoleic acid and cis-9-dococtitriene preference experiment, based on their dwell time around the two compounds; Odor preference index of distance traveled around the two compounds after injection of 390 (red bar, middle) and eticlopride (blue bar, right); (O) Odor preference index of time spent around the two compounds after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) in the linoleic acid and cis-9-tetratriene preference experiment; (P) Odor preference index of distance traveled around the two compounds after local injection of saline (black bar, left), SCH23390 (red bar, middle), and eticlopride (blue bar, right) in the linoleic acid and cis-9-tetratriene preference experiment. Two different groups of mice were used in the experiment. n = 11 mice in (EL) and n = 12 mice in (MP). Data are expressed as mean ± standard error. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. No significant difference was observed in ns.

[0033] Figure 18During the bidirectional odor preference test, D1 and D2 type intermediate spinous neurons (SPNs) expressing GCaMP6s in mOT responded to linoleic acid (LA) and cis-9-tetratriene ((Z)-9-TE). (A) Schematic diagram of fiber optic photometric recording experiment of D1 or D2 SPNs in mOT (left), and schematic diagram of viral injection / fiber optic implantation sites in D1-Cre mice (middle) and D2-Cre mice (right). Scale bar: 100 μm; (B) Mean calcium signal response of neurons in D1-Cre mice when smelling linoleic acid or mineral oil (left), heatmap (68 events for linoleic acid, 62 events for mineral oil) shows the calcium signal response induced by a single test in D1 SPNs (middle), as well as the quantification and differential comparison of calcium signal changes (far right, area under the curve (AUC), 0 to 5 seconds; peak ΔF / F); (C) Mean calcium signal response of neurons in D1-Cre mice when smelling cis-9-tetratriene or mineral oil (left), heatmap (77 events for linoleic acid, 71 events for mineral oil) shows the D1 (middle) Calcium signaling response induced by a single test in SPNs, and quantification and differential comparison of calcium signal changes (far right, area under the curve (AUC), 0 to 5 seconds; peak ΔF / F); (D) Average calcium signaling response of neurons in D1-Cre mice when smelling linoleic acid or cis-9-tetratriene (left), heatmap (total number of linoleic acid events: 37, cis-9-tetratriene events: 71) shows the calcium signaling response induced by a single test in D1 SPNs, and quantification and differential comparison of calcium signal changes (far right, area under the curve (AUC), 0 to 5 seconds; peak ΔF / F); (E) Average calcium signaling response of neurons in D2-Cre mice when smelling linoleic acid or mineral oil (left), heatmap (total number of linoleic acid events: 114, mineral oil events: 90) shows the average calcium signaling response of neurons in D2-Cre mice when smelling linoleic acid or mineral oil. Calcium signaling responses induced by a single trial in SPNs (middle), and quantification and differential comparison of calcium signaling changes (far right, area under the curve (AUC), 0 to 5 seconds; peak ΔF / F); (F) Average calcium signaling response of neurons in D2-Cre mice when smelling cis-9-tetratriene or mineral oil (left), heatmap (total number of cis-9-tetratriene events: 87, mineral oil events: 95) showing D2 The calcium signaling response induced by a single test in SPNs (middle), and the quantification and differential comparison of calcium signal changes (far right, area under the curve (AUC), 0 to 5 seconds; peak ΔF / F); the mean calcium signaling response of neurons in (G)D2-Cre mice when smelling linoleic acid or cis-9-dococtitriene (left), heatmap (64 total events for linoleic acid, 69 events for cis-9-dococtitriene) shows the calcium signaling response induced by a single test in D2SPNs (middle), and the quantification and differential comparison of calcium signal changes (far right, area under the curve (AUC), 0 to 5 seconds; peak ΔF / F).Data were collected from 8 D1-Cre mice in the (BD) and 8 D2-Cre mice in the (EG). Data are presented as mean ± standard error. *P < 0.05, **P < 0.01. No significant difference was observed within ns.

[0034] Figure 19 D1 and D2 type intermediate-spinous neurons (SPNs) expressing GCaMP6s in the medial olfactory tubercle (mOT) showed similar responses to 3,5-di-tert-butylphenol, n-pentane, oleic acid, and n-heptadecane. (A) Mean calcium signaling response of neurons in D1-Cre mice upon smelling the compounds (left), heatmap showing the calcium signaling response induced by a single test in D1 SPNs (middle), (B), and quantitative and differential comparison of calcium signaling changes (AUC, 0 to 5 seconds; peak ΔF / F); (C) Mean calcium signaling response of neurons in D2-Cre mice upon smelling the compounds (left), heatmap showing the calcium signaling response induced by a single test in D2 SPNs (middle), (D), and quantitative and differential comparison of calcium signaling changes (AUC, 0 to 5 seconds; peak ΔF / F). Data were collected from 4 D1-Cre and 4 D2-Cre mice. In (A), the number of events for mineral oil, 3,5-di-tert-butylphenol, n-pecapane, oleic acid, and n-heptadecane were 43, 29, 29, 19, and 29, respectively. For D2-Cre mice, the number of events for the corresponding odor analyses in (C) were 45, 36, 29, 25, and 30, respectively. The dashed line indicates the start of olfactory behavior. The shaded area of ​​the curve in the figure represents the standard error (SEM). Data are presented as mean ± standard error. No significant difference was observed in ns.

[0035] Figure 20In the medial olfactory tubercle (mOT), D1 and D2 type intermediate spiny neurons (SPNs) expressing enhanced green fluorescent protein (EGFP) showed no significant response to mineral oil, linoleic acid, cis-9-tetratriene, 3,5-di-tert-butylphenol, n-pentane, oleic acid, and n-heptadecane. (A) Mean calcium signal response map (left) and heatmap (right) of mice when smelling mineral oil (39 events in D1-Cre mice, 39 events in D2-Cre mice) or linoleic acid (42 events in D1-Cre mice, 40 events in D2-Cre mice); (B) Mean calcium signal response map (left) and heatmap (right) of neurons in mice when smelling mineral oil (42 events in D1-Cre mice, 40 events in D2-Cre mice) or cis-9-dococtitriene (45 events in D1-Cre mice, 42 events in D2-Cre mice); (C) Mean calcium signal response map (left) and heatmap (right) of neurons in mice when smelling linoleic acid (33 events in D1-Cre mice, 35 events in D2-Cre mice) or cis-9-dococtitriene (33 events in D1-Cre mice, 35 events in D2-Cre mice). (D) Mean calcium signaling response of neurons (left) and heatmap (right) during 6 events (32 events in D2-Cre mice); (D) Mean calcium signaling response of neurons (left) and heatmap (right) during mice smelling single odors, including mineral oil (24 events in D1-Cre mice, 26 events in D2-Cre mice), 3,5-di-tert-butylphenol (40 events in D1-Cre mice, 41 events in D2-Cre mice), n-pecapane (41 events in D1-Cre mice, 43 events in D2-Cre mice), oleic acid (42 events in D1-Cre mice, 40 events in D2-Cre mice), and n-heptadecane (40 events in D1-Cre mice, 45 events in D2-Cre mice). Dashed lines indicate the start of smelling. The shaded areas of the curves in the figures represent standard errors (SEM). The data came from four D1-Cre mice and four D2-Cre mice.

[0036] Figure 21 A working model of the VTA-mOT dopaminergic pathway coordinating a "seesaw" effect in mice’s preference for fed larvae over starved larvae. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] Example 1: Screening of substances with attractant and repellent activity

[0040] 1) Both non-fasted and fasted mice preferred to be fed bollworm larvae.

[0041] To determine whether mice feed on bollworm larvae under laboratory conditions, we simulated a natural foraging environment using a plastic cage divided into two equal-sized chambers. Twenty larvae, treated with either satiated or starved conditions, were evenly distributed within the cage. Then, mice that were neither starved nor fasted were introduced into the center of the cage. Figure 1 A) The number of larvae consumed by mice within 20 minutes was recorded. Feeding behavior was quantified by measuring the number of larvae consumed and calculating the percentage of larvae consumed, defined as the proportion of larvae consumed relative to the total number of larvae introduced into the cage. Both non-fasted and fasted mice consumed larvae treated with either satiety or starvation, but non-fasted mice showed a clear preference for satiety-treated larvae, consuming a larger quantity and at a higher rate compared to starved larvae. Figure 1 D, E). All fasted mice will consume cotton bollworm larvae ( Figure 1 K), but prefers well-fed larvae (K). Figure 1 J).

[0042] To further investigate whether larval stage affects their feeding preferences in mice, we conducted a two-way selection feeding preference experiment. Twenty larvae, treated with either satiated or starved conditions, were placed diagonally opposite each other in a cage, and the feeding behavior of the mice was assessed. Figure 1 B). Compared to starved larvae, non-starved mice consumed more full-fed larvae and showed a significantly higher percentage of larvae consumed. Figure 1 F, G). Fasted mice also consumed more satiated larvae, and there was no statistically significant difference in the percentage of satiated or starved larvae consumed by non-fasted and fasted mice. Figure 1 These results indicate that both non-fasted and fasted mice preferred well-fed larvae, possibly because they provided better nutritional quality than starved larvae.

[0043] To rule out the potential influence of larval movement on mouse behavior, we fixed the larvae in a petri dish covered with a wire pencil tube. Figure 1 C). Non-fasted mice showed spatial preference for satiated larvae, spending more time and moving a greater distance around the culture dish containing satiated larvae. Figure 1H,I). Similarly, fasted mice showed the same spatial preference for satiated larvae (H,I). Figure 1 These findings suggest that, regardless of satiety, mice prefer well-fed larvae to starved larvae.

[0044] 2) Visual cues are not a necessary condition for mice to prefer feeding on bollworm larvae.

[0045] Next, we explored the sensory mechanisms driving spatial preference in mice towards satiated larvae through a series of experiments. To systematically evaluate the role of visual cues, we designed a two-way preference test system. In the experiment, a cylindrical wire pencil tube was placed over a culture dish containing bollworm larvae—the 5cm diameter tube was double-wrapped in medical gauze. This innovative design ensured the free diffusion of volatile odor molecules while effectively blocking the mice's visual signals to the larvae, and physically preventing the mice from contacting them. See the experimental diagram below (…). Figure 2 A, B, G).

[0046] During the 15-minute observation period, regardless of whether the mice were fasted or not, the time spent around satiated bollworm larvae and the total time spent on one side of satiated bollworm larvae were significantly longer than those spent on starved bollworm larvae. Figure 2 C, D, H, I,). Regardless of whether the mice were fasted or not, the distance they traveled around satiated bollworm larvae and the total distance they traveled to one side of satiated bollworm larvae were significantly greater than those in the starved bollworm larvae treatment. Figure 2 (E, F, J, K). This series of data strongly supports the following conclusion: even in the absence of visual input, mice can still accurately identify and maintain their preference for satiated larvae through other sensory modalities (such as olfaction). This indicates that visual cues are not necessary for mice's spatial preference for satiated larvae.

[0047] 3) The primary olfactory system mediates the preference of mice for satiated bollworm larvae.

[0048] Given the crucial role of smell in foraging, we assess the contribution of the olfactory system ( Figure 3 A specific chemical lesion paradigm was used to functionally analyze the main olfactory pathway. A selective olfactory epithelial injury model was established by intraperitoneal injection of methimazole solution (dissolved in 0.9% saline). The study showed that methimazole solution could selectively ablate olfactory epithelial cells without affecting the morphology of the vomeronasal organ. For the control group, intraperitoneal injection of saline was performed. Behavioral tests showed that the time spent and distance traveled by non-fasted or fasted mice treated with saline were significantly increased compared to those treated with starvation when surrounded by satiated bollworm larvae. Figure 3Conversely, in non-fasted or fasted mice treated with methimazole, there was no significant difference in the time spent or distance traveled around saturated bollworm larvae compared to those treated with starvation. Figure 3 F, G, J, K). Mice treated with saline showed more intensive movement around satiated bollworm larvae, while mice treated with methimazole showed no significant difference in movement patterns around satiated or starved bollworm larvae. Figure 3 (B, C). This indicates that the mouse's primary olfactory system mediates this preference behavior.

[0049] 4) Feed odor is not the main factor mediating mice's preference for feeding on bollworm larvae.

[0050] To identify the specific olfactory signaling molecules mediating mouse preferences, we systematically analyzed potential odor sources in the larval environment. Given that artificial feed formulations contain wheat bran, wheat germ, and tomato paste matrix, we examined the effect of residual feed odors on mice's preference for feeding on bollworm larvae. Figure 4 ).

[0051] In the two-way selection experiment, filter paper soaked in residual feed odor (experimental group) and untreated filter paper (control group) were placed at opposite ends of a symmetrically distributed experimental box. Figure 4 A). The results showed that, regardless of whether the mice were fasted or not, there was no significant difference in the time spent in the vicinity of the experimental group compared to the control group. Figure 4 C, F); there was no significant difference in the distance moved around the experimental group compared to the control group in either the fasted or non-fasted mice. Figure 4 Similarly, there was no significant difference in the movement trajectories of either non-fasted or fasted mice between the experimental and control groups. Figure 4 (B, E). This indicates that the odor of residual feed is not a factor in mice's preference for feeding on bollworm larvae.

[0052] 5) Excrement is not the main factor mediating mice's preference for feeding on bollworm larvae.

[0053] In our experiment, we found a large amount of excrement in the environment where bollworm larvae live. Therefore, we examined the effect of excrement odor on mice's preference for feeding on bollworm larvae. Figure 5 ).

[0054] We collected excrement from both satiated and starved bollworm larvae and stored it in a 4°C refrigerator. In the two-way selection experiment, we placed the excrement from satiated and starved bollworm larvae at opposite ends of a symmetrically distributed experimental chamber. Figure 5A). The results showed that, regardless of whether the mice were not fasted or fasted, there was no significant difference in the time they spent around the excrement of satiated bollworm larvae compared to the excrement of starved bollworm larvae. Figure 5 C, F); there was no significant difference in the distance that non-fasted or fasted mice moved around the excrement of cotton bollworm larvae in the cases of satiated or starved mice. Figure 5 Similarly, there was no significant difference in the movement trajectories of mice, whether they were not fasted or fasted, around the excrement of cotton bollworm larvae under satiated or starved conditions. Figure 5 (B, E). This indicates that the odor of excrement is not a factor in mice's preference for feeding on bollworm larvae.

[0055] 6) GC-MS analysis

[0056] Based on the above experimental results, we used gas chromatography-mass spectrometry (GC-MS) to systematically analyze the volatile compounds on the surface of fourth- and fifth-instar larvae of the cotton bollworm. Three biological replicates were set up, with 20 cotton bollworm larvae in each group. GC-MS analysis identified approximately 30 compounds (…). Figure 6 The amounts of compounds A and B differ between larvae subjected to satiated or starved conditions. Among these, the following compounds show significant differences: linoleic acid, (Z)-9-tricosene, pentacosane, heptacosane, and 3,5-di-tert-butylphenol.

[0057] We conducted a detailed analysis of compounds that showed significant differences in the body surface of bollworm larvae under satiated or starved treatments in GC-MS analysis: linoleic acid (LA, Figure 7 A) 3,5-di-tert-butylphenol Figure 7 C) Pentacosane Figure 7 E) and 3-ethyl-5-(2-ethylbutyl)octadecane, Figure 7 F) was found in significantly higher relative amounts in satiated larvae, while cis-9-tetratriene ((Z)-9-TE) was present. Figure 7 B) and heptacosane (B) Figure 7 D) The phase content was significantly higher in larvae subjected to starvation treatment.

[0058] 7) Bidirectional selection test of mice for linoleic acid and cis-9-tetratriene

[0059] To verify the effects of these compounds on mouse preference behavior, we conducted a two-way odor selection preference test. Figure 8 (A, B, C above). First, we conducted a single-compound preference test for linoleic acid, using mineral oil as a control group. The results showed that, regardless of whether the mice were fasted or not, they spent significantly more time exploring and sniffing around linoleic acid than around mineral oil. Figure 8 Similarly, compared to mineral oil, unfasted mice moved significantly longer distances around linoleic acid. Figure 8 E). Interestingly, the fasted mice did not show a significant difference in the distance they moved around linoleic acid or mineral oil. This suggests that the mice exhibit a strong spatial preference for linoleic acid, which is also evidenced by the trajectory maps of the mice moving around linoleic acid and mineral oil, where the movement trajectories around linoleic acid were more dense (E). Figure 8 A, below). Next, we conducted a single-compound preference test for cis-9-docosahexaene, using mineral oil as a control group. The results showed that, regardless of whether the mice were fasted or not, they spent significantly less time exploring and sniffing around cis-9-docosahexaene than around mineral oil. Figure 8 H,J); similarly, compared to mineral oil, fasted mice exhibited significantly less movement distance around cis-9-tetratriene (H,J); Figure 8 K). Unfasted mice showed no significant difference in the distance they moved around cis-9-dococtitriene or mineral oil. This indicates that the mice exhibited a clear avoidance behavior towards cis-9-dococtitriene, which is also evidenced by their trajectory maps around cis-9-dococtitriene and mineral oil, showing a sparser trajectory around cis-9-dococtitriene. Figure 8 B, below). Based on the preceding GC-MS data, we hypothesize that higher levels of linoleic acid and lower levels of cis-9-docosahexaene in fed bollworm larvae drive mouse preference in a two-way larval feeding test. To verify this, we conducted two odor preference tests during mouse movement, comparing linoleic acid with cis-9-docosahexaene (B). Figure 8 C, upper side). Compared to (Z)-9-TE, mice spent more time around linoleic acid. Figure 8 L, N) and the increase in movement distance ( Figure 8 Correspondingly, the mice exhibited more dense movement patterns around linoleic acid (M, O). Figure 8 (C, below). This indicates that mice have a significantly higher preference for linoleic acid than for cis-9-tetratriene.

[0060] Next, we tested the effects of several other compounds that showed significant differences in GC-MS analysis on mouse preference behavior. First, we performed a single-compound preference test, using mineral oil as a control group. The results showed that, compared to mineral oil, mice showed a greater preference for 3,5-di-tert-butylphenol (…). Figure 9 A,B), n-Petalane ( Figure 9 C,D) or n-hexadecane ( Figure 9 E and F) did not show any preference behavior. Furthermore, we identified oleic acid, a monounsaturated fatty acid structurally similar to the polyunsaturated fatty acid LA. To determine whether the degree of fatty acid saturation affects preference, we tested the mice's response to oleic acid. Mice showed a higher preference for oleic acid than for mineral oil, but the difference was not statistically significant. Figure 9 G,H).

[0061] 8) Regarding the volatile compounds linoleic acid and cis-9-tetratriene exuded on the larval surface, their ratio differed between larvae subjected to satiated and starved treatments. To investigate this, we examined the effect of this difference in ratio on preference behavior in mice. Results are shown below. Figure 10 The results showed that, compared with mineral oil, mice exhibited a preference for a mixture of linoleic acid (LA:(Z)-9-TE = 1.32:1), in which linoleic acid constituted the majority of the mixture. Figure 10 In the D and E groups, no preference was observed for a mixture of linoleic acid and cis-9-tetraterene (LA:(Z)-9-TE = 1:2), in which cis-9-tetraterene was the predominant component. Figure 10 The mixture of linoleic acid and cis-9-docosahexaene (with linoleic acid as the main component) showed a clear preference for the mixture of linoleic acid and cis-9-docosahexaene (with cis-9-docosahexaene as the main component). Figure 10 (H,I).

[0062] 9) Two-way selection test of Brandt's vole and brown rat for linoleic acid and cis-9-tetratriene

[0063] To determine whether linoleic acid (LA) and cis-9-tetratriene elicit similar responses in wild rodents, we tested Brandt's voles (Lasiopodomys brandtii), a grassland pest, and brown rats (Rattus norvegicus), an agricultural pest. In the experiments, we observed that both animals exhibited behavioral responses to LA and cis-9-tetratriene similar to those in mice. Brandt's voles and brown rats showed a preference for LA, while exhibiting avoidance behavior towards cis-9-tetratriene ((Z)-9-TE). Figure 11This suggests that the attraction effect of linoleic acid and the repulsion effect of cis-9-tetratriene are likely to be generalized across all rodent species, highlighting their potential ecological significance in foraging and feeding behavior.

[0064] Example 2: Mechanism Study of Influencing Rodent Tendency Behavior

[0065] 1) Chemogenetic manipulation of the VTA-mOT dopaminergic pathway

[0066] The VTA contains a high proportion of dopaminergic neurons, and the VTA-mOT dopaminergic pathway plays a key role in mediating odor preferences in mice. We hypothesize that this pathway regulates preference for linoleic acid and avoidance of cis-9-tetratriene. To verify this, we expressed pAAV-retro-hSyn-cre-wpre and cre-dependent inhibitory design receptors (activated only by the design drug, DREADDs) rAAV-hSyn-DIO-hM4D(Gi)-P2A-EGFP in mOT and VTA, respectively, and implanted a drug delivery catheter in mOT. We then tested the preference behavior in mice using a bidirectional odor preference assay. Figure 12 A). In the bidirectional odor preference test (LAvs.(Z)-9-TE) ( Figure 12 (B above), the movement trajectory of hM4Di mice treated with physiological saline is shown in the figure ( Figure 12 As shown in (B), their trajectories around linoleic acid are more concentrated, and they spend more time around linoleic acid. Figure 12 C) and traveled a longer distance ( Figure 12 D). This indicates that the mice still maintained a preference for linoleic acid. However, CNO treatment eliminated this preference, and CNO-treated hM4Di mice showed fairly similar dwell times around linoleic acid and cis-9-tetratriene. Figure 12 E) and distance of movement ( Figure 12 F). This is also illustrated in the mouse trajectory plots, where their trajectory densities around linoleic acid and cis-9-tetratriene are similar (F). Figure 12 (B below). This indicates that, in non-fasted mice, inhibition of the VTA-mOT dopaminergic pathway eliminated the mice's strong preference for linoleic acid over cis-9-tetratriene.

[0067] To further investigate the role of the VTA-mOT pathway in regulating bidirectional selection for linoleic acid and cis-9-tetratriene in mice, we activated this pathway in mOT and VTA by expressing pAVV-retro-hsyn-cre-wpre and cre-dependent excitatory DREADDs rAAV-hSyn-DIO-hM3D(Gq)-P2A-EGFP, respectively. Figure 12G), and a catheter was implanted in the mOT to track the movement trajectory of non-fasted mice in a bidirectional selection odor preference test. Figure 12 H). The movement trajectory of hM3Dq mice treated with physiological saline is shown in the figure ( Figure 12 As shown in H), their trajectories around linoleic acid are more concentrated, and they spend more time around linoleic acid. Figure 12 I) and traveled a longer distance ( Figure 12 J). This indicates that the mice still maintained a preference for linoleic acid. Notably, treatment with CNO reversed this preference in hM3Dq mice, which instead spent more time around cis-9-tetratriene. Figure 12 K) and traveled a longer distance ( Figure 12 L). This is also reflected in the movement trajectory diagrams of mice, where their movement trajectories around cis-9-tetratriene are more concentrated compared to linoleic acid. Figure 12 (H below). This indicates that in non-fasted mice, activation of the VTA-mOT dopaminergic pathway altered the mice's preference for linoleic acid and instead caused them to develop a preference for cis-9-tetratriene.

[0068] To further investigate whether this reversal of odor preference was due to changes in the mice's preference for linoleic acid and cis-9-dococtitriene, we conducted single-compound preference experiments on linoleic acid or cis-9-dococtitriene, using mineral oil as a control group. In the single-compound preference experiment on linoleic acid, mice expressing hM3Dq treated with saline still showed a significant preference for linoleic acid. Specifically, compared to being around mineral oil, these mice spent significantly more time around linoleic acid and moved significantly longer distances. Figure 13 (A, B, left). Furthermore, we treated hM3Dq-expressing mice with CNO, altering their preference for linoleic acid. Specifically, mice spent longer periods and moved significantly longer distances around linoleic acid compared to around mineral oil. Figure 13 (A, B, right).

[0069] Next, we conducted a single-compound preference experiment with cis-9-dococaryne. Mice expressing hM3Dq, treated with saline, still exhibited avoidance behavior towards cis-9-dococaryne. Specifically, compared to being around mineral oil, these mice spent significantly less time around cis-9-dococaryne and moved significantly shorter distances. Figure 13(C, D, left). Subsequently, we conducted a CNO treatment experiment. The results showed that mice expressing hM3Dq, treated with CNO, exhibited significantly altered avoidance behavior towards cis-9-dococtitriene. Specifically, the time mice spent near mineral oil was roughly the same as their time spent near cis-9-dococtitriene, and the distance they moved was also almost identical (see...). Figure 13 C, D (right). This indicates that activation of the VTA-mOT pathway reverses the initial preference for linoleic acid and eliminates the avoidance of cis-9-tetratriene (C). Figure 13 In summary, this pathway modulates the titer assessment of these odors in mice, which may be achieved through D1- / D2 SPNs in mOT.

[0070] In addition, we performed the same experiment using mCherry mice (mOT and VTA mice expressing pAAV-retro-hSyn-cre-WPRE and cre-dependent rAAV-hSyn-DIO-mCherry, respectively) as controls. Figure 14 Similar to hM3Dq- and hM4Di mice, mCherry mice injected with saline maintained their preference for linoleic acid and avoidance of cis-9-docosahexaene. Specifically, these mice spent significantly more time around linoleic acid and moved significantly farther than they did around mineral oil or cis-9-docosahexaene. Figure 14 (C, E, G, left). Unlike the results observed in hM3Dq- and hM4Di mice, mCherry mice in the CNO-treated group maintained their preference for linoleic acid and avoidance behavior towards cis-9-dococtitriene. Specifically, compared to being around mineral oil or cis-9-dococtitriene, these mice spent significantly more time around linoleic acid and moved significantly farther. Figure 14 (C, E, G on the right). These findings confirm that the observed phenotypes are specifically caused by chemical genetic manipulation of the VTA-mOT pathway, rather than by the non-specific effects of CNO or its metabolites.

[0071] To further verify the potential impact of the VTA-mOT dopaminergic pathway on larval preference, we performed chemogenetic manipulation of this pathway as described above. Mice expressing hM3Dq or hM4Di, even after saline treatment, still exhibited a significant preference for satiated bollworm larvae. Specifically, these mice spent significantly longer periods around satiated bollworm larvae compared to those around starved bollworm larvae. Figure 15 (A, E), although the distance of movement was not significantly different ( Figure 15B, F). We then treated mice with CNO, and the preference for satiated bollworm larvae changed in CNO-treated mice expressing hM3Dq or hM4Di. Specifically, these mice spent a longer time around starved bollworm larvae compared to around satiated bollworm larvae. Figure 15 C,G), and the movement distance is also significantly longer ( Figure 15 D,H). Chemical genetic inhibition or activation of this pathway reversed the initial preference for satiated larvae in non-fasting mice (D,H). Figure 15 This further supports the role of this pathway in regulating odor-guided feeding behavior.

[0072] To verify the key role of the VTA-mOT dopaminergic pathway in mediating odor preference in mice, we performed immunostaining. The results confirmed that neurons co-expressing hM4Di-EGFP and TH accounted for 80% of the total number of neurons expressing hM4Di. Figure 16 A), and neurons that co-express hM3Dq and TH account for 80% of the total number of neurons expressing hM3Dq. Figure 16 B). Electrophysiological experiments showed that after administration of CNO, the firing frequency of hM4Di-expressing neurons was significantly reduced ( Figure 16 C), the firing frequency of neurons expressing hM3Dq was significantly increased ( Figure 16 D). These results collectively indicate that dopaminergic neurons in the VTA-mOT pathway are involved in regulating the preference for linoleic acid and the avoidance of cis-9-tetratriene, and that the VTA-mOT dopaminergic pathway is successfully inhibited or activated by CNO.

[0073] Subsequently, we conducted a series of control experiments to eliminate various confounding factors that might interfere with the observed phenotypes. First, to verify the effect of CNO administration on the sense of smell in mice, we performed a food burial experiment. We administered saline or CNO to mice expressing mCherry-, hM3Dq-, and hM4Di, followed by the food burial experiment. The results showed that there was no significant difference in the latency of first locating food particles in the buried food locator experiment between the saline and CNO groups in mice expressing mCherry-, hM3Dq-, and hM4Di. Figure 16 E). This indicates that the chemical genetic manipulation of the VTA-mOT dopamine pathway does not affect the olfactory perception ability of mice. Next, to verify the effect of CNO administration on the motor ability of mice, we counted the total distance traveled by mice in a bidirectional odor preference test. The results showed that there was no significant difference in the distance traveled between the CNO-treated mCherry-, hM3Dq-, and hM4Di mice and the saline-treated mice. Figure 16 F) confirmed that CNO treatment did not alter the mice's motor abilities.

[0074] 2) Dopamine D1 and D2 receptors in the medial olfactory tubercle (mOT) mediate the preference for linoleic acid and the avoidance of cis-9-tetratriene in mice.

[0075] The olfactory tubercle exhibits region-dependent differences in odor processing and related behaviors. Odor modulation varies across different subregions of the olfactory tubercle (OT). In the meta-otonic tubercle (mOT), D1 SPNs are involved in regulating motivational behaviors related to attraction, feeding, and reward, while D2 SPNs show the opposite effect. Conversely, D1 and D2 SPNs in the lateral otonic tubercle are involved in regulating aversion behaviors induced by odor stimuli, while D1 and D2 SPNs in the mOT play opposite roles. Activation of D1 receptor neurons in the mOT promotes feeding behavior in mice because it enhances the mice's discrimination and attraction to food odors. Conversely, activation of D2 receptor neurons in the mOT or D1 receptor neurons in the lateral olfactory tubercle has an inhibitory effect on feeding in mice by reducing the mice's recognition of food odors and generating an aversion response.

[0076] To further determine the roles of D1 and D2 SPNs in odor preference, we locally infused mOT with either saline or dopamine receptor antagonists (D1 receptor antagonist, SCH23390 and D2 receptor antagonist, eticlopride). Figure 17 A), and conducted a bidirectional odor preference experiment on mice ( Figure 17 (B, C above). Here we use an odor preference index to evaluate the preference behavior of mice. A positive odor preference index indicates that the mice prefer the odor, while a negative odor preference index indicates that the mice avoid the odor. First, we tested the mice's preference for a single compound, linoleic acid, while using mineral oil as a control group. The results showed that compared with the group treated with physiological saline ( Figure 17 Compared to the black bars on the right (E, G, F, H), SCH23390 treatment significantly reduced the exploration time of mice around linoleic acid. Figure 17 E, G (middle red bar) and distance traveled ( Figure 17 The F and H bars (red bars in the middle) represent the odor preference index. In contrast, this difference was not observed in eticlopride-treated mice. Figure 17 E, G, H (blue bars on the right); however, in non-fasting mice, the odor preference index related to the distance moved around linoleic acid was decreased in the eticlopride treatment group ( Figure 17 (F-side blue bar chart). This indicates that mice in the saline injection group still maintained a preference for linoleic acid, while SCH23390 treatment eliminated this preference. Besides reducing the movement distance of non-fasted mice (… Figure 17Apart from F), eticlopride had no significant effect on mouse preference. This indicates that pharmacological blockade of dopamine D1 receptors (rather than D2 receptors) eliminated the preference for linoleic acid in mice; D1 SPNs are primarily responsible for mediating the preference for linoleic acid in mice. The odor preference index in terms of time or distance of movement was calculated as: (time or distance of movement of mice around linoleic acid - time or distance of movement of mice around mineral oil) / (time or distance of movement of mice around linoleic acid + time or distance of movement of mice around mineral oil).

[0077] In addition, we conducted a preference experiment for a single compound, cis-9-tetratriene, while using mineral oil as a control group. The results showed that, compared to the saline injection group ( Figure 17 Compared to the black bars on the right (I, J, K, L), SCH23390 treatment altered the exploration time of mice around cis-9-tetratriene. Figure 17 I, K (the red bars in the middle) and the distance moved ( Figure 17 The odor preference index (J, L, middle red bars) is related to this. Although the exploration time of mice around cis-9-tetratriene remained negative, the mean value of this value increased from approximately -0.5 to approximately -0.2 in non-fasted mice and from approximately -0.3 to approximately -0.2 in fasted mice. Correspondingly, the preference index for movement distance decreased from approximately -0.3 to approximately -0.17 in non-fasted mice and from approximately -0.23 to approximately -0.1 in fasted mice. Notably, after administering eticlopride to mice (compared to the saline group, the exploration time of mice around cis-9-tetratriene decreased significantly), the odor preference index for movement distance decreased significantly. Figure 17 (I, K, right-hand blue bars) and movement distance ( Figure 17The odor preference index (as shown in the blue bars on the right of J and L) changed from negative to positive. Specifically, the mean preference index for exploration time around cis-9-dococcasione in non-fasting mice increased from approximately -0.5 to approximately 0.4, while the mean value in fasting mice increased from approximately -0.3 to approximately 0.12. Correspondingly, the preference index for movement distance increased from approximately -0.4 to approximately 0.26 in non-fasting mice and from approximately -0.23 to approximately 0.06 in fasting mice. This indicates that the avoidance of cis-9-dococcasione in mice has turned into a preference. Although D1 SPNs played a role in the avoidance behavior of mice towards cis-9-dococcasione, it is clear that D2 SPNs are essential for mediating the avoidance of cis-9-dococcasione in mice. The odor preference index related to time or distance of movement is calculated as follows: (time or distance of movement of mice around cis-9-tetratriene - time or distance of movement of mice around mineral oil) / (time or distance of movement of mice around cis-9-tetratriene + time or distance of movement of mice around mineral oil).

[0078] Finally, in a bidirectional odor preference test, we evaluated the behavior of mice simultaneously exposed to linoleic acid and cis-9-tetratriene. Mice treated with saline still showed a stronger preference for linoleic acid, exhibiting a positive odor preference index (...). Figure 17 M, N, O, P (black bars on the right). However, mice in the SCH23390 treatment group showed a significantly reduced preference, with significantly lower preference index scores for both time spent around linoleic acid and distance traveled. Figure 17 (M, N, O, P are represented by the red bars in the middle). Specifically, the mean preference index for exploration time around linoleic acid in non-fasting mice decreased from approximately 0.2 to approximately -0.1, while the mean value in fasting mice decreased from approximately 0.25 to approximately 0. Correspondingly, the preference index for movement distance decreased from approximately 0.2 to approximately 0 in both non-fasting and fasting mice, and from approximately 0.27 to approximately 0. Eticlopride treatment had no effect on the preference in non-fasting mice. Figure 17 M, N (blue bars on the right), but it has an impairing effect on the preference of fasted mice ( Figure 17 O, P (right side blue bars). The odor preference index related to time or distance of movement is calculated as: (time or distance of movement of mice around linoleic acid - time or distance of movement of mice around cis-9-tetratriene) / (time or distance of movement of mice around linoleic acid + time or distance of movement of mice around cis-9-tetratriene).

[0079] 3) The reaction of D1- and D2-SPNs expressing GCaMP6s in mOT to linoleic acid and cis-9-tetratriene

[0080] To investigate the response patterns of dopamine D1 and D2 polyspinous projection neurons (SPNs) to linoleic acid (LA) and cis-9-tetraterene ((Z)-9-TE) in mice under free-movement conditions, we employed fiber optic spectrophotometry based on calcium signal detection. Specifically, we injected cre-dependent gene-encoded calcium ion indicators, rAAV-hSyn-DIO-GCaMP6s (rAAV-CMV-DIO-EGFP-WPRE-hGH PA served as a control) into the medial olfactory tubercle (mOT) region of D1-Cre and D2-Cre mice, respectively. In a bidirectional odor preference test, when mice were exposed to contrast combinations of linoleic acid or cis-9-tetraterene with mineral oil (MO), and a direct contrast combination of linoleic acid and cis-9-tetraterene, the dynamic changes in calcium signals within D1 SPNs or D2 SPNs were monitored in real time (e.g., ...). Figure 18 (As shown).

[0081] Experimental results showed that D1 SPNs expressing GCaMP6s exhibited a stronger response during the inhalation of linoleic acid compared to mice inhaling mineral oil or cis-9-tetratriene. During the linoleic acid inhalation phase, these neurons showed a higher area under the curve (AUC) value, and the peak ΔF / F value was also significantly increased (e.g., ...). Figure 18 (As shown in B and D). In contrast, D1 SPNs produce a more similar reaction when smelling cis-9-tetratriene or mineral oil. Figure 18 C).

[0082] Similarly, D2 SPNs expressing GCaMP6s also exhibit unique response characteristics. When mice smelled linoleic acid, the response intensity of D2 SPNs was significantly higher than that when they smelled mineral oil. These neurons showed a higher area under the curve (AUC) value, and the peak ΔF / F value was also significantly increased (e.g., Figure 18 E). Of particular note is that, compared to mineral oil and linoleic acid, D2 SPNs exhibited a significantly stronger response upon olfaction of cis-9-tetratriene. These neurons displayed a higher area under the curve (AUC) value, and the peak ΔF / F value was also significantly increased. Figure 18 F,G).

[0083] To verify the responses of D1 and D2 SPNs to other odors, we recorded calcium signals in D1 and D2-Cre mice expressing GCaMP6s when they smelled other odors. The results showed that the responses of D1 and D2 SPNs expressing GCaMP6s to 3,5-di-tert-butylphenol, n-pecapane, oleic acid, and n-heptadecane were similar to those to mineral oil. The mean calcium signal response plot, thermogram, AUC, and peak value (ΔF / F) of D1 and D2 SPNs were also similar to those to mineral oil. Figure 19 (A, B). This indicates that the reactions of D1 and D2SPNs with linoleic acid and cis-9-tetratriene are specific.

[0084] To rule out the effects of viral injection and fiber optic implantation on the behavioral phenotype of mice, we conducted a control experiment. In contrast, D1- or D2-Cre mice expressing EGFP (data pooled) did not show significant changes in calcium signaling after exposure to the tested odor. Figure 20 The results (AD) confirmed that the signal observed in GCaMP6s mice is a specific dynamic response of calcium signaling in D1 and D2 SPNs.

[0085] Our current research indicates that mice rely on their primary olfactory system to detect and locate insect larvae for feeding by sensing odors originating from larvae. Specifically, the different proportions of LA and (Z)-9-TE released in larvae under satiated or starved conditions elicited approach and avoidance behaviors in mice, respectively. The VTA-mOT dopaminergic pathway plays a crucial role in coordinating these odor-mediated behaviors by modulating D1 and D2 SPNs in the mOT. D1 SPNs primarily mediate preference for LA, while D2 SPNs primarily regulate avoidance of (Z)-9-TE. In this study, we found that D1 and D2 SPNs in the mOT play different roles in modulating odor preference. D1 SPNs primarily regulate preference for LA, while D2 SPNs primarily regulate avoidance of (Z)-9-TE. Our results suggest that blocking D1 receptors, rather than D2 receptors, eliminates the initial preference for LA, indicating that D1 SPNs, rather than D2 SPNs, are the primary necessary factor mediating the initial preference for LA. Conversely, blocking D2 receptors, rather than D1 receptors, reversed avoidance of (Z)-9-TE, suggesting that D2 SPNs, rather than D1 SPNs, are the primary mediator of initial (Z)-9-TE avoidance. Furthermore, in the comparison of LA and (Z)-9-TE, blocking dopamine D1 or D2 receptors significantly reduced initial preference for LA and avoidance of (Z)-9-TE, with D1 receptor blocking showing a more pronounced effect. These findings, combined with our fiber optic photometric experimental data, indicate that D1 and D2 SPNs are involved in encoding odor properties and odor valence, but play different roles: D1 SPNs primarily encode positive odor valence, while D2 SPNs primarily encode negative odor valence.

[0086] We also confirmed that linoleic acid and cis-9-dococtitriene elicit similar behavioral responses in wild rodents. Behavioral experiments on Brandt's voles and brown rats showed that both animals exhibited similar behavioral responses to linoleic acid and cis-9-dococtitriene as mice, with Brandt's voles and brown rats showing a preference for linoleic acid and an avoidance of cis-9-dococtitriene.

[0087] Mice prefer well-fed larvae to starved larvae because well-fed larvae contain relatively high levels of linoleic acid and low levels of cis-9-dococtitriene. Under physiological conditions, the VTA-mOT dopaminergic pathway releases dopamine into the mOT. The D1 and D2 SPNs in the mOT respond to the dopamine released by the VTA, creating an unbalanced "seesaw" state of preference for linoleic acid and avoidance of cis-9-dococtitriene. Inactivating this pathway, or blocking dopamine D1 receptors (inactivating D1 SPNs), disrupts the imbalance, eliminating the preference for linoleic acid and restoring balance to a state where there is no preference for either linoleic acid or cis-9-dococtitriene. Conversely, activating this pathway, or blocking dopamine D2 receptors (activating D2 SPNs), reverses the preference for linoleic acid and the avoidance of cis-9-dococtitriene, creating the opposite "seesaw" state observed under physiological conditions. Figure 21 ).

[0088] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of cis-9-tetratriene as a rodent repellent or in the preparation of rodent repellents.

2. The application of mixtures with cis-9-tetratriene as the main component as rodent repellents or in the preparation of rodent repellents.

3. The application according to claim 1 or 2, characterized in that, The rodents mentioned are: mouse, Brandt's vole, and brown rat.

4. The application according to any one of claims 1-3, characterized in that, In rodents, the VTA-mOT dopaminergic pathway coordinates odor-guided behavior by regulating D1 and D2 SPNs in mOT, with D2 SPNs primarily regulating avoidance of cis-9-tetratriene.

5. A rodent repellent, characterized in that, The rodent repellent includes cis-9-tetratriene and excipients.

6. A rodent repelling device, said rodent repelling device comprising a repelling device body, characterized in that, The rodent repellent agent as described in claim 5 is placed inside the body of the repellent device.

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