Novel pest control method targeting the respiratory organ formation mechanism of insect pests

Abstract

The present invention provides a new pest control means that has a small environmental load and excellent versatility and that is for controlling pests in the phylum Arthropoda by using a component that disrupts the respiratory organ forming mechanism that is widely common in the phylum Arthropoda.　The present invention inhibits the activity of dual oxidase (Duox) and / or eliminates or suppresses generation of reactive oxygen species (ROS) in the body of a pest to be controlled. As a result, the trachea forming capability is reduced and / or the intestinal symbiotic microorganism flora is disrupted in the pest, and the survival rate of the pest is reduced.

Description

Technical Field

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[0001] The present invention relates to a pest control composition containing a component that disturbs the respiratory organ formation mechanism commonly found in the phylum Arthropoda, and a method for controlling pests of the phylum Arthropoda using the component.

Background Art

[0002] Pests impose significant economic costs in modern agriculture. Current agricultural systems require growing one or several crop or plant species over large areas. Such ecologically unbalanced systems are vulnerable to pest damage, and it is said that currently one-third of agricultural crops are lost to pests.

[0003] On the other hand, some pests are also harmful to the health of animals including humans. For example, mosquitoes are known to transmit various diseases such as malaria, Zika fever, dengue fever, and chikungunya fever. In particular, the number of malaria patients still exceeds 200 million annually, mainly in tropical regions, and it is said to be one of the three major infectious diseases in the world that cause great health damage to humanity, along with tuberculosis and AIDS.

[0004] Pest control has mainly been carried out using chemical insecticides that act on various physiologies of the target pests. However, the impact of such chemical insecticides on the surrounding environment other than the target pests, that is, the environmental load, has become a problem in recent years. In the "Green Food System Strategy" (Ministry of Agriculture, Forestry and Fisheries) formulated in May 2021, a 50% reduction (in terms of risk) in the use of chemical pesticides by 2050 was set as a goal.

[0005] In recent years, general consumers have also started to pay attention to chemical residues and their effects on the health of animals and plants as well as the environment, and insecticides that consider the environmental load are favorably received in general society.

[0006] One example of a pest control technology with a low environmental impact is biological control. Biological control provides an alternative means of pest control that reduces reliance on chemicals. A wide range of biological control agents, including bacteria, yeasts, and fungi, are being considered for use in pest control. For example, Bacillus thuringiensis (Bt) is a well-known biological control agent, commercially available as products such as Thuricide® and Dipel® (Non-Patent Literature 1).

[0007] Attempts are also being made to utilize insecticidal compounds that are highly safe and have a low environmental impact. Examples of such insecticides include higher fatty acid esters such as lauric acid ester and palmitic acid ester, which exist naturally and are easily decomposed by soil bacteria, as well as higher alcohols such as octanol and undecanol (Patent Document 1), or fatty acids such as caproic acid and capric acid (Patent Document 2), which have been reported to be used as wood-boring insect control agents.

[0008] Furthermore, pests can develop resistance to insecticides. Therefore, there is always a high demand for novel pest control methods that complement or replace pest control using major chemicals, preferably with low environmental impact. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] : Japanese Patent Application Publication No. 8-133909 [Patent Document 2] : Japanese Patent Application Publication No. 10-067607 [Non-patent literature]

[0010] [Non-Patent Document 1] Glare TR, O'Callaghan M. 1998. Environmental and health impacts of Bacillus thuringiensis israelensis. Report prepared for the New Zealand Ministry of Health. New Zealand Ministry of Health, Wellington, New Zealand. [Non-Patent Document 2] Jang S, Mergaert P, Ohbayashi T, Ishigami K, Shigenobu S, Itoh H, Kikuchi Y.: Dual oxidase enables insect gut symbiosis by mediating respiratory network formation. Proc. Natl. Acad. Sci. USA 118: e2020922118 (2021) [Overview of the project] [Problems that the invention aims to solve]

[0011] This invention provides a novel pest control method that is environmentally friendly and highly versatile, using components that disrupt the respiratory system formation mechanism, which is widely common in arthropods, to control arthropod pests. [Means for solving the problem]

[0012] The inventors have revealed that reactive oxygen species (ROS) are involved in the crosslinking of proteins that make up the trachea during its formation, which is the respiratory organ of terrestrial arthropods, and play an important role in its hardening and morphological maintenance (Non-Patent Literature 2). The inventors anticipated that it would be possible to control pests of terrestrial arthropods that have tracheas by inhibiting this newly discovered mechanism of tracheal formation and morphological maintenance by ROS. When they applied antioxidants that scavenge ROS and inhibitors of dual oxidase (Duox), which generates ROS in the pest's body, to various pests, they confirmed significant tracheal formation failure and a decrease in survival rate. This invention relates to the novel pest control composition and pest control method discovered by the inventors.

[0013] Therefore, this application provides the following invention.

[0014] 1. A composition for controlling pests, comprising one or more compounds having a dual oxidase (Duox) activity inhibitory effect and / or a reactive oxygen species (ROS) generation suppression or scavenging effect in the body of the target pest. 2. The composition according to item 1, wherein the compound is selected from antioxidants and Duox inhibitors. 3. The antioxidants include: Vitamin A / retinol, Vitamin E / α-tocopherol, Coenzyme Q10, N-acetylcysteine ​​(NAC), S-methylcysteine, glutathione, L-cysteine, D-cysteine, cysteine ​​persulfide, cysteine ​​trisulfide, allicin, alliin, allyl disulfide, cyanidin chloride, cyanidin-3,5-diglucoside, isoflavone, quercetin, catechin, theaflavin, sesamin, sesaminol, resveratrol, curcumin, chlorogenic acid, ferulic acid, A composition as described in item 2, selected from the group consisting of 3,4-dihydroxycinnamic acid, β-carotene, lycopene, astaxanthin, zeaxanthin, L-cysteine ​​methyl hydrochloride (LCM), L-cysteine ​​ethyl hydrochloride (LCE), D-cysteine ​​hydrochloride monohydrate (DCH), D-cysteine ​​methyl hydrochloride (DCM), D-penicillamine (DPA), 2-aminoethanethiol (2-AET), L-methionine, L-methionine methyl hydrochloride (LMM), and two or more combinations thereof. 4. The composition according to item 2, wherein the antioxidant is a thiol or a sulfide. 5. The composition according to item 1, wherein the target pest is an arthropod having a trachea. 6. The aforementioned arthropods include Polyphagotarsonemus latus, Phytonemus pallidus, Penthaleus major, Brevipalpus lewisi, Brevipalpus phoenicis, Panonychus citri, Panonychus ulmi, Tetranychus urticae, Tetranychus kanzawai, Amphitetranychus viennensis, Oligonychus ununguis, Bryobia eharai, Eotetranychus kankitus, and Bryobia praetiosa), apple rust mite (Aculus Schlechtendali), citrus rust mite (Aculops pelekassi), Ryukyu citrus rust mite (Phyllocoptruta citri), false pear rust mite (Eriophyes chibaensis), tulip rust mite (Aceria tulipae), grape leafminer mite (Colomerus vitis), peach rust mite (Aculus fockeui), tea rust mite (Calacarus carinatus), long-haired flour mite (Tyrophagus putrescentiae), robin mite (Rhizoglyphus robini), long-haired flour mite (Tyrophagus putrescentiae), sorghum mite (Lardoglyphus konoi), sugar mite (Carpoglyphus lactis), cedar spider mite (Oligonichus (hondoensis), fir tree mite (Oligonichus ununguis), honeybee mite (Varroa jacobsoni), two-spotted mite (Haemaphysalis longicornis), Japanese mite (Ixodes ovatus), cattle mite (Boophilus)microplus), Amblyomma testudinarium, Ornithonyssus sylvialum, Dermanyssus gallinae, Demodex phylloides, Sarcoptes scabiei bovis, Knemidocoptes mutans, Otodectes cynotis, Psoroptes communis, Ixodes persulcatus, Ornithonyssus bacoti, Chelacaropsis moorei, Pyemotes ventricosus, Demodex folliculorum, Dermotophagoides Pteronyssinus), Sarcoptes scabiei, Trombicula akamushi, Chiracanthium japonicum, Heteropoda venatoria, Spermophora senoculata, Pholcus phalangioides, Uroctea compactilis, Plexippus paykulli, Plexippus adansoni, Isometrus europaeus, Scolopendra subspinipes mutilans, Scolopendra subspinipes japonica, Scolopendra multidens), centipede (Bothropolys asperatus), house centipede (Thereuronema hilgendofi), millipede (Oxidus gracilis), aedes aegypti, Asian tiger mosquito (Aedesalbopictus), Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freeborni, Culex species, Culiseta melanura, Mediterranean fruit fly (Ceratitis capitata), Mexican fruit fly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera Fruit flies selected from the following: Bactrocera oleae, melon fly (Bactrocera oleae), Natal fruit fly (Ceratitis rosa), cherry fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni), peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), citrus fruit fly (Bactrocera dorsalis), and West Indian fruit fly (Anastrepha obliqua), New World spiral insect (Cochliomyia hominivorax), Old World spiral insect (Chrysomya bezziana), Australian sheep blowfly / copper green blowfly (Lucilia cuprina), pink moth larva (Pectinophora gossypiella), European gypsy moth (Lymantria dispar), navel orange insect (Amyelois Transitella, Peach branch borer (Anarsia lineatella), Rice stem borer (Tryporyza incertulas), Noctuidae moths, Heliothinae tobacco budworms, Japanese beetle (Papilla japonica), White beetle (Graphognathus)(spp.), cotton boll weevil (Anthonomous grandis), Colorado leaf beetle (Leptinotarsa ​​decemlineata), vine mealybug (Planococcus ficus), Asian citrus psyllid (Diaphorina citri), spotted-wing fruit fly (Drosophila suzukii), blue-green sharpshooter (Homalodisca vitripennis), glass-winged sharpshooter (Homalodisca vitripennis), light brown apple moth (Epiphyas postvittana), Bagrada worm (Bagrada hilaris), Asian gypsy moth (Lymantria dispar asiatica), Japanese gypsy moth (Lymantria dispar japonica), white-skinned snail (Lymantria albescens), Ezo snail (Lymantria umbrosa), and small white-skinned snail (Lymantria Selected from the group of (postalba) are the Asian gypsy moth, Asian longhorn beetle (Anoplophora glabripennis), coconut beetle (Oryctes rhinoceros), emerald ash borer (Agrilus planipennis), European grapevine moth (Lobesia botrana), European gypsy moth (Lymantria dispar), pseudocodling moth (Thaumatotibia leucotreta), red imported fire ant (Solenopsis invicta Buren and S. richteri Forel), Old World moth larva (Helicoverpa armigera), spotted lantern fly (Lycorma delicatula), African honeybee (Apis mellifera scutellata), fruit shoot borer (Leucinodes orbonalis), corn root beetle (Diabrotica spp.), and Western corn root beetle (Diabrotica Whiteflies (Bemisia tabaci), houseflies (Musca domestica), green bottle flies (Lucilia cuprina), silkworms (Bombyx)Cattle flies selected from redscale flies (Aonidiella aurantia), canine heartworms (Dirofilaria immitis), southern pine beetles (Dendroctonus frontalis), avocado thrips (Thysanoptera Spp.), Oestridae spp., and human botflies (Dermatobia hominis), horse flies (Tabanus sulcifrons), horned flies (Haematobia irritans), spiral flies selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi or C. minima, tsetse flies (Glossina spp.), warble flies selected from cattle flies (Hypoderma bovis) or striped cattle flies (Hypoderma lineatum), and spotted lantern flies (Lycorma Delicatula, Capra beetle (Trogoderma granarium), honeybee mite (Varroa destructor), termite (Coptotermes formosanus), hemlock woolly aphid (Adelges tsugae), walnut branch beetle (Pityophthorus juglandis), European hornet (Sirex noctilio), pink spotted moth larva (Pectinophora scutigera), two-spotted spider mite (Tertanychus urticae), diamondback moth (Plutella xylostella), Taro caterpillar (Spodoptera litura), confused flour beetle (Tribolium castaneum), green peach aphid (Myzus persicae), cotton aphid (Aphis gossypii), brown planthopper (Nilaparvata lugens), beet armyworm (Spodoptera exigua), Western thrips (Frankliniella occidentalis), codling moth (Cydia pomonella), four-spotted bean weevil (Callosobruchus maculatus), pea aphid (Acyrthosiphon)The composition described in item 5, selected from the group consisting of pisum, tomato leaf borer (Tuta absoluta), onion thrips (Thrips tabaci), and cottony moth larva (Helicoverpa armigera). 7. The composition according to item 1, having a dosage form selected from the group consisting of granules, hydrated powder, water-dispersible granules (powder), water-soluble granules, soluble concentrate, oil-in-water emulsion, microemulsion, aqueous suspension concentrate, aqueous capsule suspension, oil-based suspension concentrate, and aqueous suspension emulsion. 8. The composition described in item 1, applied by a method selected from the group consisting of immersion, fumigation, dusting, aerial spraying, aerosol spraying, coating, spraying, solid spraying, and injection. 9. A method for controlling insect pests, comprising inhibiting the activity of dual oxidase (Duox) in the body of the target insect pest, and / or suppressing or eliminating the generation of reactive oxygen species (ROS). 10. The control method according to item 9, further comprising reducing the survival rate of the pest by inhibiting the activity of Duox and / or suppressing or eliminating the generation of ROS, thereby causing a decrease in the tracheal formation ability of the pest and / or disruption of the intestinal symbiotic microbiota. 11. The control method according to item 9, comprising applying one or more compounds having an inhibitory effect on Duox activity and / or an inhibitory or scavenging effect on the generation of ROS, or a composition according to any one of items 1 to 8 containing such compounds, to the pest, to the place where it may live, or to a place or object that is to be protected from damage by the pest. [Effects of the Invention]

[0015] The pest control composition and pest control method of the present invention utilize components that disrupt the respiratory system formation mechanism, which is widely common in arthropods, and therefore have a low environmental impact and excellent versatility. [Brief explanation of the drawing]

[0016] [Figure 1]Figure 1 shows that the amount of symbiotic microorganisms in the intestine of the striped bug (Riptortus pedestris) is significantly reduced when Duox-RNAi is applied as an inhibitor of Duox or N-acetylcysteine (NAC) is applied as an antioxidant.

[0017] [Figure 2] Figure 2 shows that the density of tracheae in the intestinal tissue is significantly reduced in the striped bug to which Duox-RNAi is applied as an inhibitor of Duox or NAC is applied as an antioxidant, compared with the control. The tracheae were observed by immunostaining with a dityrosine antibody.

[0018] [Figure 3] Figure 3 shows that the amount of symbiotic microorganisms in the intestine of the striped bug is reduced by the application of Duox-RNAi, and the amount of microorganisms is restored under high oxygen conditions.

[0019] [Figure 4] Figure 4 shows that the density of tracheae in the intestinal tissue is significantly reduced in the striped bug to which RNAi of Bnl, Trh, and Sima is applied as inhibitors of tracheal formation, and in germ-free individuals lacking symbiotic microorganisms in the intestine, compared with the control. The tracheae were observed by immunostaining with a dityrosine antibody.

[0020] [Figure 5] Figure 5 shows that the expression level of Duox in the tracheae is significantly higher than that in other organs in any of the model insect species of coorogi, silkworm, Asian corn borer, and Drosophila melanogaster that are systematically isolated from each other.

[0021] [Figure 6] Figure 6 shows that the amount of symbiotic microorganisms in the intestine of the striped bug (Riptortus pedestris) and the narrow-headed bug (Cletus punctiger) is reduced by the application of NAC, and their survival rate is significantly reduced compared with the control by the application of NAC.

[0022] [Figure 7] Figure 7 shows that the survival rates of Plautia stali and Dolycoris baccarum are significantly reduced by the application of NAC compared to the control group.

[0023] [Figure 8] Figure 8 shows that the survival rates of Stenotus binotatus and Stenodema calcarata are significantly reduced by the application of NAC compared to the control group.

[0024] [Figure 9] Figure 9 shows that the survival rate of the green stink bug (Apolygus spinolae) is significantly reduced by the application of NAC compared to the control group.

[0025] [Figure 10] Figure 10 shows a list of antioxidants used in the insecticidal tests.

[0026] [Figure 11] Figure 11 shows the mortality rate (A) of slender stink bugs treated with the antioxidants shown in Figure 10 14 days after treatment, and the Kaplan-Meier survival curve (B) for 20 days after treatment.

[0027] [Figure 12] Figure 12 shows the changes in the number of surviving stink bugs and their age over 15 days after treatment with the antioxidant shown in Figure 10.

[0028] [Figure 13] Figure 13 shows images of the trachea surrounding the digestive tract of the slender stink bug (Reptilia japonica) treated with ascorbic acid, LCM, L-cysteine, or NAC (10 mg / ml, pH 6.8), visualized by dityrosine antibody immunostaining.

[0029] [Figure 14] Figure 14 shows the mortality rate of slender stink bugs 7 days after treatment with ascorbic acid, NAC, L-cysteine, LCM, LCE, DCH, DCM, DPA, 2-AET, L-methionine, and LMM, adjusted to a low concentration of 1 mg / ml (pH 6.8).

[0030] [Figure 15] Figure 15 shows the mortality rates of Apolygus spinolae, Blattere lateralis, Tenebrio molitor, and Drosophila melanogaster after treatment with ascorbic acid, NAC, L-cysteine, and LCM, 2 days (Drosophila melanogaster), 7 days (Apolygus spinolae), or 14 days (Blattea lateralis and mealworms). [Modes for carrying out the invention]

[0031] The present invention will be described below based on specific embodiments, but the present invention is not limited in any way to these embodiments. All documents, including patent publications, patent application publications, and non-patent publications, referenced herein are incorporated herein by reference in their entirety for any purpose.

[0032] Most terrestrial arthropods breathe through their tracheae. The tracheae are tubular structures lined with a cuticle, extending throughout the arthropod's entire body and connecting to openings (spiracles) on the body surface. The trachea is an organ that terrestrial arthropods evolved to efficiently breathe air after moving onto land. While the trachea is thought to have played a crucial role in arthropods' adaptation to land, its formation mechanism, particularly the mechanism of hardening that gives it its robustness, has remained largely unknown. Understanding the unique physiological ecology of insects, and the genetic and molecular biological mechanisms that underlie them, is of academic importance.

[0033] Dual oxidase (Duox) is known as an enzyme that produces reactive oxygen species (ROS). In some insects, it has been reported that ROS produced by Duox expressed in the epithelial cells of the digestive tract are released into the lumen of the digestive tract and play an important role in maintaining intestinal symbiotic bacteria. However, much remains unknown about the role that Duox plays in maintaining intestinal symbiotic bacteria. The inventors diligently studied the mechanism by which Duox and the ROS it produces maintain intestinal symbiotic bacteria and found that ROS produced by Duox promote dityrosine crosslinking of proteins that make up the trachea in the intestinal tissue, thereby acting on the formation and maintenance of the trachea, ensuring oxygen supply to the intestinal tract, and thus achieving the maintenance of intestinal symbiotic bacteria (Non-Patent Literature 2). An outline of this research is shown below.

[0034] The symbiotic bacteria in the gut of the slender stink bug live in sac-like tissues (blind sacs) that have developed in the digestive tract. When the expression of Duox in the slender stink bug was suppressed by RNAi, the amount of symbiotic bacteria decreased significantly (Figure 1).

[0035] It was previously known that ROS produced by Duox cross-link tyrosine in proteins, forming dityrosine bonds (DTNs), which play an important role in maintaining tissue morphology and hardening. Therefore, the inventors observed the digestive tract of the slender stink bug by immunostaining with a dityrosine antibody and showed that dityrosine is specifically found in the trachea (Figure 2). Furthermore, inhibiting Duox by RNAi or applying the antioxidant N-acetylcysteine ​​(NAC) to the slender stink bug to suppress ROS production significantly inhibited dityrosine formation, i.e., tracheal formation (Figure 2).

[0036] Since symbiotic bacteria are aerobic, it is possible that the impaired formation of the trachea in the digestive tract tissue disrupted the supply of oxygen to the digestive tract lumen, inhibiting and reducing the growth of symbiotic bacteria. In fact, the symbiotic bacteria in individuals in which Duox expression was suppressed showed a characteristic gene expression pattern under oxygen deficiency. Furthermore, when slender rice bugs in which trachea formation was inhibited by suppressing Duox expression via RNAi were reared in a chamber with an oxygen concentration of 40%, the amount of symbiotic bacteria recovered despite the inhibition of trachea formation (Figure 3). These results indicate that Duox plays an important role in trachea formation, which increases the oxygen concentration in the intestines and stably maintains symbiotic bacteria.

[0037] Furthermore, it was found that tracheal development was significantly reduced in individuals not infected with symbiotic bacteria (Figure 4). This indicates that symbiotic bacteria colonized in the gut induce tracheal formation. It is likely that tracheal formation in the gut is activated in order to efficiently supply oxygen to the symbiotic bacteria in the gut. In addition, when the expression of transcription factors involved in tracheal formation induction (Bnl, Trh, Sima) was suppressed by RNAi, tracheal formation was significantly inhibited, similar to when Duox expression was suppressed (Figure 4), and the amount of symbiotic bacteria also decreased significantly at the same time.

[0038] The findings in this study that ROS and Duox play important roles in tracheal formation suggested that inhibiting the tracheal formation pathway mediated by ROS and Duox in some way could control terrestrial arthropods that form and maintain tracheas through this pathway. Therefore, when NAC was applied as an antioxidant that scavenges ROS in various species of true bugs, the survival rate after NAC application was significantly reduced compared to the control in all insect species, as expected (Figures 6-9).

[0039] The present invention is based on such findings, and one aspect thereof relates to a composition for controlling pests, which contains one or more compounds having a Duox activity inhibitory effect and / or a ROS generation suppression or scavenging effect in the body of the target pest (hereinafter referred to as "the pest control composition of the present invention" as appropriate).

[0040] Another aspect of the present invention relates to a method for controlling pests, which includes inhibiting the activity of dual oxidase (Duox) in the body of the target pest and / or suppressing or eliminating the generation of reactive oxygen species (ROS) (hereinafter referred to as "the pest control method of the present invention" as appropriate). The pest control method of the present invention may optionally further include reducing the survival rate of the pest by inhibiting Duox activity and / or suppressing or eliminating the generation of ROS, thereby causing a decrease in the tracheal formation ability of the pest and / or disruption of the intestinal symbiotic microbiota.

[0041] In this invention, "pest" refers to arthropods that cause some kind of harm to human society in agriculture, forestry, horticulture, public health, and material protection, as well as arthropods that are harmless but evoke feelings of aversion. "Control" means reducing the damage caused by pests by suppressing or preventing the occurrence, arrival, activity, or reproduction of pests, or by reducing their survival rate or causing them to die. The control method in this invention can control pests in all of the above senses by inhibiting the formation and maintenance of respiratory organs as described above.

[0042] Duox was shown to be highly expressed in the trachea regardless of the insect species (Figure 5), which suggests that the tracheal formation pathway is commonly used in all of these species, and therefore the pest control method of the present invention, which is based on inhibiting such a tracheal formation pathway, is effective against all terrestrial arthropods that have trachea.

[0043] The phylum Arthropoda includes the subphyla Chelicerata, Myriapoda, Crustacea, and Hexapoda (insects), which employ different respiratory modes depending on their habitat. Most terrestrial arthropods, excluding crustaceans, breathe through tracheae, and the tracheal formation pathway is thought to be common among these arthropods. Therefore, the control method of the present invention, which inhibits the respiratory system formation mechanism essential for survival common to terrestrial arthropods, can target any terrestrial arthropod that possesses a trachea.

[0044] The Chelicerata pests that may be controlled by the method of the present invention are not limited to, but include: Polyphagotarsonemus latus, Phytonemus pallidus, Penthaleus major, Brevipalpus lewisi, Brevipalpus phoenicis, Panonychus citri, Panonychus ulmi, Tetranychus urticae, Tetranychus kanzawai, Amphitetranychus viennensis, Oligonychus ununguis, Bryobia eharai, and Eotetranychus Kankitus), clover spider mite (Bryobia praetiosa), apple rust mite (Aculus Schlechtendali), citrus rust mite (Aculops pelekassi), Ryukyu citrus rust mite (Phyllocoptruta citri), false pear rust mite (Eriophyes chibaensis), tulip rust mite (Aceria tulipae), grape leafminer mite (Colomerus vitis), peach rust mite (Aculus fockeui), tea rust mite (Calacarus carinatus), long-haired mite (Tyrophagus putrescentiae), robin mite (Rhizoglyphus robini), long-haired mite (Tyrophagus putrescentiae), sorghum mite (Lardoglyphus konoi), sugar mite (Carpoglyphus (Lactis), Japanese spider mite (Oligonichus hondoensis), fir tree spider mite (Oligonichus ununguis), honeybee mite (Varroa jacobsoni), two-spotted tick (Haemaphysalis longicornis), Japanese tick (Ixodes ovatus), Boophilusmicroplus), Amblyomma testudinarium, Ornithonyssus sylvialum, Dermanyssus gallinae, Demodex phylloides, Sarcoptes scabiei bovis, Knemidocoptes mutans, Otodectes cynotis, Psoroptes communis, Ixodes persulcatus, Ornithonyssus bacoti, Chelacaropsis moorei, Pyemotes ventricosus, Demodex folliculorum, Dermotophagoides This includes species such as pteronyssinus, Sarcoptes scabiei, Trombicula akamushi, Chiracanthium japonicum, Heteropoda venatoria, Spermophora senoculata, Pholcus phalangioides, Uroctea compactilis, Plexippus paykulli, Plexippus adansoni, and Isometrus europaeus.

[0045] Pests of the Myriapoda subphylum that can be controlled by the method of the present invention include, but are not limited to, Scolopendra subspinipes mutilans, Scolopendra subspinipes japonica, Scolopendra multidens, Bothropolys asperatus, Thereuronema hilgendofi, and Oxidus gracilis.

[0046] The Hexapoda pests that may be controlled by the method of the present invention are not limited to, but include: Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freeborni, Culex species, Culiseta melanura, Mediterranean fruit fly (Ceratitis capitata), and Mexican fruit fly (Anastrepha). Fruit flies selected from the following: Ludens, Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fruit fly (Bactrocera oleae), Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasis), Queensland fruit fly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental fruit fly (Bactrocera dorsalis), and West Indian fruit fly (Anastrepha obliqua), New World spiral insect (Cochliomyia hominivorax), Old World spiral insect (Chrysomya bezziana), Australian sheep blowfly / copper green blowfly (Lucilia cuprina), Pink moth larva (Pectinophora gossypiella, European gypsy moth (Lymantria dispar), navel orange worm (Amyelois transitella), peach branch borer (Anarsia lineatella), rice stem borer (Tryporyza)Incertulas), Noctuidae moths, Heliothinae tobacco budworms, Papilla japonica beetles, Graphognatus spp. beetles, Anthonomous grandis weevils, Leptinotarsa ​​decemlineata leaf beetles, Planococcus ficus, Diaphorina citri citrus psyllids, Drosophila suzukii fruit flies, Homalodisca vitripennis blue-green sharpshooters, Homalodisca vitripennis glass-winged sharpshooters, Epiphyas postvittana apple moths, Bagrada hilaris, Lymantria dispar asiatica Asian gypsy moths, Lymantria dispar asiatica Japanese gypsy moths Asian gypsy moths selected from the group of Lymantria japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba; Asian longhorn beetles (Anoplophora glabripennis); coconut beetles (Oryctes rhinoceros); emerald ash wood-boring beetles (Agrilus planipennis); European grapevine moths (Lobesia botrana); European gypsy moths (Lymantria dispar); pseudocodling moths (Thaumatotibia leucotreta); red imported fire ants selected from Solenopsis invicta Buren and S. richteri Forel; Old World moth larvae (Helicoverpa armigera); spotted lantern flies (Lycorma delicatula); African honeybees (Apis mellifera) scutellata), fruit shoot borer (Leucinodes orbonalis), corn root worm (Diabrotica spp.), Western corn root worm (DiabroticaCattle flies, horse flies (Tabanus sulcifrons), horned flies (Haematobia irritans), spiral flies (Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi or C. minima), and tsetse flies (Glossina japonica). Warble flies selected from cattle flies (Hypoderma bovis) or striped cattle flies (Hypoderma lineatum), spotted lantern flies (Lycorma delicatula), capra beetles (Trogoderma granarium), honeybee mites (Varroa destructor), termites (Coptotermes formosanus), hemlock woolly aphids (Adelges tsugae), walnut branch beetles (Pityophthorus juglandis), European hornets (Sirex noctilio), pink-spotted moth larvae (Pectinophora scutigera), two-spotted spider mites (Tertanychus urticae), diamondback moths (Plutella xylostella), Taro caterpillars (Spodoptera litura), confused flour beetles (Tribolium) Castaneum, green peach aphid (Myzus persicae), cotton aphid (Aphis gossypii), brown planthopper (Nilaparvata lugens), beet armyworm (Spodotera exigua), and thrips (Frankliniella).This includes *Occidentalis*, *Cydia pomonella*, *Callosobruchus maculatus*, *Acyrthosiphon pisum*, *Tuta absoluta*, *Thrips tabaci*, and *Helicoverpa armigera* larvae.

[0047] In the pest control method of the present invention, a substance that inhibits tracheal formation or hardening pathways by Duox is used as the active control component. Examples of substances used as such active control components include Duox inhibitors or antioxidants that have ROS generation suppression or scavenging activity.

[0048] Duox inhibitors include, for example, means of inhibiting Duox gene expression, means of inhibiting Duox enzymatic activity, and means of selectively disrupting Duox genes or enzymes. One example of a means of inhibiting Duox gene expression is Duox-RNAi, which, as mentioned above, has been confirmed in Non-Patent Literature 2 to inhibit tracheal formation and reduce the amount of intestinal commensal microorganisms (Figures 1 and 2). In addition to ROS (hydrogen peroxide) production discussed above, Duox also possesses peroxidase activity, which is also thought to contribute to the formation of dityrosine bonds in tracheal tissue, and consequently to tracheal formation and maintenance. Therefore, two types of enzymatic activity, ROS production activity and peroxidase activity, can be targets for inhibiting tracheal formation in the present invention. Substances that inhibit any of these Duox tracheal formation pathways can be obtained by those skilled in the art through normal screening without requiring excessive trial and error.

[0049] In the following examples, N-acetylcysteine ​​(NAC) is used as an antioxidant that has an inhibitory or scavenging effect on ROS generation. Antioxidants usable in the present invention include, but are not limited to, vitamins such as vitamin A / retinol, vitamin E / α-tocopherol, coenzyme Q10, sulfur compounds such as N-acetylcysteine, S-methylcysteine, glutathione, L-cysteine, D-cysteine, cysteine ​​persulfide, cysteine ​​trisulfide, allicin, alliin, allyl disulfide, flavonoid polyphenols such as cyanidin chloride, cyanidin-3,5-diglucoside, isoflavones, quercetin, catechin, theaflavin, sesamin, sesaminol, and le This includes sveratrol, non-flavonoid polyphenols such as curcumin, chlorogenic acid, ferulic acid, 3,4-dihydroxycinnamic acid, carotenoids such as β-carotene, lycopene, astaxanthin, zeaxanthin, L-cysteine ​​methyl hydrochloride (LCM), L-cysteine ​​ethyl hydrochloride (LCE), D-cysteine ​​hydrochloride monohydrate (DCH), D-cysteine ​​methyl hydrochloride (DCM), D-penicillamine (DPA), 2-aminoethanethiol (2-AET), L-methionine, and L-methionine methyl hydrochloride (LMM). All antioxidants listed here have been confirmed to be non-toxic to humans, animals, and plants. Antioxidants are common substances in nature, and the pest control compositions and methods of the present invention that use them as active ingredients have an extremely low environmental impact.

[0050] As shown in the examples, among the antioxidants mentioned above, thiols having an -SH group and sulfides having an -SR group (where R represents a monovalent substituent) have been demonstrated to have potent insecticidal activity in the insecticidal control method of the present invention. Therefore, preferably, thiols or sulfides are used as antioxidants in the present invention.

[0051] The pest control method of the present invention inhibits the tracheal formation mechanism, which is essential for survival in terrestrial arthropods, and therefore may be effective against all terrestrial arthropods that possess tracheae. Conversely, this suggests that the control method has low selectivity, or that it may affect insects in the surrounding environment other than the target pest. However, these drawbacks can be appropriately resolved by condition studies and optimizations that are normally carried out in the field of pest control, such as selecting the substance used as the active ingredient, combining it with other compounds, and devising the means of application and the amount to be applied.

[0052] The present invention provides a pest control method comprising applying one or more compounds having an inhibitory effect on Duox activity and / or an inhibitory or scavenging effect on ROS generation, or a pest control composition of the present invention containing such compounds, to the pest, to the place where the pest may live, or to a place or object that is to be protected from damage by the pest.

[0053] The amount and means of application of the pest control composition of the present invention sufficient to achieve desired pest control can be determined by a person skilled in the art through normal condition studies, depending on the species of pest to be controlled, the degree of control, the type of pest control composition to be applied, and the environment in which it is applied. The pest control composition of the present invention can be prepared as a composition in any suitable dosage form, such as aqueous solution or suspension, oily solution or suspension, emulsion, aerosol, powder, granules, wettable powder, water-dispersible granules (powder), water-soluble granules, soluble concentrate, oil-in-water emulsion, microemulsion, aqueous suspension concentrate, aqueous capsule suspension, oil-based suspension concentrate, and aqueous suspension emulsion. If such a pest control composition of the present invention is a liquid, it can be used as is, or diluted, dissolved, or suspended in a predetermined liquid. How the pest control composition of the present invention is prepared can be appropriately determined by a person skilled in the art, taking into account the conditions under which it is actually applied.

[0054] The pest control composition of the present invention can be applied by any means, such as immersion, fumigation, dusting, aerial spraying, aerosol spraying, coating, spraying, solid application, injection, etc.

[0055] The pest control method of the present invention can be used to control pests in all situations in which terrestrial arthropods cause harm, such as in agriculture, forestry, horticulture, public health, material protection, and control of nuisance insects. In each application, those skilled in the art can design a method to achieve the desired pest control by considering the species of pest to be controlled, the degree of control, the type of pest control composition to be applied, the form of the pest control composition, and the environment in which it is applied.

[0056] Accordingly, a person skilled in the art who intends to implement the pest control method of the present invention can identify the target pest species, determine the effective dose of an insecticidal component for controlling the desired pest species through a simple survival test, and apply the pest control composition containing the insecticidal component in an amount and by means sufficient to ensure that the pest species comes into contact with that dose of the insecticidal component under the implementation environment. [Examples]

[0057] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative examples provided for explanatory purposes, and the present invention is not limited in any sense to these examples.

[0058] Example 1. Insecticidal test of Hemiptera insects using antioxidants In this embodiment, N-acetylcysteine ​​(NAC) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the active ingredient in the pest control method of the present invention. NAC is a typical antioxidant and is commonly used in experimental systems for testing the effects of antioxidants. Therefore, those skilled in the art will naturally understand that the results obtained using NAC in this embodiment mean that all other antioxidants used as active ingredients in the present invention should act similarly, and that by combining these with the descriptions in Non-Patent Document 2 and Figures 1-5 of this application, Duox inhibitors should also act similarly.

[0059] NAC was prepared as an aqueous solution (10 mg / ml; adjusted to pH 6.8). Appropriate numbers of Riptortus pedestris, Cletus punctiger, Plautia stali, Dolycoris baccarum, Stenotus binotatus, Stenodema calcarata, and Apolygus spinolae, collected in the field, were placed in plastic containers. Plant seeds (soybean, sunflower seeds, wheat seeds) were given as food, and sterile water was soaked into 3 cm square cotton balls and given to the insects. They were then maintained in an illuminated incubator (manufactured by Tokyo Rikakikai) at 25°C under long-day conditions (16 hours in light, 8 hours in darkness).

[0060] The control effect of NAC on stink bugs was determined by counting the number of surviving stink bugs daily for 10 or 14 days after the start of rearing, and calculating the ratio of surviving individuals to the initial number of individuals. These results are shown in Figures 6-9. In all stink bugs tested, it was confirmed that the survival rate of stink bugs given NAC decreased rapidly and significantly compared to the control group.

[0061] Example 2. Insecticidal tests of various antioxidants Ascorbic acid, N-acetylcysteine ​​(NAC), L-cysteine, L-cysteine ​​methyl hydrochloride (LCM), L-cysteine ​​ethyl hydrochloride (LCE), D-cysteine ​​hydrochloride monohydrate (DCH), D-cysteine ​​methyl hydrochloride (DCM), D-penicillamine (DPA), 2-aminoethanethiol (2-AET), L-methionine, L-methionine methyl hydrochloride (LMM), red cabbage pigment, and uric acid (all manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were used in the experiment (Figure 10). The aqueous solutions of the antioxidants were adjusted to a final concentration of 10 mg / ml and pH 6.8, and each solution was changed once every 5 days. The control group was fed sterile water and aqueous ascorbic acid solution. The number of dead individuals was counted after checking the rearing containers.

[0062] For ascorbic acid, NAC, L-cysteine, LCM, LCE, DCH, DCM, DPA, 2-AET, L-methionine, and LMM, the concentration was adjusted to 1 mg / ml (pH 6.8) as a low-concentration treatment, and the number of deaths was measured in the same manner.

[0063] In this study, the slender stink bug (Riptortus pedestris) was used.

[0064] For the slender stink bug, larvae (nymphs) were used in the experiment. Ten stink bugs were placed in a plastic petri dish and given plant seeds (soybeans, wheat, sunflower) as food. At this time, the antioxidant solution mentioned above was added to cotton swabs and used as water for hydration.

[0065] result Measurement of the mortality rate of insects treated with each antioxidant. Screening of various antioxidants (at a concentration of 10 mg / ml) using the slender stink bug revealed that red cabbage pigment and uric acid showed no insecticidal effect, and ascorbic acid had a limited insecticidal effect. However, compounds containing thiol groups (-SH) or sulfide groups (-SCH3) showed high insecticidal effects (Figure 11A).

[0066] Among the antioxidants used, 2-AET, L-cysteine, L-cysteine ​​methyl hydrochloride (LCM), D-cysteine ​​methyl hydrochloride (DCM), and L-methionine resulted in the death of all individuals within 7 days, demonstrating an early effect (Figure 11B). On the other hand, the insecticidal effects of N-acetylcysteine ​​(NAC), D-cysteine ​​hydrochloride monohydrate (DCH), D-penicillamine (DPA), and L-methionine methyl hydrochloride (LMM) were slower (Figure 11B). Furthermore, mortality rates increased with NAC, DPA, and LMM when the insects changed ages due to molting (Figure 12).

[0067] Immunostaining with dityrosine antibody was used to observe a decrease in tracheal formation in the digestive tract of slender stink bugs treated with LCM, L-cysteine, or NAC (10 mg / ml, pH 6.8) (Figure 13). Application of LCM and NAC significantly reduced tracheal formation compared to the control (ascorbic acid). Furthermore, application of L-cysteine ​​resulted in the disappearance of large tracheae.

[0068] The insecticidal activity of ascorbic acid, NAC, L-cysteine, LCM, LCE, DCH, DCM, DPA, 2-AET, L-methionine, and LMM was tested at low concentrations, adjusted to 1 mg / ml. As a result, LCM showed the highest insecticidal effect, with a mortality rate of over 80% observed at day 7 (Figure 14).

[0069] Example 3. Insecticidal tests against various insect species Based on these results, we also investigated the effectiveness of the treatment on other insects besides the slender stink bug mentioned above, including the green stink bug (Apolygus spinolae), red roach (Blattea lateralis), mealworm (Tenebrio molitor), and yellow fruit fly (Drosophila melanogaster).

[0070] For the *Lymantria dispar* stink bug, adult specimens were used in the experiment. Ten stink bugs were placed in a plastic petri dish and given plant seeds (soybeans, wheat, sunflower) as food. At this time, the antioxidant solution mentioned above was added to cotton swabs and used as water for hydration.

[0071] For the red roach, larvae (nymphs) were used in the experiment. Ten red roaches were placed in a plastic cup and fed with tropical fish food. At this time, the antioxidant solution mentioned above was added to cotton swabs and used as water for feeding.

[0072] For mealworms, larvae were used in the experiment. Ten mealworms were placed in a plastic petri dish and fed with tropical fish food. The antioxidants mentioned above were mixed into the food, and the final concentration was adjusted to 10 mg / ml.

[0073] Adult Drosophila melanogaster were used in the experiment. Ten flies were placed in a plastic cup and fed a 10% honey solution. The antioxidant was suspended in the honey solution and adjusted to a final concentration of 10 mg / ml.

[0074] As a result, the insecticidal effects of compounds containing thiol groups were observed in insects other than the slender stink bug, and the most effective antioxidant differed depending on the insect species (Figure 15). Specifically, L-cysteine ​​and LCM showed high insecticidal effects in the green stink bug, while NAC showed the highest insecticidal effect in the red roach. In addition, L-cysteine ​​showed the highest insecticidal effect in mealworms, and LCM showed the highest insecticidal effect in fruit flies.

Claims

1. A composition for controlling insects, comprising one or more antioxidants, wherein the antioxidants are thiols or sulfides, and which have an inhibitory or scavenging effect on the generation of reactive oxygen species (ROS) in the body of the insect to be controlled, thereby reducing the tracheal formation ability of the insect and reducing the survival rate of the insect.

2. The composition according to claim 1, wherein the antioxidant is selected from the group consisting of N-acetylcysteine ​​(NAC), S-methylcysteine, glutathione, L-cysteine, D-cysteine, cysteine ​​persulfide, cysteine ​​trisulfide, allicin, alliin, allyl disulfide, L-cysteine ​​methyl hydrochloride (LCM), L-cysteine ​​ethyl hydrochloride (LCE), D-cysteine ​​hydrochloride monohydrate (DCH), D-cysteine ​​methyl hydrochloride (DCM), D-penicillamine (DPA), 2-aminoethanethiol (2-AET), L-methionine, L-methionine methyl hydrochloride (LMM), and two or more combinations thereof.

3. The composition according to claim 1, wherein the insect expresses dual oxidase (Duox) in its trachea.

4. The composition according to claim 1, wherein the insect is selected from the group consisting of Hemiptera insects including Riptortus pedestris, Cletus punctiger, Plautia stali, Dolycoris baccarum, Stenotus binotatus, Stenodema calcarata, and Apolygus spinolae; Blattodea insects including Blatta (Shelfordella) lateralis and Reticulitermes speratus; Coleoptera insects including Tenebrio molitor; Diptera insects including Drosophila melanogaster; and Lepidoptera insects.

5. The composition according to claim 1, having a dosage form selected from the group consisting of granules, hydrated powder, water-dispersible granules (powder), water-soluble granules, soluble concentrate, oil-in-water emulsion, microemulsion, aqueous suspension concentrate, aqueous capsule suspension, oil-based suspension concentrate, and aqueous suspension emulsion.

6. The composition according to claim 1, applied by a method selected from the group consisting of immersion, fumigation, dusting, aerial spraying, aerosol spraying, coating, spraying, solid spraying, and injection.

7. A method for controlling insects, comprising using the composition described in any one of claims 1 to 6 to suppress or eliminate the generation of reactive oxygen species (ROS) in the body of the insect to be controlled, thereby reducing the tracheal formation ability of the insect and reducing the survival rate of the insect.

8. The method for controlling insects according to claim 7, wherein the method comprises applying the composition according to any one of claims 1 to 6 to a place where the insects may live, or to a place where contact with the insects is to be avoided.

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