Method for controlling insects by oral administration of trehalase inhibitor

Abstract

To provide an insect control composition and an insect control method utilizing a novel mechanism of action of a trehalase inhibitor.SOLUTION: A trehalase inhibitor is orally administered to an insect to be controlled that carries trehalose-requiring symbiotic bacteria in symbiotic organs of the intestinal tract, thereby suppressing the trehalose-requiring symbiotic bacteria in the intestinal tract to control the host insect.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an insect control composition containing a trehalase inhibitor, and a method for controlling insects using the trehalase inhibitor. [Background technology]

[0002] Invertebrates that have some harmful effect on human social activities are generally referred to as "pests." Pests, particularly in the agricultural sector, are considered a problem that imposes enormous economic costs on modern agriculture. Current agricultural systems require the cultivation of one or several crops or plant species over vast areas. Such ecologically unbalanced systems are prone to damage by pests, and it is said that one-third of agricultural crops are currently 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. Malaria, in particular, currently affects more than 200 million people annually, mainly in tropical regions, and is said to be one of the world's three major infectious diseases, along with tuberculosis and AIDS, that pose a serious health risk to humanity.

[0004] The most common method of pest control is the use of chemical insecticides, which are compounds that have a detrimental effect on various physiological functions of target pests. For example, chemical insecticides that target various physiological functions of pests have been developed, such as neurotransmitter inhibitors of pests (e.g., organophosphate, carbamate, and neonicotinoid insecticides), electron transport chain I-III inhibitors, intracellular respiration inhibitors (e.g., uncoupling agents), growth regulators (e.g., chitin synthesis inhibitors and hormone-like substances), metabolic inhibitors (e.g., tetronic acid or tetramic acid derivatives, and dihalopropenes).

[0005] Chemical insecticides have a direct and powerful control effect on target pests and are easy to obtain and use, so they are widely used in various environments where pest control is necessary. However, there are technical issues that hinder the use of insecticides, such as ecosystem disruption in the surrounding environment (environmental burden), the possibility of adverse effects on the human body, and the development of resistance among pests, and these have not all been resolved. Therefore, there is always a high demand for new pest control methods that target the physiological functions of pests that are not targeted by conventional chemical insecticides, as they will increase the options to complement or replace the use of existing chemical insecticides.

[0006] Validamycin (also written as validamycin A or VMA), a type of antibiotic, and its aglycone validoxylamine (also written as validoxylamine A or VaA) are commercially available as pesticides for fungal plant pathogens. VMA and VaA are structurally similar to trehalose and act as inhibitors of the trehalase enzyme (EC 3.2.1.28). Thus, VMA and VaA control trehalose-requiring plant pathogens through their trehalase inhibitory activity.

[0007] The trehalase inhibitors VMA and VaA also have a control effect against other organisms that require trehalose as an essential substance for survival (trehalose auxotrophy), such as the above-mentioned plant pathogens. Because insects use trehalose as their primary blood sugar, trehalase inhibition by VMA and VaA is expected to be effective in controlling insects. The effects of VMA and VaA on various insects have been tested, and it has been reported that VMA and VaA inhibit insect trehalase, resulting in growth inhibition, malformation, and other problems (Non-Patent Documents 1 to 4).

[0008] Based on such reports, one might think of using VMA and VaA as chemical insecticides to control agricultural pest insects, but the above reports have shown that VMA and VaA are ineffective when administered percutaneously or orally, and injection into the body, which has been the main method of administration, is difficult to use as a means of administering insecticides. Therefore, attempts to develop chemical insecticides for insect control that contain VMA and VaA as active ingredients have not been successful. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Tatun, N., Tungjitwitayakul, J. & Sakurai, S. Developmental and Lethal Effects of Trehalase Inhibitor (Validamycin) on the Tribolium castaneum (Coleoptera: Tenebrionidae). Ann. Entomol. Soc. Am. 109, 224-231 (2016). [Non-patent document 2] Zhang, L. et al. Study on the Effect of Wing Bud Chitin Metabolism and Its Developmental Network Genes in the Brown Planthopper, Nilaparvata lugens, by Knockdown of TRE Gene. Front. Physiol. 8, 750 (2017). [Non-patent document 3] Tang, B. et al. Suppressing trehalase activity with validamycin disrupts the trehalose and chitin biosynthesis pathways in the rice brown planthopper, Nilaparvata lugens. Pestic. Biochem. Physiol. 137, 81–90 (2017).

Outdoor Tools 4

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Outdoor Configuration6

Direct Environment 7

[0010] The present invention provides a novel means for controlling insects by orally ingesting a trehalase inhibitor, which inhibits symbiotic bacteria present in symbiotic organs in the intestinal tract, thereby controlling insects. [Means for solving the problem]

[0011] The inventors of the present invention focused on the fact that symbiotic bacteria present in the symbiotic organs of the intestinal tract of stink bugs and other insects are trehalose-requiring, and through research into the carbon source utilization of these symbiotic bacteria and their effects on the host, they discovered that compounds that act on the symbiotic bacteria in the intestinal tract, rather than on the stink bugs themselves, can be used as insecticides and pesticides.In other words, they discovered that it is possible to provide chemical insecticides with a new mechanism of action that suppress the symbiotic bacteria necessary for stink bug survival from the stink bug, thereby ultimately eliminating the stink bug host.This led to the present invention.

[0012] Therefore, the present application provides the following inventions.

[0013] 1. A composition for controlling insects, which contains a trehalase inhibitor and is applied to insects to be controlled that carry symbiotic bacteria in symbiotic organs in their intestinal tracts by orally ingesting the composition so that the symbiotic bacteria are exposed to the trehalase inhibitor. 2. The composition according to item 1, wherein the trehalase inhibitor is validamycin or validoxylamine A. 3. The composition according to Item 1, wherein the control is achieved by one or more of the following effects: inhibition of growth or death of larvae of the insect to be controlled, or suppression of egg-laying in adult insects and reduction of the hatchability of laid eggs, by the trehalase inhibitor. 4. The composition according to item 1, wherein the symbiotic bacterium is a trehalose-requiring symbiotic bacterium. 5. The composition described in item 1, wherein the symbiotic bacteria comprise one or more species of bacteria belonging to any one of the classes Gammaproteobacteria, Betaproteobacteria, or Actinobacteria. 6. The composition according to item 1, wherein the symbiotic bacteria comprise one or more bacteria belonging to any one of the genera Pantoea, Rhodococcus, and Tachikawaea. 7. The composition according to item 4, wherein the trehalose-requiring symbiotic bacterium has a gene encoding trehalase. 8. The composition according to item 7, wherein the trehalase is a prokaryotic periplasmic trehalase. 9. The composition according to item 7, wherein the gene encoding the trehalase is treA. 10. The composition according to item 1, wherein the insect belongs to any one of the orders Hemiptera, Coleoptera, or Blattodea. 11. The insects are selected from the families Pentatomidae, Scutelleridae, Urostylidae, Dinidoridae, Cydnidae, Coreidae, Berytidae, Largidae, Alydidae, Lygaeidae, Pyrrhocoridae, and the like. Item 1. The composition according to item 1, wherein the beetle belongs to any one of the following families: Rhocoridae, Plataspidae, Acanthosomatidae, Rhopalidae, Miridae, Tingidae, Chrysomelidae, Isoptera, Triatominae, or Curculionidae. 12. The composition according to item 1, wherein the insect belongs to any one of the families Pentatomidae, Scutelleridae, or Urostylidae. 13. The insects are selected from the group consisting of green stink bugs (Nezara antennata), red stink bugs (Pygomenida bengalensis), red-striped stink bugs (Graphosoma rubrolineatum), striped stink bugs (Piezodorus hybneri), rice stink bugs (Lagynotomus elongatus), black rice stink bugs (Scotinophara lurida), cow bugs (Alcimocoris japonensis), Siberian green stink bugs (Palomena angulosa), shrimp-colored stink bugs (Gonopsis affinis), large spur-spotted white spotted stink bugs (Eysarcoris lewisi), yellow spotted stink bugs (Erthesina fullo), brown marmorated stink bugs (Halyomorpha mista), white-spotted stink bug (Eysarcoris ventralis), brown-winged green bug (Plautia stali), horned green bug (Pentatoma japonica), white-spotted stink bug (Menida violacea), shiny green bug (Glaucias subpunctatus), shiny white-spotted stink bug (Eysarcoris annamita), thorny stink bug (Carbula humerigera), thorny white-spotted stink bug (Eysarcoris parvus), spotted stink bug (Lelia decempunctata), long-legged stink bug (Eurydema rugosum), small long-legged stink bug (Eurydema pulchrum), spotted stink bug (Dolycoris baccarum), white-spotted stink bug (Eysarcoris guttiger), citrus spiny stink bug (Rhynchocoris humeralis), southern green stink bug (Nezara viridula), purple spotted stink bug (Carpocoris purpureipennis), purple spotted stink bug (Eysarcoris annamita), four-spotted stink bug (Homalogonia obtusa), red-striped stink bug (Poecilocoris Lewisii), large golden stink bug (Eucorysses grandis), brown stink bug (EurygasterItem 2. The composition according to item 1, wherein the insecticide is selected from the group consisting of: insecticides against stink bugs (Urochela luteovaria), and insecticides against stink bugs (Urochela luteovaria). 14. The composition according to item 1, wherein the insects are selected from the group consisting of the brown winged stink bug (Plautia stali), the brown marmorated stink bug (Halyomorpha halys), and the shiny green stink bug (Nezara antennata). 15. The composition according to item 1, wherein the composition is applied so that the trehalase inhibitor is contained in food or water ingested by the insects to be controlled. 16. A method for controlling insects, comprising orally ingesting the composition according to any one of items 1 to 15 to an insect to be controlled, the insect carrying a trehalose-requiring symbiotic bacterium in a symbiotic organ in the intestinal tract, thereby exposing the symbiotic bacterium to a trehalase inhibitor. 17. The method according to item 16, comprising applying the composition according to any one of items 1 to 15 so that the trehalase inhibitor is contained in food or water ingested by the insect to be controlled. 18. The method according to item 16, comprising inhibiting trehalase activity in the insect's intestinal tract, thereby suppressing trehalose-requiring symbiotic bacteria in the intestinal tract. [Effects of the Invention]

[0014] The present invention provides a novel insect control composition and method based on a new mechanism of action in which a trehalase inhibitor is orally ingested by insects, such as stink bugs, that harbor symbiotic bacteria in symbiotic organs in their intestinal tracts, thereby killing the symbiotic bacteria in the intestinal tract. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the VMA and VaA susceptibility assessment of culturable symbiotic bacteria of the German-winged green stink bug in the presence of glucose or trehalose.

[0016] [Figure 2]Figure 2 shows the survival rate of newly hatched German-winged green stink bug nymphs 26 days after oral administration of VMA. In the box-and-whisker plot, the horizontal bar indicates the median, the top and bottom of the box indicate the third and first quartiles, respectively, and the whiskers indicate the maximum and minimum values. Statistically significant differences were observed (P < 0.05 in the pairwise Wilcoxon test).

[0017] [Figure 3] Figure 3 shows the survival rate of newly hatched German-winged stink bug larvae orally administered with VaA 26 days after administration. A statistically significant difference was observed (P<0.05 in the pairwise Wilcoxon test).

[0018] [Figure 4] Figure 4 shows the survival rate of newly hatched brown marmorated stink bug larvae 26 to 28 days after oral administration of VMA. A statistically significant difference was observed (P<0.05 in the Wilcoxon test).

[0019] [Figure 5] Figure 5 shows the survival rate of newly hatched shiny green stink bug larvae orally administered VMA 24 to 27 days after administration. A statistically significant difference was observed (P<0.05 in the Wilcoxon test).

[0020] [Figure 6] Figure 6 shows the total number of eggs laid by adult German-winged green stink bugs that were orally administered VMA after pairing, one month after administration. A statistically significant difference was observed (Wilcoxon test, P<0.05).

[0021] [Figure 7] Figure 7 shows the evaluation of egg hatchability obtained from the evaluation of total egg production in Figure 6. Statistically significant differences were observed (P<0.05 in the Wilcoxon test).

[0022] [Figure 8]Figure 8 shows the survival rate of newly hatched German-winged stink bug nymphs, from which the symbiotic bacteria had been removed, 26 days after oral administration of VMA. No statistically significant difference was observed (Pairwise Wilcoxon test). DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be described below based on specific embodiments, but the present invention is not limited to these embodiments. All documents cited in this specification, including patent publications, patent application publications, and non-patent publications, are incorporated herein by reference in their entirety for all purposes.

[0024] In the present invention, "symbiotic bacteria" refers to bacteria that form a symbiotic relationship with a host insect and are essential for the normal growth, reproduction, or survival of the host insect. Symbiotic relationships between insects and these symbiotic bacteria are widely observed in insects that utilize nutritionally unbalanced food resources, and the symbiotic bacteria supplement the nutrients necessary for the normal growth and reproduction of the host insect that are lacking in these food resources, while the bacteria are also protected within the host body and can obtain the necessary nutrients from the insect body. Host insects that have lost their symbiotic bacteria exhibit significant growth deficiencies due to a lack of the essential nutrients provided by the symbiotic bacteria.

[0025] For example, between aphids and the Buchnera bacteria that live symbiotically in their intestines, the Buchnera bacteria supply essential amino acids, and the aphids provide the symbiotic bacteria with a living environment and essential amino acids and other nutrients (Non-Patent Document 5).Furthermore, between bedbugs and Wolbachia bacteria, the Wolbachia bacteria supply B vitamins, which are difficult for bedbugs to obtain from their blood, and the bedbugs provide the Wolbachia bacteria with a living environment and various nutrients (Non-Patent Document 6).

[0026] The inventors of the present invention aim to elucidate the mutualistic system between insects and microorganisms, in particular, using the symbiotic system between the German-winged green stink bug (Plautia stali) and its intestinal symbiotic bacteria as a model to empirically elucidate the function of the symbiotic bacteria, the molecular basis for maintaining the symbiotic system, and the evolutionary pathway. The carbon source used by the symbiotic bacteria of the German-winged green stink bug within the host body was previously unknown, and no specific research had been conducted on this topic.

[0027] The German-winged green bug is known as a pest that damages mandarin oranges and other fruit trees with its proboscis, and when it infests an area in large numbers, it can cause severe damage to agricultural production in that area. Many insects in the Hemiptera order, in addition to the German-winged green bug, are pests of agricultural crops, and appropriate and effective control of them is required in agriculture.

[0028] The German-winged green bug is highly dependent on its symbiotic bacteria, and cannot develop normally without them. When the symbiotic bacteria were removed, most German-winged green bug larvae died at the fourth or fifth instar, and the few adults that did emerge were small, brown, and had lost the ability to reproduce.

[0029] As mentioned above, it was previously unknown which carbon source the symbiotic bacteria of stink bugs use to survive. The main blood sugar of insects, including stink bugs, is trehalose. Sugars ingested by insects are digested into glucose in the digestive tract and absorbed into the body, but are immediately converted into trehalose in the fat body and released into the body fluids. In organs and tissues that use the sugars, trehalase converts them back into glucose. While vertebrates use glucose from blood sugar directly, insects maintain a system that converts glucose into trehalose, suggesting that trehalose plays some important role within the insect body.

[0030] Furthermore, in an evolution experiment of an artificial gut symbiotic system constructed by infecting stink bugs with E. coli, the inventors found that mutations in the carbon catabolite repression (CCR) regulatory system caused E. coli to become a symbiotic bacterium. The fact that a deficiency in the CCR regulatory system was necessary for E. coli to become a symbiotic bacterium means that glucose is not available (is not present in sufficient amounts) in the symbiotic organ in the stink bug's intestinal tract, suggesting that the symbiotic bacterium in stink bugs uses a carbon source other than glucose.

[0031] Furthermore, genome analysis of the stinkbug-derived symbiotic bacteria SymA and SymB revealed that although they had lost many glycosidases due to genome degeneracy (the reduction in genome size of the symbiotic bacterium due to symbiotic evolution between the host and the symbiotic bacterium), they retained the gene encoding trehalase. In particular, SymB's genome size had shrunk significantly compared to other symbiotic bacteria, and it contained almost no glycosidases other than those required for trehalose degradation.

[0032] From the above, it was inferred that the symbiotic bacteria of stink bugs use trehalose as a carbon source. To verify this, stink bugs were orally administered a trehalase inhibitor and reared, and the growth-inhibitory effect was evaluated.

[0033] The trehalase inhibitors used here were validamycin (VMA) and its aglycone, validoxylamine (VaA). VMA and VaA are well-known trehalase inhibitors with structures similar to trehalose, and are commonly used as control agents for trehalose-requiring plant pathogens.

[0034] Trehalose utilization and the antibacterial activity of validamycin against the cultivable symbiotic bacterium SymC derived from stink bugs were previously tested. SymC grew vigorously on M9 medium supplemented with trehalose, but its growth was significantly inhibited by the addition of 10 μg / mL validamycin.

[0035] In fact, the insecticidal effect of validamycin on the German winged green bug was tested, and when the bugs were reared in water containing 100 ppm validamycin, more than 80% of the larvae died by the time they reached the third instar.

[0036] To verify whether the insecticidal effect of validamycin is due to the suppression of symbiotic bacteria or to a direct effect on stink bugs, we created "symbiotic bacteria-free insects" that lack symbiotic bacteria and evaluated the effects of oral administration of validamycin in the same manner as described above. The larval growth of the symbiotic bacteria-free insects was delayed, and no individuals emerged. However, there was no significant difference in the mean survival rate between the control group, the 10 ppm VMA-treated group, and the 20 ppm VMA-treated group. On the other hand, when the symbiotic bacteria were not removed, the mean survival rate decreased in a VMA concentration-dependent manner. These results indicate that the insecticidal effect of oral administration of validamycin on stink bugs is due to the suppression of symbiotic bacteria.

[0037] In the prior art, it was believed that validamycin was ineffective when orally ingested by insects, and the effect of validamycin on insects was tested by injection into the body cavity. The findings of the present invention are contrary to such prior art. This is presumably because, in the insects targeted for control in the present invention, symbiotic bacteria reside in symbiotic organs developed in the posterior part of the digestive tract, and orally ingested validamycin can reach the symbiotic bacteria.

[0038] Therefore, one aspect of the present invention relates to a composition for controlling insects, which contains a trehalase inhibitor and is applied to target insects that carry symbiotic bacteria in symbiotic organs in their intestinal tracts by orally ingesting the composition so that the symbiotic bacteria are exposed to the trehalase inhibitor (hereinafter referred to as the "insect control composition of the present invention").

[0039] Another aspect of the present invention relates to a method for controlling insects, which comprises orally ingesting the insect control composition of the present invention to an insect to be controlled, the insect carrying symbiotic bacteria in a symbiotic organ in its intestinal tract, thereby exposing the symbiotic bacteria to a trehalase inhibitor (hereinafter appropriately referred to as the "insect control method of the present invention").

[0040] The insect control composition and method of the present invention control host insects by orally ingesting a trehalase inhibitor that inhibits trehalose-requiring symbiotic bacteria in the intestinal tract.

[0041] In the present invention, "control" means reducing the damage caused by insects by suppressing or preventing the emergence, arrival, growth, activity, or reproduction of the insects to be controlled, or by reducing their survival rate or killing them. The insect control composition and insect control method of the present invention damage the host insect by suppressing the symbiotic bacteria in the intestinal tract, and can control insects in one or more of the above senses. Furthermore, in the present invention, "suppression" of symbiotic bacteria means that the symbiotic bacteria are reduced, the balance of the bacterial population is changed, or their activity is reduced, making it impossible to supply substances necessary for maintaining the normal physiological functions of the host insect.

[0042] The following examples demonstrate that the trehalase inhibitors validamycin and validoxylamine A have the effect of inhibiting the growth of or killing stink bug larvae, as well as the effect of suppressing egg-laying in adult stink bugs and reducing the hatchability of laid eggs.

[0043] Insects to be controlled in the present invention must harbor trehalose-requiring symbiotic bacteria in their intestinal symbiotic organs, and this characteristic allows orally ingested trehalase inhibitors to reach the symbiotic bacteria and inhibit the activity of trehalase, which is necessary for the survival of the symbiotic bacteria, thereby suppressing the symbiotic bacteria.

[0044] Preferably, the symbiotic bacteria inhibited by the composition or method of the present invention have a gene encoding trehalase (EC 3.2.1.28). Particularly preferably, the trehalase is a prokaryotic periplasmic trehalase or is encoded by the trehalase gene treA.

[0045] The symbiotic bacteria inhibited by the compositions or methods of the present invention include, but are not limited to, one or more bacteria belonging to the classes Gammaproteobacteria, Betaproteobacteria, or Actinobacteria. Alternatively, the symbiotic bacteria include, but are not limited to, one or more bacteria belonging to the genera Pantoea, Rhodococcus, or Tachikawaea. Alternatively, the symbiotic bacteria include, but are not limited to, Rhodococcus rhodnii.

[0046] Whether an insect harbors symbiotic bacteria in a symbiotic organ in its intestinal tract and whether the symbiotic bacteria harbored are trehalose-requiring is known or can be easily determined by histological testing. Insects that can be controlled by the compositions or methods of the present invention include, but are not limited to, insects belonging to the orders Hemiptera, Coleoptera, and Blattodea.

[0047] Insects that can be controlled by the composition or method of the present invention include, but are not limited to, Pentatomidae, Scutelleridae, Urostylidae, Dinidoridae, Cydnidae, Coreidae, Berytidae, and O. nigricans. These include insects belonging to the families Largidae, Alydidae, Lygaeidae, Pyrrhocoridae, Platyceridae, Heteroptera, Pyrrhocoridae, Miridae, Tingidae, Chrysomelidae, Isoptera, Triatominae, and Curculionidae. Preferably, the insects are from the Pentatomidae, Scutelleridae, Urostylidae, Dinidoridae, Cydnidae, Coreidae, Berytidae, Largidae, Alydidae, and Lygaeidae families. ), more preferably, Pentatomidae, Scutelleridae, Urostylidae, Dinidoridae, and Cydnidae, and even more preferably, Pentatomidae, Scutelleridae, and Urostylidae.

[0048] Insects that can be controlled by the composition or method of the present invention include, but are not limited to, the green stink bug (Nezara antennata), the red stink bug (Pygomenida bengalensis), the red-striped stink bug (Graphosoma rubrolineatum), the spotted stink bug (Piezodorus hybneri), the rice stink bug (Lagynotomus elongatus), the black rice stink bug (Scotinophara lurida), the cow bug (Alcimocoris japonensis), the Siberian green stink bug (Palomena angulosa), the shrimp-colored stink bug (Gonopsis affinis), the large spiny spotted stink bug (Eysarcoris lewisi), the yellow spotted stink bug (Erthesina fullo), the brown marmorated stink bug (Halyomorpha mista), white-spotted stink bug (Eysarcoris ventralis), brown-winged green bug (Plautia stali), horned green bug (Pentatoma japonica), white-spotted stink bug (Menida violacea), shiny green bug (Glaucias subpunctatus), shiny white-spotted stink bug (Eysarcoris annamita), thorny stink bug (Carbula humerigera), thorny white-spotted stink bug (Eysarcoris parvus), spotted stink bug (Lelia decempunctata), long-legged stink bug (Eurydema rugosum), small long-legged stink bug (Eurydema pulchrum), spotted stink bug (Dolycoris baccarum), white-spotted stink bug (Eysarcoris guttiger), citrus spiny stink bug (Rhynchocoris humeralis), southern green stink bug (Nezara viridula), purple spotted stink bug (Carpocoris purpureipennis), purple spotted stink bug (Eysarcoris annamita), four-spotted stink bug (Homalogonia obtusa), red-striped stink bug (Poecilocoris Lewisii), large golden stink bug (Eucorysses grandis), brown stink bug (Eurygastertestudinaria, Japanese stink bug (Urochela luteovaria), sawtoothed stink bug (Megymenum gracilicorne), round-toothed stink bug (Microporus nigrita), foot-striped stink bug (Leptoglossus gonagra), adzuki bean stink bug (Homoeocerus marginiventris), large spider stink bug (Anacanthocoris striicornis), Japanese rice stink bug (Hygia opaca), broad-toothed stink bug (Homoeocerus dilatatus), ground cherry stink bug (Acanthocoris sordidus), spotted stink bug (Homoeocerus (Tliponius) unipunctatus), narrow-toothed stink bug (Cletus punctiger), southern spiny stink bug (Paradasynus spinosus) Hsiao, Yellow-stem bug (Yemma exilis), Spider bug (Leptocorisa chinensis), Bean-spotted bug (Leptocorisa acuta), Large spotted bug (Physopelta gutta), Small spotted bug (Physopelta parviceps), Brown-spotted bug (Riptortus clavatus), Strawberry long-horned bug (Stigmatonotum geniculatum), Candy spotted bug (Cavelerius saccharivorus), White-spotted bug (Panaorus japonicus), Brown long-horned bug (Neolethaeus dallasi), Cabbage long-horned bug (Panaorus albomaculatus), Red spotted bug (Dysdercus cingulatus), two-spotted stink bug (Pyrrhocoris sibiricus), large stink bug (Piocoris varius), long-legged stink bug (Tropidothorax cruciger), small long-legged stink bug (Tropidothorax sinensis), rice-striped stink bug (Chauliops fallax), and long-legged stink bug (Arocatusmelanostomus, Nysius plebeius, Coptosoma semiflavum, Megacopta cribraria, Coptosoma biguttulum, Megacopta punctatissimum, Acanthosoma denticaudum, Elasmucha signoreti, Aeschynteles maculatus, Liorhyssus hyalinus, Creontiades pallidifer, Arbolygus rubripes, Apolygus spinolae (Meyer-Dur)), Pale-striped grass turtle (Taylorilygus pallidulus), Large black-striped grass turtle (Ectometopterus micantulus), Black-striped grass turtle (Halticiellus insularis), Green grass turtle (Apolygus lucorum), Small green grass turtle (Apolygus lucorum), White-striped artemisiae grass turtle (Europiella artemisiae), Tobacco grass turtle (Cyrtopeltis tennuis), Black-striped green grass turtle (Apolygus spinolai), Black-striped grass turtle (Adelphocoris suturalis), Black-striped green grass turtle (Apolygus nigronasutus), Long-whiskered grass turtle (Adelphocoris lineolatus), Two-striped grass turtle (Adelphocoris variabilis), Spotted Black Migratory Bug (Adelphocoris triannulatus), Spiky Migratory Bug (Lygus disponsi), Monk's Black Migratory Bug (Deraeocoris ater), Apple Black Migratory Bug (Heterocordylus flavipes), Chrysanthemum Rootworm (Galeatus spinifrons), Pear Rootworm (Stephanitisnashi), Triatominae insects (Rhodnius prolixus, Triatoma dimidiata, Triatoma infestans, Triatoma rubrofasciata, Panstrongylus megistus), and bonito weevils (Lixus impressiventris). Preferably, the insects include the green stink bug (Nezara antennata), the red stink bug (Pygomenida bengalensis), the red striped stink bug (Graphosoma rubrolineatum), the striped stink bug (Piezodorus hybneri), the rice stink bug (Lagynotomus elongatus), the black rice stink bug (Scotinophara lurida), the cow bug (Alcimocoris japonensis), the Hokkaido green stink bug (Palomena angulosa), the shrimp-colored stink bug (Gonopsis affinis), the large spur-spotted white spotted bug (Eysarcoris lewisi), the yellow spotted stink bug (Erthesina fullo), the brown marmorated stink bug (Halyomorpha mista), and the white spotted stink bug (Eysarcoris ventralis, brown-winged stink bug (Plautia stali), horned green bug (Pentatoma japonica), white-spotted stink bug (Menida violacea), shiny green bug (Glaucias subpunctatus), shiny spotted stink bug (Eysarcoris annamita), thorny stink bug (Carbula humerigera), thorny spotted stink bug (Eysarcoris parvus), spotted stink bug (Lelia decempunctata), long-legged stink bug (Eurydema rugosum), small long-legged stink bug (Eurydema pulchrum), spotted stink bug (Dolycoris baccarum), spotted stink bug (Eysarcoris guttiger), orange spiny stink bug (Rhynchocoris humeralis), southern green stink bug (Nezara viridula), purple stink bug (Carpocorispurpureipennis, purple spotted stink bug (Eysarcoris annamita), four-spotted stink bug (Homalogonia obtusa), red-striped stink bug (Poecilocoris Lewisii), large golden stink bug (Eucorysses grandis), brown stink bug (Eurygaster testudinaria), and white-spotted stink bug (Urochela luteovaria), and more preferably, brown stink bug (Plautia stali), brown marmorated stink bug (Halyomorpha halys), and shiny green stink bug (Nezara antennata).

[0049] In the insect control composition and insect control method of the present invention, a trehalase inhibitor is used as an active ingredient for suppressing symbiotic bacteria in the insect gut. In a preferred embodiment of the present invention, validamycin (also written as validamycin A or VMA) or its aglycone, validoxylamine (also written as validoxylamine A or VaA) is used as the trehalase inhibitor of the present invention. In the examples described below, validamycin and validoxylamine A are used as trehalase inhibitors. However, the technical scope of the present invention should not be limited by such examples. Rather, these examples support the high probability that other compounds having trehalase inhibitory activity can function as active ingredients for insect control in the present invention, similar to the exemplified validamycin and validoxylamine A, when combined with the mechanism of action of trehalase inhibitors for insect control revealed for the first time in the present invention.

[0050] Validamycin was identified as an antibiotic produced by the actinomycete Streptomyces hygroscopicus var. limoneus collected in Akashi City, Hyogo Prefecture, and is commercially available as a pesticide against various fungal and bacterial plant pathogens. Validamycin is structurally similar to trehalose and has been shown to act as a trehalase inhibitor.

[0051] Since its launch in 1972, validamycin has been used as an agricultural fungicide to control a wide variety of plant diseases in various crops, and safe and efficient methods for its use have been established. Therefore, it should be easy to use validamycin as the active ingredient of an insecticide for insect control in the present invention. Despite this, the fact that attempts to use validamycin as an insecticide targeting insects have not been successful to date is evidence that such conversion is not a mere compilation of known technologies, but rather requires the discovery, first discovered by the present invention, that orally ingested validamycin controls host insects by suppressing trehalose-requiring symbiotic bacteria in the intestinal tract.

[0052] The trehalase inhibitor used as an active ingredient in the insect control composition and control method of the present invention may also affect the species of organisms in the surrounding environment that utilize trehalose. However, such inconveniences can be appropriately resolved by examining and optimizing the conditions, such as the type of trehalase inhibitor used, the application amount, the combination with other compounds, and the application method, which are commonly performed in the technical field of pest control.

[0053] The insect control method of the present invention comprises applying the pest control composition of the present invention containing a trehalase inhibitor so that the trehalase inhibitor is contained in food or water ingested by the insect.

[0054] The amount and application means of the pest control composition of the present invention sufficient to achieve the desired insect control can be determined by those skilled in the art through routine consideration of the type of insect to be controlled, the degree of control, the form of the pest control composition to be applied, and the environment in which it will be applied. The insect control composition of the present invention can be prepared in any suitable form, such as an aqueous solution or suspension, an oily solution or suspension, an emulsion, an aerosol, a powder, a granule, a wettable powder, a water-dispersible granule (powder), a water-soluble granule, a soluble concentrate, an oil-in-water emulsion, a microemulsion, an aqueous suspension concentrate, an aqueous capsule suspension, an oil-based suspension concentrate, and an aqueous suspoemulsion. When the pest control composition of the present invention is a liquid, it can be used as is, or it can be diluted, dissolved, or suspended in a predetermined liquid before use. How to prepare the insect control composition of the present invention is a matter that can be appropriately determined by those skilled in the art, taking into account the actual application conditions.

[0055] The pest control composition of the present invention can be applied by any means, such as dipping, fumigation, dusting, aerial application, aerosol application, painting, spraying, solid application, and the like.

[0056] The insect control method of the present invention can be used to control insects in any situation where the insects targeted by the present invention cause harm, such as agriculture, forestry, horticulture, public health, material protection, control of nuisance pests, etc. For each application, a person skilled in the art can design a method to achieve the desired insect control, taking into consideration the type of insect to be controlled, the degree of control, the form of the insect control composition to be applied, the form of the pest control composition, and the environment in which it is applied.

[0057] Therefore, a person skilled in the art who intends to carry out the control method of the present invention can identify the type of insect to be controlled, determine the dose of the insect control composition of the present invention that is effective for the desired control of the insect, if necessary, through a simple survival test, and apply the insect control composition in an amount and by a means sufficient for the insect to orally ingest that dose of the control composition in the implementation environment. [Example]

[0058] The present invention will be described in more detail below with reference to examples, but these examples are merely illustrative and are not intended to limit the scope of the present invention in any way.

[0059] Test insects and bacterial strains For the German-winged green stink bug (Plautia stali), we used a laboratory-reared strain established from several female individuals collected in Tsukuba City, Ibaraki Prefecture in 2012. For the brown marmorated stink bug (Halyomorpha halys), we used a laboratory-reared strain established from multiple female individuals collected in Tsukuba City, Ibaraki Prefecture in 2017. For the shiny green stink bug (Nezara antennata), we used a laboratory-reared strain established from multiple female individuals collected in Kyoto City, Kyoto Prefecture in 2019.

[0060] The rearing containers were 9 cm diameter plastic petri dishes lined with autoclave-sterilized filter paper. The food consisted of peanuts for the German winged green bug, peanuts for the brown marmorated stink bug, and almonds for the shiny green bug. Rearing water contained 0.05% ascorbic acid. In experiments evaluating the effects of trehalase inhibitors, rearing water supplemented with validamycin (VMA) (Dr. Ehrenstorfer, #DRE-C17899900) or validoxylamine (VaA) (Toronto Research Chemicals, #V943450) was used.

[0061] The animals were reared under long-day conditions with a 16-hour light period and an 8-hour dark period in an incubator (PHCBI, MLE-352-PJ) adjusted to a temperature of 25±1°C and a relative humidity of 50±5%. During rearing, the rearing containers were replaced as necessary if they became soiled with feces or other contaminants. All rearing conditions in the following experiments, including the evaluation of the effects of trehalase inhibitors, were carried out under these same conditions.

[0062] Six phylogenetically distinct types of symbiotic bacteria, designated SymA through F, have been identified in Japanese field populations of the German-winged green stink bug, and each individual stink bug possesses only one of these symbiotic bacteria. These symbiotic bacteria are thought to be interdependent, providing the host stink bug with vitamins and essential amino acids, while the host stink bug provides the symbiotic bacteria with carbon sources and dispensable amino acids. These symbiotic bacteria are functionally equivalent and can be substituted for each other. Furthermore, four of these six types, designated SymC through F, are known to be capable of being artificially cultivated. Previous research has revealed that laboratory-reared strains of German-winged green stink bugs used to evaluate the insecticidal effects of oral administration of VMA and VaA (described below), possess the symbiotic bacterium SymA through whole-genome sequencing. Furthermore, the genome of this SymA has been found to contain the treA gene, which encodes trehalase. The amino acid sequence of this treA was identical to that of a known treA (GenBank accession number, BAN98376). The symbiotic bacterium SymC, used in the in vitro evaluation of the growth inhibitory effects of trehalase inhibitors (described below), is closely related to SymA and Pantoea dispersa and was isolated from a female specimen collected in Ishigaki City (Ishigaki Island), Okinawa Prefecture, in September 2009. Glycerol stocks were prepared according to standard procedures and stored frozen until use.

[0063] Evaluation of the in vitro growth inhibitory effects of trehalase inhibitors VMA and VaA on the symbiotic bacteria of the German green stink bug Because VMA and VaA have molecular structures very similar to trehalose, they are known to inhibit the action of trehalose-degrading enzymes (trehalases) found in many organisms, from microorganisms to mammals (Non-Patent Document 7). However, whether trehalase inhibitors have an inhibitory effect on the growth of the symbiotic bacteria of the German green stink bug has not yet been investigated. Therefore, the following experiment was conducted to evaluate the effects of the trehalase inhibitors VMA and VaA on the survival and growth of the symbiotic bacteria of the German green stink bug.

[0064] A stock of C-type symbiotic bacteria (SymC), one of the artificially cultivable symbiotic bacteria of the German stink bug, was inoculated onto an LB agar plate (MP Biomedicals, #3002121). Single colonies obtained after overnight incubation were picked and cultured overnight in LB liquid medium at 25°C. The culture was diluted appropriately with sterile water and plated onto M9 minimal agar plates (3 mg / mL KH2PO4, 3.2 mg / mL Na2HPO4, 0.5 mg / mL NaCl, 1 mg / mL NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, 1.5% agar) supplemented with glucose or trehalose as a carbon source. For the experimental groups supplemented with VMA or VaA, the diluted culture containing the C-type symbiotic bacteria was similarly plated onto M9 minimal agar plates supplemented with VMA or VaA. The concentrations and combinations of carbon source and trehalose inhibitor were as follows: (1) 0.4% glucose only; (2) 0.4% glucose and 10 μg / mL VMA; (3) 0.4% glucose and 10 μg / mL VaA; (4) 0.4% trehalose only; (5) 0.4% trehalose and 10 μg / mL VMA; (6) 0.4% trehalose and 10 μg / mL VaA; The experiment was conducted using plates containing the above agar medium. The above agar medium plates were cultured statically in an incubator (TAITEC, BioShaker BR-41FL) at 25±1°C for 3 days, and the effect on the growth of the symbiotic bacteria was evaluated based on the size of the colonies formed. Photographs of each cultured plate were taken using a scanner (EPSON, GY-X830).

[0065] Evaluation of the insecticidal effect of oral administration of VMA and VaA against stink bugs Previously, oral ingestion of VMA or VaA was thought to have no insecticidal effect on insects, but its effects on stink bug development had not been fully investigated. Therefore, the effects of oral ingestion of trehalase inhibitors (VMA and VaA) on stink bugs were evaluated as follows. A single egg mass containing 10–15 eggs was placed in a rearing container, and after hatching, rearing water and food in a small rectangular container were added. Rearing water containing 0 (control group), 10, 20, or 100 ppm VMA, or 0 (control group), 10, or 100 ppm VaA was used. The number of surviving insects was counted 26 days after hatching for German winged green bugs, and 24–28 days after hatching for brown marmorated stink bugs and shiny green stink bugs. The survival rate was calculated by dividing the number by the number of hatched eggs.

[0066] Evaluation of the effects of trehalase inhibitors on oviposition by the German green bug, Plagioclase gracilis To evaluate the effects of VMA and VaA on oviposition by the German-winged green bug, newly emerged female and newly adult male German-winged green bugs were paired and reared in water containing 0 (control) or 100 ppm VMA. Egg masses laid by the mated females were collected for 36 days and the total number of eggs laid was measured. The resulting egg masses were then reared individually, and the hatched larvae were counted approximately one week later. The hatchability of eggs laid by a single female was calculated by dividing the total number of hatched larvae by the total number of eggs laid. The Wilcoxon rank-sum test was used for survival rate, total egg number, hatchability, and statistical testing. For multiple comparisons of three or more groups, p values ​​were corrected using the Holm method. Furthermore, the mortality rate was calculated by dividing the number of newly adult males and females who died during the egg collection period by the total number.

[0067] Evaluation of the difference in the effect of trehalase inhibitors on the german-winged green bug symbiotic bacteria infection Like many insects, stink bugs transmit symbiotic bacteria vertically between mother and offspring. This occurs when the hatched larvae inhale the symbiotic bacteria, which the female applies to the surface of the eggs during oviposition. To evaluate the effect of symbiotic bacteria on stink bug development, we immersed German stink bug egg masses within one day of oviposition in 4% formalin for 20 minutes and washed them twice with sterile water to obtain symbiotic-free egg masses, which sterilized the symbiotic bacteria on the egg surface. Symbiotic-free egg masses and unsterilized egg masses were individually reared in rearing water containing 0 (control), 10, or 20 ppm VMA. The hatched larvae were counted and rearing continued. The number of surviving individuals 26 days after hatching was counted and divided by the number of hatched larvae to calculate the "survival rate." The log-rank test was used to test this survival rate and the mortality rate of newly hatched adults.

[0068] result Trehalase inhibitors validamycin and validoxylamine suppress the growth of the symbiotic bacteria of the German green stink bug by inhibiting its ability to utilize trehalose As mentioned above, six species of Pantoea symbiotic bacteria have been detected in field populations of the German-winged green stink bug (Non-Patent Document 8). Of these, we investigated the in vitro growth of artificially culturable type C symbiotic bacteria by using the trehalase inhibitors validamycin (VMA) and validoxylamine (VaA). When 0.4% glucose was used as the sole carbon source, the C-type symbiotic bacteria formed clear colonies regardless of the presence or absence of VMA and VaA. This indicates that VMA and VaA do not inhibit the bacteria's ability to utilize glucose or suppress its growth (Fig. 1, top panel). On the other hand, when 0.4% trehalose was used as the sole carbon source, the addition of VMA and VaA significantly suppressed colony formation (Fig. 1, bottom panel). This result confirmed that when the bacteria used trehalose as a carbon source, VMA and VaA suppressed their growth. This indicates that the German-winged green stink bug symbiotic bacteria can be targeted by the trehalase inhibitors VMA and VaA.

[0069] Oral administration of trehalase inhibitors to stink bugs produces insecticidal and oviposition-suppressing effects Many previous studies have attempted to use VMA as an insecticide, but external administration, especially oral administration, has proven ineffective. It was generally accepted that VMA administered dermally or orally has no insecticidal effect on insects. However, unlike aphids and leafhoppers, which harbor symbiotic bacteria in a bacteriocyte mass (a symbiotic organ located in the body cavity), many stinkbugs harbor symbiotic bacteria in a specialized symbiotic organ located in the posterior part of their digestive tract. Therefore, if trehalase inhibitors are orally ingested and enter the digestive tract, these symbiotic bacteria can be exposed to the trehalase inhibitor. As described above, trehalase inhibitors have been shown to inhibit the growth of symbiotic bacteria in vitro. This suggests that even when ingested orally, trehalase inhibitors may reach the symbiotic bacteria in the digestive tract and inhibit their activity, thereby indirectly inhibiting the growth of host insects and reducing their survival rate, potentially resulting in an insecticidal effect through a novel mechanism of action.

[0070] Newly hatched larvae of various stink bug species were orally administered a trehalase inhibitor via rearing water, and the effects on growth and survival were evaluated. When VMA or VaA was administered to newly hatched larvae of the German-winged green stink bug, the median survival rate in the control group to which no VMA was added was 1, while the median survival rate in the group treated with 10 ppm VMA was 0.82, and the median survival rate in the group treated with 20 ppm VMA was 0.39 (Table 1, Figure 2). In the box-and-whisker plot in Figure 2, the horizontal bar indicates the median, the top and bottom of the box indicate the third and first quartiles, respectively, and the whiskers indicate the maximum and minimum values. This is also true for the following figures. [Table 1] *Significant differences were observed among all three experimental groups (p<0.05).

[0071] Similarly, the median survival rate for the control group was 0.93, the median survival rate for the 10 ppm VaA-treated group was 0.28, and the median survival rate for the 100 ppm VaA-treated group was 0 (all individuals died) (Table 2, Figure 3). [Table 2] *Significant differences were observed among all three experimental groups (p<0.05).

[0072] In brown marmorated stink bugs, the median survival rate in the control group was 0.87, while in the 100 ppm VMA-treated group it was 0 (all individuals died) (Table 3, Figure 4). [Table 3] *Significant difference was observed between the control group and the VMA-treated group (p<0.05).

[0073] For the shiny green stink bug, the median survival rate for the control group was 0.75, while the mean survival rate for the 100 ppm VMA-treated group was 0 (all individuals died) (Table 4, Figure 5). [Table 4] *Significant difference was observed between the control group and the VMA-treated group (p<0.05).

[0074] As shown above, VMA and VaA had a strong insecticidal effect on all stink bug larvae (Tables 1-4).

[0075] Next, to evaluate the effects of trehalase inhibitors on adult reproductive ability, newly adult male and female German-winged green bugs were paired, and the total number of eggs laid by each female and the hatchability of the eggs were measured over 36 days after treatment in a control group that did not receive a trehalase inhibitor, and in a group that was orally administered VMA. The median total number of eggs laid per female in the control group was 292, while the median total number of eggs laid in the group that was administered 100 ppm VMA was 57.5 (Table 5, Figure 6). [Table 5] *Significant difference was observed between the control group and the VMA-treated group (p<0.05).

[0076] In the above cases, the median hatchability of the eggs laid was 0.77 in the control group, while the number of hatched individuals in the 100 ppm VMA-treated group was 0 (all eggs unhatched) (Table 6, Figure 7). [Table 6] *Significant difference was observed between the control group and the VMA-treated group (p<0.05).

[0077] Furthermore, the combined mortality rate of newly adult stink bugs (male and female) over the approximately one-month rearing period in this experiment was 0.06 in the control group and 0.11 in the VMA-treated group, showing no significant difference, making it clear that VMA administration does not affect the survival of newly adult stink bugs, at least for approximately one month. This is consistent with conventional technical common sense (Table 7). [Table 7] *No significant difference was observed by log-rank test. These results confirmed that VMA not only affects the growth and survival of stink bug larvae, but also affects the reproductive ability and egg-laying of adults. Oral administration of a trehalase inhibitor to stink bugs was shown to inhibit the growth of larval stink bugs and suppress egg-laying in reproductive adults, reducing the hatchability of laid eggs.

[0078] Orally administered trehalase inhibitors inhibit stink bug growth indirectly through suppression of symbiotic bacteria To evaluate the difference in the effect of trehalase inhibitors on the growth of stink bugs depending on whether or not they contained symbiotic bacteria, we created "symbiotic bacteria-free insects" that did not contain symbiotic bacteria, and evaluated the effect of oral intake of VMA in the same manner as above.

[0079] Because the symbiotic bacteria were not transmitted from mother to larvae in the symbiotic bacteria-free insects, larval growth was delayed and no adults emerged in any group, including the control group. However, the median survival rates for the control group, the 10 ppm VMA-treated group, and the 20 ppm VMA-treated group were 0.93, 0.93, and 0.93, respectively, and no significant differences were observed among any of the three experimental groups (Table 8, Figure 8). [Table 8] *No significant difference was observed by log-rank test. On the other hand, when the symbiotic bacteria were not removed, the survival rate in the VMA-treated group decreased in a VMA concentration-dependent manner, as shown in Table 1 and Figure 2. These results demonstrate that the insecticidal effect of oral administration of trehalase inhibitors on stink bugs is mediated by the symbiotic bacteria.

Claims

1. A composition for controlling insects, which contains a trehalase inhibitor and is applied to insects to be controlled that carry symbiotic bacteria in symbiotic organs in their intestinal tracts by orally ingesting the composition so that the symbiotic bacteria are exposed to the trehalase inhibitor.

2. 2. The composition of claim 1, wherein the trehalase inhibitor is validamycin or validoxylamine A.

3. 2. The composition according to claim 1, wherein the control is achieved by one or more of the following: an effect of inhibiting the growth of or killing larvae of the insect to be controlled, or an effect of suppressing egg-laying in adult insects and an effect of reducing the hatchability of laid eggs, by the trehalase inhibitor.

4. The composition according to claim 1 , wherein the symbiotic bacterium is a trehalose-requiring symbiotic bacterium.

5. 2. The composition of claim 1, wherein the probiotic bacteria comprise one or more bacteria belonging to any one of the classes Gammaproteobacteria, Betaproteobacteria, or Actinobacteria.

6. The composition of claim 1 , wherein the probiotic bacteria comprise one or more bacteria belonging to any one of the genera Pantoea, Rhodococcus, or Tachikawaea.

7. The composition according to claim 4, wherein the trehalose-requiring symbiotic bacterium has a gene encoding trehalase.

8. The composition according to claim 7, wherein the trehalase is a prokaryotic periplasmic trehalase.

9. The composition according to claim 7, wherein the gene encoding the trehalase is treA.

10. 2. The composition of claim 1, wherein the insect belongs to any one of the orders Hemiptera, Coleoptera, or Blattodea.

11. The insects include those of the Pentatomidae, Scutelleridae, Urostylidae, Dinidoridae, Cydnidae, Coreidae, Berytidae, Largidae, Alydidae, Lygaeidae, Pyrrhocoridae, 2. The composition of claim 1, wherein the insect belongs to any one of the following families: Hocoridae, Plataspidae, Acanthosomatidae, Rhopalidae, Miridae, Tingidae, Chrysomelidae, Isoptera, Triatominae, or Curculionidae.

12. 2. The composition of claim 1, wherein the insect belongs to any one of the families Pentatomidae, Scutelleridae, or Urostylidae.

13. The insects include green stink bugs (Nezara antennata), red stink bugs (Pygomenida bengalensis), red-striped stink bugs (Graphosoma rubrolineatum), striped stink bugs (Piezodorus hybneri), rice stink bugs (Lagynotomus elongatus), black rice stink bugs (Scotinophara lurida), cow bugs (Alcimocoris japonensis), Siberian green stink bugs (Palomena angulosa), shrimp-colored stink bugs (Gonopsis affinis), large spur-spotted white-spotted stink bugs (Eysarcoris lewisi), yellow-spotted stink bugs (Erthesina fullo), brown marmorated stink bugs (Halyomorpha mista), white-spotted stink bugs (Eysarcoris ventralis), brown-winged stink bug (Plautia stali), horned green bug (Pentatoma japonica), white-spotted stink bug (Menida violacea), shiny green stink bug (Glaucias subpunctatus), shiny spotted stink bug (Eysarcoris annamita), thorny stink bug (Carbula humerigera), thorny spotted stink bug (Eysarcoris parvus), spotted stink bug (Lelia decempunctata), long-legged stink bug (Eurydema rugosum), small long-legged stink bug (Eurydema pulchrum), spotted stink bug (Dolycoris baccarum), spotted stink bug (Eysarcoris guttiger), citrus spiny stink bug (Rhynchocoris humeralis), southern green stink bug (Nezara viridula), purple stink bug (Carpocoris purpureipennis), purple spotted stink bug (Eysarcoris annamita), four-spotted stink bug (Homalogonia obtusa), red-striped stink bug (Poecilocoris Lewisii), large golden stink bug (Eucorysses grandis), brown stink bug (Eurygaster2. The composition of claim 1, wherein the insect pest is selected from the group consisting of: the Japanese stink bug (Urochela luteovaria), and the Asian stink bug (Urochela luteovaria).

14. 2. The composition of claim 1, wherein the insect is selected from the group consisting of the brown winged stink bug (Plautia stali), the brown marmorated stink bug (Halyomorpha halys), and the shiny green stink bug (Nezara antennata).

15. The composition according to claim 1, wherein the composition is applied so that the trehalase inhibitor is contained in food or water ingested by the insects to be controlled.

16. A method for controlling insects, comprising orally ingesting the composition according to any one of claims 1 to 15 to an insect to be controlled, the insect carrying a trehalose-requiring symbiotic bacterium in a symbiotic organ in its intestinal tract, thereby exposing the symbiotic bacterium to a trehalase inhibitor.

17. The method according to claim 16, comprising applying the composition according to any one of claims 1 to 15 so that the trehalase inhibitor is contained in food or water ingested by the insects to be controlled.

18. 17. The method of claim 16, comprising inhibiting trehalase activity in the insect's gut, thereby suppressing trehalose-requiring symbiotic bacteria in the gut.

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