Method for creating area-restricted search type arthropod

Abstract

Developed and provided is a method for creating an area-restricted search type arthropod which belongs to the class Insecta or Arachnida and has high selection efficiency.　An area-restricted search type arthropod having high selection efficiency is created by measuring the expression level of a cGMP-dependent protein kinase gene in each test individual in a homogeneous individual group and selecting a test individual of which the expression level is significantly higher than that of a wild-type control individual.

Description

Region-intensive exploration type arthropod creation method

[0001] The present invention relates to a method for producing a local concentration-searching arthropod, a kit for detecting a local concentration-searching arthropod, and a nucleic acid preparation for producing a local concentration-searching arthropod.

[0002] Thrips are difficult-to-control pests that cause serious damage to various agricultural crops, including vegetables, fruit trees, and tea. Traditionally, chemical pesticides containing active ingredients such as thiamethoxam, emamectin benzoate, and fenitrothion have been used to control these insects. However, in recent years, issues such as the emergence of pesticide-resistant individuals, chemical damage to pollinating insects, environmental pollution, and the suspension of agricultural product shipments due to pesticide residues have become a problem, and alternative control methods to chemical pesticides are needed.

[0003] In recent years, there has been a demand for a shift to sustainable control techniques that are in harmony with the environment, and biological control has attracted attention. Biological control is a method of controlling pests, pathogenic microorganisms, weeds, etc. by using natural enemies as biological pesticides (natural enemy formulations) based on the predator-prey or host-parasite relationships in natural ecosystems.

[0004] Traditionally, Amblyseius swirskii has been the most commonly used natural enemy for biological control of thrips, but this species becomes less active at temperatures below 15°C, significantly reducing its effectiveness.

[0005] Insects of the Anthocoridae family can also be used as natural enemy formulations that are effective in controlling thrips. In particular, the strigicollis (Orius strigicollis), a natural enemy native to Japan, preys on a large number of thrips and is effective under high temperatures. Furthermore, because this species has a shallow diapause under short days (Non-Patent Document 1), it has been put to practical use as a natural enemy formulation in greenhouse horticulture, primarily forcing cultivation.

[0006] However, this species has a major problem of low initial establishment and reproduction rates after release (Non-Patent Document 2). This is thought to be primarily due to the recommendations in the field, such as releasing natural enemies at the early stage of target pest emergence (Non-Patent Document 3) and suppressing target pest density in advance by spraying chemical pesticides before releasing natural enemies (Non-Patent Document 4). These recommended methods result in a shortage of thrips to feed on immediately after release, resulting in the death of this species or its escape from the facility. In order to use Orius strigiformes and other insects as natural enemies for effective biological control of thrips and other insects, a major challenge is to improve the establishment rate immediately after release.

[0007] To solve the above problems, the present inventors conceived a method for enhancing the functionality of natural enemies themselves to increase the rate of land colonization after release, and have completed an invention based on this method. Specifically, this method improves colonization by selecting individuals with behavioral characteristics specialized for local intensive searching rather than switching to wide-area searching even under conditions of low target pest density, and then creating a lineage of such individuals (Patent Document 1, Non-Patent Document 5). This method is based on the negative correlation between the local intensive searching time of Orius strigiformes and the amount of walking and / or flying activity, which the present inventors discovered. By extracting individuals with low walking and / or flying activity from a population, individuals with long local intensive searching times can be easily and efficiently selected.

[0008] However, because this selection method is based on behavioral characteristics, it is not possible to completely eliminate individuals that happen to exhibit the same behavior as the target behavior from the selection targets, which reduces the selection efficiency.

[0009] Patent Publication No. 2014-207872

[0010] Kakimoto, K. et al., 2003, Journal of the Japanese Society of Applied Entomology and Zoology 47(1): 19-28. Kakimoto, K. et al., 2007, Journal of the Japanese Society of Applied Entomology and Zoology 51(1): 29-37. Japan Plant Protection Association, 2002, Biological Pesticide Handbook 2002, Tokyo, 205pp. Umekawa, M. et al. (eds.), 2005, IPM Manual - Integrated Pest Management Techniques for Reducing Environmental Impact, National Agricultural Research Center, Tsukuba, 236pp. Seko and Miura, 2024, Journal of Pest Science, https: / / doi.org / 10.1007 / s10340-023-01696-4

[0011] The object of the present invention is to develop and provide a method for producing local concentration-exploring arthropods with high selection efficiency, which does not mix in individuals whose behavior coincides with the behavioral characteristics of local concentration-exploring arthropods.

[0012] To solve the above problems, the present inventors focused on and searched for genes related to the behavioral characteristics of area-focused exploration arthropods that had not been identified before, i.e., genes involved in the amount of walking activity in insects. Genes directly involved in area-focused exploration can be selected based on their expression levels, regardless of behavioral characteristics, so there is no risk of mistakenly selecting individuals whose behavior coincides by chance.

[0013] As a result of intensive research, the present inventors isolated a cGMP-dependent protein kinase gene (often referred to herein as the "PKG gene") as a candidate gene. They then compared the expression levels of this gene in strains of Orius strigiformes selected using a conventional method based on locomotion activity with those in unselected strains. They found that the expression levels of the gene in the selected strain were significantly higher than those in the unselected strain. These results suggested that increased expression of the PKG gene reduces locomotion activity. They then performed gene knockdown by introducing RNAi of the gene into wild-type Orius strigiformes, resulting in a significant increase in locomotion activity, demonstrating that the PKG gene is a gene involved in insect locomotion activity. Based on these results, the present inventors have successfully developed a method for producing area-focused, foraging arthropods with high accuracy and selection efficiency by using the PKG gene as a selection marker and selecting based on its expression level. The present invention is based on these development results and provides the following:

[0014] (1) A method for producing a local-searching arthropod belonging to the class Insecta or the class Arachnida, the method comprising: a nucleic acid extraction step of extracting nucleic acid from each test individual in a homogeneous population; a measurement step of measuring the expression level of the PKG gene in the nucleic acid extracted from each test individual; and a selection step of selecting a local-searching arthropod based on the expression level of the gene in the test individual measured in the measurement step. (2) The method according to (1), comprising: a mating step of using the local-searching arthropod produced in (1) as at least one of the parent individuals for mating; and a repetitive step of repeating the nucleic acid extraction step, measurement step, and selection step described in (1) for the hybrid population obtained in the mating step. (3) The method according to (2), comprising a line production step of repeating the mating step and the repetitive step multiple times. (4) The method according to any one of (1) to (3), wherein the PKG gene is a polynucleotide consisting of any one of the following nucleotide sequences (a) to (c): (a) a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 34, (b) a nucleotide sequence comprising one or more base additions, deletions, and / or substitutions in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 34, and (c) a nucleotide sequence having 90% or more nucleotide identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 34. (5) The method according to any one of (1) to (4), wherein the local-searching arthropod is a natural enemy organism for biological control. (6) The method according to (5), wherein the natural enemy belongs to any one of the group consisting of Anthocoridae, Miridae, Lygaeidae, Coccinellidae, and Phytoseiidae. (7) A kit for detecting arthropods in a localized area, comprising a nucleic acid probe capable of specifically binding to a polynucleotide or a fragment thereof derived from the mRNA of the PKG gene, which is a localized arthropod marker, and / or a primer that specifically recognizes and amplifies the same.(8) The kit according to (7), wherein the probe and / or primer is a polynucleotide selected from the group consisting of any one of the following polynucleotides (a) to (e): (a) a polynucleotide consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 34, or a fragment thereof containing 15 or more consecutive nucleotides, (b) a polynucleotide containing a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 34, (c) a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 34, or a fragment thereof containing 15 or more consecutive nucleotides, (d) a polynucleotide containing a nucleotide sequence complementary to a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 34, and (e) a polynucleotide that hybridizes under stringent conditions with any one of the polynucleotides (a) to (d). (9) The kit according to (7) or (8), wherein the local-concentration-searching arthropod is a natural enemy organism for biological control. (10) A nucleic acid formulation for producing a local-concentration-searching arthropod, comprising an expression vector capable of expressing a PKG gene or an active fragment thereof in a host cell. (11) The nucleic acid formulation according to (10), wherein the PKG gene is a polynucleotide consisting of any of the following nucleotide sequences (a) to (c): (a) a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 34, (b) a nucleotide sequence comprising one or more base additions, deletions, and / or substitutions in the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 34, and (c) a nucleotide sequence having 90% or more nucleotide identity with the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 34. (12) A local-concentrated searching arthropod obtained by the method set forth in any of (1) to (6) or comprising the nucleic acid formulation set forth in (10) or (11). (13) The local-concentrated searching arthropod according to (12), wherein the local-concentrated searching arthropod is a natural enemy organism for biological control. This specification incorporates the disclosures of Japanese Patent Application No. 2024-152535, from which the present application claims priority.

[0015] According to the method of the present invention for producing local-concentrated-exploring arthropods, it is possible to produce local-concentrated-exploring arthropods simply and with high accuracy, completely eliminating the mixing in of non-local-concentrated-exploring individuals that coincidentally exhibit behavior consistent with local-concentrated-exploring arthropods.

[0016] According to the kit for detecting local concentration-searching arthropods of the present invention, local concentration-searching arthropods can be easily detected from populations of various species belonging to the class Insecta or the class Arachnida.

[0017] According to the nucleic acid preparation for producing a local-searching arthropod of the present invention, a local-searching arthropod can be easily produced from any wild-type arthropod belonging to the class Insecta or Arachnida.

[0018] 1 is a flow diagram of the production method of the present invention. It is a diagram showing the expression level of the PKG gene in a local concentration searching-type Orius strigiformes (selected line) produced by a method based on behavioral characteristics. In this diagram, the expression level is shown as a relative value to the expression level of the PKG gene in a wild-type Orius strigiformes (non-selected line). It is a boxplot showing the walking activity level in a local concentration searching-type Orius strigiformes population when the PKG gene and EGFP gene are knocked down by RNAi. A shows the results for the female population, and B shows the results for the male population. It is a diagram showing the correlation between the expression level of the PKG gene and walking activity level in a male population of Orius strigiformes. It is a diagram showing the change over time in the population of a PKG-selected line selected based on the expression level of the PKG gene and an activity-selected line selected based on walking activity level in Orius strigiformes after release into the field. A shows the results for a strawberry field, and B shows the results for an eggplant field. 0 indicates the time of release into the field. This figure shows the change in the number of thrips over time in an eggplant field containing populations of PKG-selected and activity-selected Orius strigiformes. This figure shows the change in expression level over each generation in selection based on PKG gene expression levels in Orius strigiformes. A indicates the PKG gene expression level in males, and B indicates the PKG gene expression level in females. This figure shows the settlement of selected strains of Orius strigiformes after release. This figure shows the change in the number of confirmed Orius strigiformes individuals over time relative to the number of released individuals. In the figure, the solid line indicates the PKG-expressing strain, the dashed line indicates the activity-selected strain, and the dotted line indicates the non-selected strain. This figure shows the control effect of thrips after release of selected strains of Orius strigiformes. This figure shows the change over time in the number of thrips present on flowers and young fruits in the field after the release of Orius strigiformes. In the figure, the solid line indicates the PKG-expressing line, the dashed line indicates the activity-selected line, and the dotted line indicates the unselected line.

[0019] 1. Method for Producing Locally-Searching Arthropods 1-1. Overview A first aspect of the present invention is a method for producing locally-searching arthropods. In this invention, the expression level of a cGMP-dependent protein kinase gene in an arthropod belonging to the class Insecta or Arachnida is measured as a biomarker, and individuals with a significantly higher expression level of the gene than control individuals of the same species that are not locally-searching are selected as locally-searching arthropods, thereby producing the desired locally-searching arthropods. This invention makes it possible to completely eliminate the inclusion of individuals that accidentally exhibit behavior consistent with local-searching behavior, thereby enabling the production of locally-searching arthropods easily and with high accuracy.

[0020] 1-2. Definitions of Terms The following terms used in this specification are defined below.

[0021] As used herein, "localized exploration" is a term that is the counterpart of "regional exploration." It refers to a behavioral pattern in arthropods belonging to the classes Insecta and Arachnida in which, when food is plentiful, they carefully explore the area around the food source without wandering around much. On the other hand, as used herein, "regional exploration" refers to a behavioral pattern in arthropods in which, when food is scarce, they move quickly in a straight line to another food source. These exploration behaviors are known to switch in many organisms depending on food density (Jander, 1975, Annual Review of Ecology and Systematics, 6:171-188). Furthermore, the timing of switching exploration behaviors has been confirmed to vary between individuals in some insects (Ferran et al., 1994, Journal of Insect Behavior, 7(5): 633-647).

[0022] In this specification, unless otherwise specified, the term "arthropod" refers to an organism belonging to the class Insecta or Arachnida.

[0023] As used herein, the term "locally-searching arthropod" refers to an organism belonging to the class Insecta or Arachnida that does not switch to wide-area searching even under conditions of low food density, but instead has behavioral characteristics specialized for localized searching. While not limited to this, it essentially refers to a mutant that possesses the behavioral characteristics as a trait. Due to such behavioral characteristics, localized arthropods have a high initial settlement rate when released, and can therefore be used as natural enemies for biological control.

[0024] As used herein, "biological control" refers to a method of controlling target organisms such as agricultural pests by utilizing a predator-prey relationship or a host-parasite relationship in a natural ecosystem, as described above.

[0025] As used herein, "natural enemy (organism)" refers to an organism that preys on prey or an organism that parasitizes a host (including parasitic predators that parasitize a host and ultimately kill the host). Furthermore, as used herein, "natural enemies for biological control" refer to natural enemies that can be used for biological control of the target organism as prey or host.

[0026] As used herein, "useful insects" refers to insects that are beneficial to human life. Examples include nectar-collecting insects such as honeybees, silkworms such as silkworms, vector insects as pollinators, food insects such as mealworms and crickets, edible insects such as locusts and wasp larvae, and medical insects such as maggots used in maggot therapy. While insects that are natural enemies for biological control are also considered useful insects, useful insects in this specification refer to insects other than natural enemies for biological control.

[0027] As used herein, the term "subject" refers to an organism belonging to the class Insecta or Arachnida that is subjected to the method for producing a localized searching arthropod of the present invention.

[0028] "Cyclic-GMP-dependent protein kinase (PKG)" (often referred to as "PKG" herein) is a serine / threonine kinase activated by cGMP. This enzyme phosphorylates target proteins and regulates their activity, thereby participating in important biological control mechanisms such as smooth muscle relaxation, cell division, and nucleic acid synthesis.

[0029] The term "cGMP-dependent protein kinase gene (PKG gene)" refers to a gene encoding PKG. In this specification, the PKG gene serves as a biomarker for producing a localized search-type arthropod.

[0030] As used herein, the term "active fragment thereof" refers to a PKG fragment having the activity of PKG, ie, protein kinase activity, or a PKG gene fragment encoding the same.

[0031] As used herein, "plurality" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Additionally, "several" refers to 2 to 3.

[0032] As used herein, "base identity" refers to the percentage (%) of the number of matching bases between two base sequences relative to the total number of bases in one base sequence, when the two base sequences are aligned so that the degree of base identity between the two sequences is highest.

[0033] 1-3. Method The flow of the present invention is shown in Figure 1. The method of the present invention includes essential steps of a nucleic acid extraction step (S0101), a measurement step (S0102), and a selection step (S0103), as well as selection steps of a mating step (S0104), a repetition step (S0105), and a line creation step (S0106). Each step will be explained below.

[0034] (1) Nucleic Acid Extraction Step The "nucleic acid extraction step" (S0101) is a step of extracting nucleic acid from each test individual in a homogeneous population of organisms belonging to the class Insecta or Arachnida.

[0035] There are no limitations on the test individuals, but when the purpose is to create area-concentrated arthropods as natural enemies for biological control or area-concentrated arthropods as useful insects, the following organisms are preferred as test individuals.

[0036] (A) Test individuals as natural enemies for biological control When the method of the present invention is intended to produce local concentrated-searching arthropods (local concentrated-searching natural enemies) as natural enemies for biological control, the test individuals may be, for example, species belonging to the family Anthocoridae, omnivorous species belonging to the family Miridae, species belonging to the family Lygaeidae, species belonging to the family Reduviidae, species belonging to the infraorder Parasitoids (Prasitica), Preference is given to species belonging to the families Tachinidae, Syrphinae, carnivorous species belonging to the families Coccinellidae, Aphelinidae, Ephemeroptera, Braconidae, Eulophidae, Araneae and Acari.

[0037] The species to be used as a natural enemy for biological control is determined appropriately depending on the type of organism to be controlled.

[0038] When the target organisms are species belonging to the order Thysanoptera or the superfamily Aphidoidea, species belonging to the family Anthocoridae may be used. For species belonging to the order Thysanoptera, species belonging to the genus Orius, such as Orius strigicollis, Orius sauteri, Orius minutus, Orius nagaii, Orius tantillus, Orius insidiosus, Orius tristicolor, or Orius laevigatus, are particularly suitable.

[0039] In the case of target organisms belonging to the superfamily Aphidoidea, species belonging to the order Ephemeroptera or the family Braconidae are preferred, such as Aphidius colemani, Aphidius gifuensis, Lysiphlebus japonicus, Diaeretiella rapae, Chrysoperla nipponensis, etc. In the case of target organisms belonging to the superfamily Aphidoidea or the family Aleyrodidae, species belonging to the family Aphelinidae are preferred, such as Eretmocerus mundus, Eretmocerus eremicus, Encarsia formosa, etc. Furthermore, in the case of target organisms belonging to the Agromyzidae family, species belonging to the Eulophidae family, such as Diglyphus isaea, Neochrysocharis formosa, and Hemiptarsenus varicornis, are suitable.

[0040] When the target organisms are species belonging to the superfamily Aphidoidea or Coccoidea, carnivorous species belonging to the family Coccidae, such as the two-spotted ladybird beetle (Harmonia axyridis), the two-spotted ladybird beetle (Adalia bipunctata), the seven-spotted ladybird beetle (Coccinella septempunctata), the striped ladybird beetle (Menochilus sexmaculatus), the red-spotted ladybird beetle (Chilocorus rubidus), the small red-spotted ladybird beetle (Chilocorus kuwanae), the small tortoiseshell ladybird beetle (Propylaea japonica), the large ladybird beetle (Synonycha grandis), the four-spotted ladybird beetle (Phymatosternus lewisii), the bed-leather ladybird beetle (Rodolia cardinalis), the red-spotted ladybird beetle (Rodolia rufocincta), the red-spotted ladybird beetle (Rodolia limbata), or the four-spotted ladybird beetle (Calvia Furthermore, when the target organisms to be controlled are species belonging to the family Tetranychidae or the order Thripidae, species belonging to the family Phytoseiidae, such as Phytoseiulus swirskii, Amblyseius californicus, Amblyseius womersleyi, Amblyseius eharai, Amblyseius cucumeris, Gynaeseius liturivorus, Neoseiulus barkeri, or Phytoseiulus persimilis, are preferred.

[0041] When the target organisms are any of species belonging to the Tetranychidae family, the Thripidae order, and the Aphidoidea superfamily, species belonging to the Lygaeidae family, such as the large stink bug (Piocoris varius), are preferred.When the target organisms are species belonging to the Aleyrodidae family, species belonging to the Miridae family, such as the tobacco mirid bug (Nesidiocoris tenuis), the black gourd mirid bug (Pilophorus typicus), or the green little mirid bug (Campylomma chinensis), are preferred.

[0042] Specific examples of organisms to be controlled include, but are not limited to, species belonging to the order Thripidae, species belonging to the superfamily Aphidoidea, species belonging to the family Aleyrodidae, species belonging to the family Tingidae, species belonging to the superfamily Coccoidea, species belonging to the family Cicadellidae, and species belonging to the family Tetranychidae.

[0043] More specific examples of species belonging to the order Thripida include Frankliniella occidentalis, Frankliniella intonsa, Heliothrips haemorrhoidalis, Thrips palmi, Thrips tabaci, Thrips nigroplosus, Scirtothrips dorsalis, and Mallotus japonica.

[0044] Examples of species belonging to the superfamily Aphidoidea include the cotton aphid (Aphis gossypii), soybean aphid (Aphis glycines), bean aphid (Aphis craccivora), pea aphid (Acyrthosiphon pisum), strawberry aphid (Aphis forbesi), snow willow aphid (Aphis spiraecola), green peach aphid (Myzus persicae), rose green aphid (Rhodobium porosum), Japanese radish aphid (Rhopalosiphum rufiabdominalis), radish aphid (Brevicoryne brassicae), false radish aphid (Lipaphis erysimi), onion aphid (Neotoxoptera formosana), Taiwan long-horned aphid (Uroleucon formosanum), and strawberry woolly aphid (Chaetosiphon fragaefolii), tulip aphid (Macrosiphum euphorbiae), corn aphid (Rhopalosiphum maidis), wheat collar aphid (Sitobion akebiae), wheat long-horn aphid (Sitobion akebiae), potato aphid (Aulacorthum solani), citrus black aphid (Toxoptera citricida), apple knob aphid (Ovatus malisuctus), and peach buttercup aphid (Hyalopterus pruni).

[0045] Examples of species belonging to the Aleyrodidae family include the tobacco whitefly (Bemisia tabaci), the silverleaf whitefly (Bemisia argentifolii), the greenhouse whitefly (Trialeurodes vaporariorum), and the citrus spiny whitefly (Aleurocanthus spiniferus).

[0046] Examples of species belonging to the superfamily Coccoidea include the cotton scale (Icerya purchasi Maskell), the ruby ​​wax scale (Ceroplastes rubens) and the pearl scale (Eulecanium kunoense).

[0047] Examples of species belonging to the family Tetranychus include Tetranychus urticae, Tetranychus kanzawai, Amphitetranychus viennensis, Panonychus citri, Panonychus ulmi, and Bryobia praetiosa.

[0048] (B) Test individuals as useful insects Furthermore, when the purpose of the method of the present invention is to produce a localized searching arthropod as a useful insect, the test individuals are preferably species belonging to the orders Hymenoptera, Diptera, Lepidoptera, or Coleoptera.

[0049] Specific examples of honey harvesters include the European honeybee (Apis mellifera) and the Japanese honeybee (Apis cerana), which belong to the order Hymenoptera.

[0050] When producing organisms that produce useful products such as silk, examples include the silkworm (Bombyx mori) and the Eri silkworm (Simia cynthia) of the order Lepidoptera.

[0051] Pollinators include species of the Hymenoptera order (e.g., European honeybee (Apis mellifera), Japanese honeybee (Apis cerana), small hornet (Vespa analis), European bumblebee (Bombus terrestris), Hokkaido giant bumblebee (Bombus hypocrita), Japanese bumblebee (Bombus ardens), Japanese bee hornet (Osmia cornifrons), Taiwan bamboo carpenter bee (Xylocopa tranquebarorum), Japanese little bumblebee (Panurginus crawfordi), tiger bumblebee (Bombus diversus), black bumblebee (Bombus ignitus), Japanese carpenter bee (Xylocopa appendiculata), common bumblebee (Osmia taurus), Japanese long-horned bee (Eucera nipponensis, Campsomeris annulata), Diptera (e.g., Episyrphus balteatus, Bombylius major, Eristalis cerealis, Helphilus virgatus, Eristalomyia tenax, Phytomia zonata, Stomorhina obsoleta, Bibio tenebrosus, Bibio rufiventris), Lepidoptera (e.g., Parnara guttata, Zizeeria maha, Pelopidas mathias, Macroglossum pyrrhosticta), Lycaena phlaeas, and species belonging to the order Coleoptera (for example, Oxycetonia jucunda and Nipponovalgus angusticollis).

[0052] The population used in this step is composed of a single species. The individuals constituting the population can be either male or female, and can be at any stage of development, including eggs, larvae, pupae, or adults.

[0053] The test individuals constituting the population used in this step may be any individuals, such as wild-collected individuals, bred individuals, or individuals with introduced mutations. Alternatively, for example, individuals with low walking and / or flying activity per unit time, selected based on conventional behavioral characteristics, as disclosed in JP 2014-207872 A, may be used. Alternatively, hybrid individuals obtained in the mating step described below may be used as test individuals.

[0054] The number of individuals constituting the population is not particularly limited, but in order to select the target local-searching arthropod, a larger number is preferable. For example, 3 to 1,000 individuals, 5 to 500 individuals, or 8 to 300 individuals may be sufficient. Usually, 10 to 200 individuals or 20 to 100 individuals are sufficient.

[0055] The nucleic acid to be extracted in this step is mRNA present in the sample, particularly PKG mRNA, which reflects the expression of the PKG gene, or a fragment thereof. PKG mRNA may be either a pre-mRNA or a mature mRNA. Typically, pre-mRNA is immediately spliced ​​in the nucleus to become a mature mRNA, so the extracted nucleic acid is essentially PKG mature mRNA.

[0056] Here, the "fragment thereof" refers to a partial fragment of PKG mRNA. The base length of the partial fragment is not particularly limited, but the lower limit may be a length that can be identified as a fragment of PKG mRNA and is detectable. For example, the length may be 100 bases or more, preferably 200 bases or more, more preferably 400 bases or more, or 600 bases or more. On the other hand, the upper limit is a length that is one base less than the full-length polynucleotide of PKG mRNA. In this specification, PKG mRNA and fragments thereof are often collectively referred to as "PKG mRNA, etc."

[0057] Any nucleic acid extraction method known in the art can be used as long as it can extract PKG mRNA from a biological sample (a test individual). Examples include methods for extracting total RNA and methods for selectively extracting only the target PKG mRNA. A typical method for extracting from all or part of a test individual involves first homogenizing or lysing tissues or cells at low temperature, removing contaminants such as protein by centrifugation or filtration as necessary, and adding a protease inhibitor to prepare a suspension. A preferred method for extracting RNA from a suspension involves extracting nucleic acids while suppressing or inactivating RNase activity in the suspension. A typical example is the AGPC (Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction) method. Specific extraction methods can be found, for example, in the nucleic acid extraction method described in Green, M.R. and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual, Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Additionally, RNA extraction kits are commercially available from various life science manufacturers, and these kits may be used. When using a kit, the specific extraction method may be performed according to or in accordance with the attached protocol. In principle, nucleic acids are extracted individually from each test subject, and the transfer process is performed individually.

[0058] In addition, a control organism that is not a localized exploratory arthropod, i.e., a conspecific organism with wild-type exploratory behavior, may be used as a control organism and subjected to this step and the measurement step (S0102) described below.

[0059] (2) Measurement step The "measurement step" (S0102) is a step of measuring the expression level of the PKG gene based on the nucleic acid extracted from each test individual in the nucleic acid extraction step (S0101). In this step, the amount of PKG mRNA, etc. contained per unit amount in the RNA sample extracted from each test individual is measured to obtain a measurement value. Here, "unit amount" refers to the amount per unit predetermined by volume or weight.

[0060] As used herein, the term "measured value" refers to a value indicating the amount of PKG mRNA measured in this step. The measured value may be an absolute value such as volume or weight, or a relative value such as concentration, ionic strength, absorbance, or fluorescence intensity. Furthermore, the measured value may be a value calculated by scoring based on values ​​such as volume or weight.

[0061] The base sequence of the PKG gene to be measured is, in principle, the base sequence of a wild-type gene encoding PKG that is present in the genome of the test individual. For example, if the test individual is Orius strigiformes, the base sequence would be the base sequence of a gene encoding PKG consisting of the amino acid sequence shown in SEQ ID NO: 35. Specifically, the wild-type PKG gene would be the base sequence shown in SEQ ID NO: 1. Furthermore, even if a mutant PKG gene is used, a gene encoding a mutant PKG having protein kinase activity equal to or greater than that of wild-type PKG can also be measured. Examples of such mutant PKG genes include a nucleotide sequence that contains one or more base additions, deletions, and / or substitutions in the nucleotide sequence of the wild-type PKG gene shown in SEQ ID NO: 1 and has protein kinase activity, or a mutant PKG gene of Orius strifei consisting of a nucleotide sequence that has 85% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more nucleotide identity with the nucleotide sequence shown in SEQ ID NO: 1 and has protein kinase activity.

[0062] Alternatively, the PKG gene may be an ortholog of another species of the wild-type PKG gene of Orius strigiforme comprising the base sequence shown in SEQ ID NO: 1. Examples include the various PKG gene orthologs shown in Table 1.

[0063]

[0064] The method for measuring PKG mRNA is not particularly limited and may be any nucleic acid quantification method known in the art. Examples include nucleic acid amplification, hybridization, and RNase protection. Each method is described below.

[0065] (a) Nucleic Acid Amplification Method. "Nucleic acid amplification method" refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a forward / reverse primer pair. Examples include PCR (including RT-PCR), NASBA, ICAN, and LAMP (registered trademark) (including RT-LAMP). Since the target nucleic acid in this step is RNA, nucleic acid amplification methods via reverse transcription (RT), such as RT-PCR or RT-LAMP, are preferred. Quantitative nucleic acid amplification methods such as real-time RT-PCR are particularly suitable for quantifying PKG mRNA. Reaction conditions for nucleic acid quantification methods using RT-PCR, such as real-time RT-PCR, are generally based on known PCR methods and vary depending on the base length of the nucleic acid region to be amplified, the amount of template nucleic acid, the base length and Tm value of the primers used, the optimal reaction temperature and optimal pH of the nucleic acid polymerase used, and other factors. Therefore, these conditions are appropriately determined based on these conditions. Various kits for real-time RT-PCR are commercially available from various life science manufacturers, and these can also be used. For example, Applied Biosystems TaqMan MicroRNA Assays Kit (Thermo Fisher Scientific) can be mentioned. When using these commercially available kits, in principle, the protocol attached to the kit should be followed.

[0066] (b) Hybridization Method The "hybridization method" is a method for detecting and quantifying a target nucleic acid or a fragment thereof by using, as a probe, a nucleic acid having a base sequence complementary to all or part of the base sequence of the target nucleic acid to be detected, and utilizing base pairing between the nucleic acid and the probe. Since the target nucleic acid in this step is RNA, for example, northern hybridization, RNA microarray, surface plasmon resonance, or quartz crystal microbalance is preferred.

[0067] (b-1) Northern Hybridization Method. "Northern hybridization" is the most common method for analyzing gene expression. RNA prepared from a sample is separated by electrophoresis using agarose gel or polyacrylamide gel under denaturing conditions, transferred (blotted) to a filter, and then the target RNA (e.g., PKG mRNA) is detected using a probe having a base sequence specific to the target nucleic acid. By labeling the probe with an appropriate labeling substance such as a fluorescent dye or radioisotope, the target RNA can be quantified based on the label intensity using a measuring device such as a chemiluminescence imaging analyzer (e.g., LightCapture), a scintillation counter, or an imaging analyzer. Northern hybridization is a well-known technique in the field; see, for example, Green, MR and Sambrook, J., 2012 (supra).

[0068] (b-2) RNA Microarray Method The "RNA microarray method" is a method that applies the DNA microarray method to RNA. Nucleic acids complementary to all or part of the base sequence of a target nucleic acid are arranged as small spots on a substrate at high density as probes and immobilized, and a sample containing the target RNA is reacted with these, and the nucleic acids hybridized to the substrate spots are detected and quantified by fluorescence or other means. Detection and quantification can be achieved by detecting and measuring the fluorescence, etc., resulting from hybridization of the target nucleic acid, etc., using a microplate reader or scanner. The RNA microarray method is also a well-known technique in the field.

[0069] (b-3) Surface Plasmon Resonance Method The "Surface Plasmon Resonance (SPR) method" is a highly sensitive method for detecting and quantifying adsorbates on a metal thin film surface. This method utilizes the surface plasmon resonance phenomenon, in which the intensity of reflected light significantly attenuates at a specific angle of incidence (resonance angle) when the incident angle of laser light irradiated onto the metal thin film is changed. In the present invention, for example, a PKG mRNA detection probe is immobilized on the metal thin film surface, and other portions of the metal thin film surface are blocked. After the nucleic acid amplification step, the amplified product is passed over the metal thin film surface, capturing the target nucleic acid through base pairing with the probe. The target nucleic acid can be detected and quantified based on the difference in measured values ​​before and after sample passage. Detection and quantification using surface plasmon resonance can be performed, for example, using an SPR sensor commercially available from Biacore. This technology is well known in the art and can be performed according to existing techniques.

[0070] (b-4) Quartz Crystal Microbalance Method The "quartz crystal microbalance (QCM) method" is a mass measurement method that utilizes the phenomenon that when a substance is adsorbed onto the surface of an electrode attached to a quartz crystal, the resonant frequency of the quartz crystal decreases in accordance with the mass of the substance, and quantitatively captures extremely small amounts of adsorbed substances based on the change in resonant frequency. Detection and quantification using this method, as with the SPR method, can also be performed using a commercially available QCM sensor. For example, target nucleic acids in the amplified products after the nucleic acid amplification step can be detected and quantified by base pairing with a PKG mRNA detection probe fixed to the electrode surface. This technique is well known in the art and can be performed according to existing techniques.

[0071] (3) Selection Step The "selection step" (S0103) is a step of selecting local concentrated search-type arthropods from the test population based on the expression level of the gene in each test individual measured in the measurement step.

[0072] "Based on the expression level of the gene" means the expression level of the PKG gene resulting from the measurement step, i.e., "based on the measured values ​​of PKG mRNA, etc." For example, whether a test individual is a local-searching arthropod can be determined based on a statistically significant difference between the measured values ​​of PKG mRNA, etc. in the test individual and the measured values ​​of PKG mRNA, etc. in a control individual that is not a local-searching type of the same species (often referred to as a "non-local-searching type" in this specification) or based on a cutoff value, and a local-searching arthropod can be selected. This step allows the production of the desired local-searching arthropod.

[0073] Below, selection based on statistically significant differences and selection based on cutoff values ​​will be explained in detail.

[0074] (i) Selection based on statistical significance In selection based on statistical significance, the measured values ​​of a test individual are compared with those of a control individual of the same species, and if the measured value of the test individual is significantly higher, the test individual is determined to be a locally intensively searching arthropod and selected.

[0075] As used herein, "significant" means "statistically significant," which applies when the risk rate (significance level) of the obtained value is small, specifically, p<0.05 (less than 5%), p<0.01 (less than 1%), or p<0.001 (less than 0.1%). Here, "p (value)" indicates the probability that the hypothesis will be correct by chance in a statistical test within the assumed distribution of statistics. Therefore, the smaller the p value, the closer the hypothesis is to the truth.

[0076] "Significantly higher" means that the difference is so large that it is statistically significant. Here, "statistically significantly different" means that there is a significant difference between the measured values ​​of the test individual and the measured values ​​of the control individual when the difference between the two is statistically processed. The test method for statistical processing is not particularly limited, and any known test method that can determine the presence or absence of significance may be used as appropriate. For example, Student's t-test can be used.

[0077] (ii) Selection based on cutoff value "Selection based on cutoff value" is a method of comparing the measurement value of the test individual with a predetermined cutoff value, and determining and selecting regionally concentrated searching arthropods based on the comparison results.

[0078] As used herein, the term "cutoff value" refers to a boundary value for classifying measured values ​​into local-searching arthropods or non-local-searching arthropods. The method for setting the cutoff value is not particularly limited and may follow methods known in the field of statistics. For example, a specific percentile in a group of measured values ​​consisting of measured values ​​of organisms known to be local-searching and organisms known to be non-local-searching can be used as the cutoff value. Specifically, for example, if almost all measured values ​​of local-searching arthropods in the group of measured values ​​are included in a value higher than the 90th percentile of the group of measured values, that corresponding measured value becomes the cutoff value. In this case, if the measured value of a test individual is higher than the cutoff value, the test individual can be determined to be a local-searching arthropod and selected.

[0079] (4) Mating Step The "mating step" (S0104) is a step of mating using the local concentration-searching arthropods produced in the selection step as at least one of the parent individuals.

[0080] The local-searching arthropods used for mating can be either male or female. The other parent individual used for mating can be a local-searching arthropod, or a non-local-searching arthropod, such as a wild-type individual, an individual with other behavioral characteristics, or an individual with a desired mutation such as low-temperature tolerance or high fecundity. Mating between local-searching arthropod individuals is preferred.

[0081] The mating method in this step may be any method known in the art and is not particularly limited. Typically, males and females are placed in the same container and allowed to mate naturally. After mating, eggs are collected by methods known in the art, and the first generation of hybrids (hereinafter often referred to as "F1") are reared by methods known in the art to obtain a hybrid population.

[0082] (5) Repetitive Step The "repetitive step" (S0105) is a step of repeating the nucleic acid extraction step, measurement step, and selection step for the hybrid population consisting of the hybrid individuals obtained in the mating step (S0104). This step constitutes the strain creation step described below, and is characterized by further selecting regional concentration-seeking arthropods from the hybrid population in order to create a mutant strain with respect to the PKG gene expression level of the present invention, i.e., a regional concentration-seeking arthropod strain.

[0083] In this step, the individuals that make up the hybrid population of interest are the hybrid individuals obtained in the hybridization step (S0104), but the nucleic acid extraction step, measurement step, and selection step to be performed are essentially the same as the methods described above in "(1) Nucleic Acid Extraction Step," "(2) Measurement Step," and "(3) Selection Step," respectively. Therefore, detailed explanations will be omitted here.

[0084] (6) Strain Creation Step The "strain creation step" (S0106) is a step in which the mating step (S0104) and the iteration step (S0105) are repeated multiple times. This step homogenizes the PKG mutant gene that causes the local concentration-seeking mutation, and the local concentration-seeking arthropod obtained in the fixation step can be made into a local concentration-seeking strain.

[0085] In this step, the regionally concentrated searching arthropods obtained after the repetition step (S0105) are subjected to the mating step (S0104) again, and the hybrid population consisting of the hybrid individuals obtained therein is subjected to the repetition step (S0105) again. This mating step (S0104) and the repetition step (S0105) are characterized by being repeated.

[0086] In this step, the cross-breeding step (S0104) and the repeating step (S0105) are counted as one unit (one time), and are performed at least twice, including the first two times. The cross-breeding step may be repeated multiple times, for example, five or more times, seven or more times, or nine or more times. There is no upper limit to the number of times, but 15 times or less is usually sufficient.

[0087] The mating step in this step can basically be carried out in accordance with the mating step (S0104) described above, but it is preferable that the individuals to be mated are inbreeding. As used herein, "inbreeding" refers to mating between individuals with the same target trait (more specifically, the causative gene for that trait). In this embodiment, mating occurs between both male and female arthropods that are localized searchers. Inbreeding also includes brother-sister mating between males and females of the same litter, in which the target traits are the same. Note that it is not necessary to perform inbreeding consecutively in each mating step.

[0088] 2. Kit for detecting arthropods by local concentration search 2-1. Overview A second aspect of the present invention is a kit for detecting arthropods by local concentration search. The kit of the present invention comprises a probe capable of specifically binding to a polynucleotide derived from PKG mRNA or a fragment thereof and / or a primer that specifically recognizes and amplifies the polynucleotide. The kit of the present invention makes it possible to easily detect arthropods by local concentration search from populations of various species belonging to the classes Insecta or Arachnida based on the expression level of the PKG gene.

[0089] 2-2. Configuration The local concentration search-type arthropod detection kit of the present invention includes, as essential components, either or both of a PKG detection probe and a PKG detection primer, and, as optional components, includes an internal control primer, an internal control probe, a detection reagent, etc. Each component will be described below.

[0090] (1) PKG detection probe The "PKG detection probe" is a probe for detecting the expression of the PKG gene, which is a regional concentration detection arthropod marker, and is capable of specifically binding to a polynucleotide derived from the mRNA of the PKG gene (PKG mRNA), or a fragment thereof.

[0091] As used herein, the term "polynucleotide derived from PKG mRNA" refers to PKG cDNA obtained by reverse transcription of PKG mRNA, or a DNA fragment amplified using the PKG cDNA as a template.

[0092] The PKG detection probe is composed of any one of the following polynucleotides (oligonucleotides are also included in the polynucleotides herein): (a) to (c)

[0093] (a) a polynucleotide consisting of a base sequence complementary to the base sequence of PKG mRNA, i.e., the base sequence of the antisense strand of the PKG gene, or a fragment thereof consisting of a sequence of 15 or more consecutive bases; (b) a polynucleotide comprising the polynucleotide described in (a) above or a fragment thereof; (c) a polynucleotide that hybridizes with all or a part of PKG mRNA under stringent conditions.

[0094] A specific example of the base sequence of the PKG mRNA is the PKG mRNA base sequence of Orius strigiformes shown in SEQ ID NO:1.

[0095] The PKG detection probe is composed of natural nucleotides and / or non-natural nucleotides. It is usually composed of natural nucleotides consisting of only DNA, only RNA, or a hybrid of DNA and RNA, but DNA alone is preferred for reasons of chemical stability and ease of chemical synthesis. Furthermore, the probe may be composed entirely or partially of non-natural nucleotides such as PNA (Peptide Nucleic Acid), BNA (Bridged Nucleic Acid) / LNA (Locked Nucleic Acid; registered trademark).

[0096] The base length and base sequence of the PKG detection probe are not particularly limited, as long as they are designed so that the Tm value is in the range of 55°C to 80°C, preferably 60°C to 75°C, so that they can hybridize specifically to PKG mRNA.

[0097] In general, the base length and base sequence of the probe are preferably such that they can hybridize to a contiguous base sequence on the template nucleic acid strand. Typically, the probe only needs to hybridize to a partial base sequence of a polynucleotide derived from PKG mRNA, and does not necessarily need to hybridize over the entire length of PKG cDNA, although this is not a limitation. For example, the probe length may be 15 to 100 contiguous bases, 15 to 80 contiguous bases, 15 to 60 contiguous bases, 16 to 50 contiguous bases, 17 to 40 contiguous bases, 18 to 30 contiguous bases, or 20 to 25 contiguous bases, which are commonly used in the art.

[0098] As described in (b) above, the PKG detection probe may contain, in addition to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of PKG mRNA or a fragment thereof consisting of a sequence of 15 or more consecutive nucleotides, another nucleotide sequence unrelated to the nucleotide sequence complementary to PKG mRNA. In this case, the other nucleotide sequence is preferably located on the 5'-end and / or 3'-end of the nucleotide sequence complementary to PKG mRNA.

[0099] The PKG detection probe may have some or all of the phosphate groups, sugars, and / or bases of the nucleotides constituting the probe labeled with a labeling substance. The labeling position of the labeling substance in the probe can be determined appropriately depending on the properties of the labeling substance and the intended use, and is not limited to, but is preferably the 5' end, 3' end, and / or the SNP site to be detected. Any substance known in the art can be used as the labeling substance. Examples include DIG, fluorescent substances, quenchers, chemiluminescent substances, radioisotopes, biotin, magnetic beads, etc. Labeling of nucleotides with each labeling substance can be performed by known methods.

[0100] The term "fluorescent substance" refers to a substance that becomes excited by absorbing excitation light of a specific wavelength and emits fluorescence when returning to its original ground state. In this specification, this refers to a fluorescent dye. Examples of such fluorescent substances include FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, ROX, TET, Bodipy493, NBD, TAMRA, Quasar (registered trademark) 670, Quasar (registered trademark) 705, CAL Fluor (registered trademark) Red 610, fluorescamine or a derivative thereof, and rhodamine or a derivative thereof.

[0101] The term "quencher" refers to a substance that absorbs the excitation energy of the fluorescent substance and suppresses its fluorescence. Examples include AMRA, DABCYL, BHQ-1, BHQ-2, and BHQ-3. Quenchers are generally used in combination with fluorescent substances. Because the wavelength range of fluorescence suppression varies depending on the type of quencher, a fluorescent substance and a quencher are combined with a quencher that can suppress the fluorescence of the fluorescent substance used for labeling. For example, when the fluorescent substance is FAM, HEX, TET, etc., BHQ1, which suppresses the wavelength range of 480 nm to 580 nm, can be used in combination. When the fluorescent substance is Cy3, Cy5, ROX, TAMRA, Texas, etc., BHQ2, which suppresses the wavelength range of 550 nm to 650 nm, can be used in combination. The arrangement of the fluorescent substance and quencher in the probe is not particularly limited, as long as both substances are positioned so that the quencher can suppress the fluorescence of the fluorescent substance. For example, one end (5' or 3' end) of the probe may be labeled with a fluorescent substance, and the other end (3' or 5' end, respectively) may be labeled with a quencher.

[0102] The term "chemiluminescent substance" refers to a substance that has the property of emitting the difference in energy as light when it returns to the ground state after being excited by a chemical reaction. Examples of such substances include luminol, lophine, and lucigenin.

[0103] The term "radioisotope" refers to an element that emits radiation among isotopes with different mass numbers. For example, 32 P, 33 P, or 35Examples include S.

[0104] The community-focused arthropod detection kit of the present invention may contain two or more types of PKG detection probes.

[0105] (2) PKG Detection Primer The "PKG detection primer" is configured to specifically recognize and amplify a polynucleotide derived from PKG mRNA.

[0106] The PKG detection primers are generally configured as a primer set consisting of a forward (Fw) primer and a reverse (Rv) primer, but may be configured to include only one of the primers. Alternatively, they may be configured as two or more primer sets capable of amplifying the same or overlapping regions, such as nested primers.

[0107] The PKG detection primer is composed of any one of the following polynucleotides (a) to (e):

[0108] (a) a polynucleotide consisting of the base sequence of PKG mRNA, or a fragment thereof containing 15 or more consecutive bases; (b) a polynucleotide containing the base sequence of PKG mRNA; (c) a polynucleotide consisting of a base sequence complementary to the base sequence of PKG mRNA, or a fragment thereof containing 15 or more consecutive bases; (d) a polynucleotide containing a base sequence complementary to the base sequence of PKG mRNA; (e) a polynucleotide selected from the group consisting of polynucleotides that hybridize under stringent conditions with any of the polynucleotides (a) to (d) above.

[0109] A specific example of the base sequence of the PKG mRNA is the PKG mRNA base sequence of Orius strigiformes shown in SEQ ID NO:1.

[0110] The primer is composed of natural nucleotides and / or non-natural nucleotides. It is usually composed of natural nucleotides of DNA or RNA. Among them, DNA is particularly preferred because it is highly stable, easy to synthesize, and inexpensive. If necessary, natural nucleotides of DNA and RNA can be combined, or non-natural nucleotides such as chemically modified nucleic acids or pseudonucleic acids can be partially combined. Examples of chemically modified nucleic acids and pseudonucleic acids include PNA, BNA / LNA, methylphosphonate DNA, phosphorothioate DNA, and 2'-O-methyl RNA.

[0111] Regarding the PKG detection primers, although not limited thereto, the Fw primer and Rv primer are each designed so that their Tm values ​​are in the range of 55°C to 80°C, preferably 60°C to 75°C. Furthermore, the base length and base sequence of each primer are not particularly limited. For example, the base length may be such that the base sequence is complementary to 15 or more, 18 or more, 20 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 35 or more, or 40 or more consecutive bases on the template nucleic acid strand.

[0112] The base length of the amplified fragment by the primer set of PKG detection primers is not limited, but may be within the range of 100 to 1500 bases, 150 to 1200 bases, 200 to 1000 bases, 250 to 800 bases, 300 to 600 bases, or 400 to 500 bases.

[0113] Each PKG detection primer can contain an additional sequence at its 5' end that is different from the base sequence of the template nucleic acid. The additional sequence can be any base sequence. For example, it can be a base sequence containing a restriction enzyme site or a new primer binding site.

[0114] Furthermore, the phosphate group, sugar, and / or base of the PKG detection primer of the present invention may be labeled with a labeling substance. The labeling position of the labeling substance in the primer can be determined appropriately depending on the properties of the labeling substance and the intended use, and is not limited thereto, but the 5' end, which does not usually contribute to the extension reaction, is preferred. Any substance known in the art can be used as a nucleic acid labeling substance. Examples include the aforementioned fluorescent dyes, chemiluminescent substances, radioisotopes, DIG, etc.

[0115] The primer set of PKG detection primers may be a single set, but may also be a set of multiple sets of primers such as nested primers.Furthermore, a control primer set capable of amplifying a control nucleic acid other than the SNP to be detected, as described below, may also be included.

[0116] (3) Internal Control Primer The area concentration type arthropod detection kit of the present invention may contain an internal control primer as a component.

[0117] The internal control primers are configured to be capable of amplifying a non-target template nucleic acid molecule present in a sample as an internal control (calibrator). The internal control primers are added as needed to monitor the success or status of the amplification reaction when detecting local concentration-searching arthropods using the local concentration-searching arthropod detection kit of the present invention.

[0118] The template nucleic acid molecule serving as an internal control may be a polypeptide (cDNA) derived from an endogenous nucleic acid molecule present in a sample such as total RNA, or an appropriate nucleic acid molecule may be added to the sample from outside and used as the template nucleic acid molecule. It is preferable to select a constitutively expressed gene, such as a housekeeping gene, as the endogenous nucleic acid molecule. In the case of the localized arthropod detection kit of the present invention, suitable internal controls include, but are not limited to, a transcription product (mRNA) of the EF1α (Elongation factor 1α) gene.

[0119] The base length of the amplified fragment of the internal control primer, and the basic structural characteristics, components, base length, and base sequence of the Fw primer and Rv primer constituting the internal control primer are not limited, but should basically be similar to the structure of the PKG detection primer.

[0120] (4) Internal Control Probe The area-focused arthropod detection kit of the present invention may also contain an internal control probe as an optional component.

[0121] The internal control probe is configured to hybridize to and detect the internal control amplified fragment amplified by the internal control primers, and is therefore used in combination with the internal control primers in the area-focused arthropod detection kit of the present invention.

[0122] The structural characteristics, constituent components, base length, and base sequence of the internal control probe are not limited, but basically may be similar to those of the PKG detection probe.

[0123] (5) Detection Reagents, etc. In addition to the above components, the community-based arthropod detection kit of the present invention may also include, for example, reagents necessary for reverse transcription reactions and nucleic acid amplification reactions (RTase, dNTPs, Taq polymerase, etc.), reagents for detecting amplified nucleic acid fragments (fluorescent / luminescent reagents, etc.), and protocols describing appropriate reaction conditions and methods for detecting SNPs.

[0124] On the other hand, the local concentration search-type arthropod detection kit of the present invention may be in the form of a device having the configuration of a nucleic acid array (including a DNA chip) to which nucleic acid array technology can be applied, such as a nucleic acid array in which multiple PKG detection probes are immobilized on a substrate (chip).

[0125] 3. Nucleic Acid Preparation for Producing Regionally Intensive Searching Arthropods 3-1. Overview The third aspect of the present invention is a nucleic acid preparation for producing regionally intensive searching arthropods (often abbreviated as "nucleic acid preparation" herein). The nucleic acid preparation of the present invention comprises an expression vector capable of expressing the PKG gene or an active fragment thereof (often referred to as "PKG gene, etc." herein) in host cells. In regionally intensive searching arthropods, the PKG gene is expressed at significantly higher levels than in control individuals (wild-type individuals). Therefore, by introducing the PKG gene exogenously into the cells of a host arthropod and causing it to be overexpressed or overexpressed, the organism becomes a regionally intensive searching arthropod. The nucleic acid preparation of the present invention utilizes this principle and can artificially produce regionally intensive searching arthropods from any wild-type arthropod.

[0126] 3-2. Structure The nucleic acid formulation of the present invention comprises an expression vector.

[0127] As used herein, the term "expression vector" refers to a single expression system unit that can deliver a gene of interest into a host cell and induce its expression. This expression vector is composed of DNA, contains a PKG gene or the like in an expressible state, and is configured so that, when introduced into a host cell, the PKG gene or the like can be expressed in large quantities or overexpressed. As used herein, "expressible state" refers to a state in which an RNAi molecule such as a PKG gene is placed under the control of a promoter within the expression vector.

[0128] The expression vector constituting the nucleic acid formulation of the present invention contains, as essential components, a base vector, a PKG gene or the like, a promoter that controls its expression, and a terminator. A marker gene may also be included as an optional component. Each component of the expression vector is specifically described below.

[0129] (a) Base Vector A "base vector" is a vector that forms the backbone of the expression vector, which is the nucleic acid formulation of the present invention, and may contain the essential components and optional components described below. Various vectors can be used as base vectors. Examples include autonomously replicating expression vectors such as plasmids or bacmids, viral vectors, expression vectors capable of homologous or non-homologous recombination into chromosomes, or parts of chromosomes into which such vectors have been inserted into the host chromosome. A shuttle vector capable of replicating in other bacteria, such as Escherichia coli, may also be used. The base vector constituting the expression vector of the present invention may consist of one or more base vectors. For example, a single expression vector may be formed, such as in the GAL4-UAS system, in which the GAL4 gene and UAS promoter are contained in separate mother nucleus vectors, or a single expression vector may be formed from a mother nucleus vector containing the inverted terminal repeats of a transposon and a mother nucleus vector acting as a helper plasmid containing a gene encoding a transposon transferase.

[0130] (b) PKG Gene, etc. The structure of the "PKG gene or an active fragment thereof" has already been described in the first aspect, so a detailed description will be omitted. A specific example of the PKG gene is the wild-type PKG gene of Orius strigiformes consisting of the nucleotide sequence set forth in SEQ ID NO: 1. Alternatively, the PKG gene may be a mutant PKG gene of Orius strigiformes having protein kinase activity equal to or greater than that of wild-type PKG. Examples of such mutant PKG genes include polynucleotides consisting of nucleotide sequences containing one or more base additions, deletions, and / or substitutions in the nucleotide sequence set forth in SEQ ID NO: 1, and nucleotide sequences having 90% or more nucleotide identity with the nucleotide sequence set forth in SEQ ID NO: 1.

[0131] The expression vector of the present invention can contain the same or different multiple PKG genes in an expressible state.

[0132] (c) Promoter A "promoter" is a gene expression regulatory region capable of controlling the expression of a gene or the like placed under its control. The promoter contained in the expression vector of the present invention may be of any type as long as it can express or overexpress a PKG gene or the like in a large amount in a host organism. Any appropriate promoter may be used. Examples include a constitutively active promoter, an overexpression promoter, and an inducible promoter.

[0133] (d) Terminator As used herein, the term "terminator" refers to a gene expression regulatory region composed of a base sequence capable of terminating the expression of a PKG gene or the like in the expression vector of the present invention.

[0134] (e) Marker gene: A marker gene is a polynucleotide consisting of a nucleotide sequence encoding a marker protein, also called a selectable marker. Examples of marker proteins include, but are not limited to, fluorescent proteins, pigment-synthesizing proteins, luminescent proteins, and exocrine proteins.

[0135] As used herein, the term "fluorescent protein" refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light of a specific wavelength. It may be either a natural or non-natural type. Furthermore, there are no particular limitations on the excitation wavelength or fluorescence wavelength. Specific examples include CFP, RFP, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, GFP (including derivatives such as EGFP and 3xP3-EGFP), and the like.

[0136] As used herein, a "pigment synthesis protein" refers to a protein, typically an enzyme, involved in the biosynthesis of a pigment. The term "pigment" as used herein refers to a low-molecular-weight compound or peptide capable of imparting a pigment to a transformant, regardless of type. Preferably, the pigment is one that appears as the external color of the individual. Examples include melanin-based pigments (including dopamine melanin), ommochrome-based pigments, and pteridine-based pigments.

[0137] As used herein, the term "photoprotein" refers to a substrate protein that can emit light without the need for excitation light or an enzyme that catalyzes the luminescence of the substrate protein. Examples of the substrate protein include luciferin or aequorin, and the enzyme luciferase.

[0138] 4. Local-concentrated searching arthropods 4-1. Overview A fourth aspect of the present invention is a local-concentrated searching arthropod. The local-concentrated searching arthropod of this aspect is a local-concentrated searching arthropod obtained by the production method described in the first aspect, or a local-concentrated searching arthropod containing the nucleic acid formulation described in the third aspect, and its progeny.

[0139] 4-2. The local-searching arthropod obtained by the production method described in the first aspect is an organism obtained by selecting and immobilizing individuals whose PKG expression level is significantly higher than that of the wild type due to a spontaneous mutation in the population, or its progeny. Furthermore, the local-searching arthropod containing the nucleic acid formulation described in the third aspect is an organism that has become a local-searching arthropod by introducing the nucleic acid formulation described in the third aspect, resulting in a significantly higher intracellular PKG gene expression level than that of the wild type, or its progeny containing an expression vector that is the nucleic acid formulation.

[0140] If the test individuals of these local concentration-searching arthropods are natural enemies for biological control, the local concentration-searching arthropods of this embodiment become natural enemies for local concentration-searching arthropod control and can be used in the agricultural field, etc.

[0141] Example 1: PKG gene expression levels in community-exploring arthropods (Objective) The present inventors isolated the PKG gene as a gene related to the behavioral characteristics of community-exploring arthropods. The present inventors will examine the expression levels of the PKG gene in community-exploring arthropods created using a conventional method based on the behavioral characteristics.

[0142] (Method) The localized search-focused arthropods used were Orius strigiformes that were created and strained based on their low locomotion activity using the method disclosed in Example 3 of JP 2014-207872 A. For control, wild-type Orius strigiformes that had not been subjected to the selection were used.

[0143] 1. Total RNA extraction: 48 individuals (24 males and 24 females) of the regional concentration search strain (selected strain) and wild-type (unselected strain) Orius strigiformes were frozen at -80°C for 10 minutes. The frozen individuals were mixed with the RNA extraction reagent ISOGEN II (Nippon Gene Co., Ltd.) and crushed using zirconia beads approximately 1.5 mm in diameter in a bead-type crusher (ShakeMaster® AUTO: Bio Medical Science Co., Ltd.), and total RNA was extracted according to the attached protocol.

[0144] 2. Quantitation of PKG mRNA by quantitative RT-PCR. cDNA synthesis was performed from the total RNA obtained using the High Capacity cDNA Reverse Transcription Kit (ABI) according to the protocol. RT-PCR was then performed using the prepared cDNA as a template and THUNDERBIRD® Next SYBR® qPCR Mix (TOYOBO) according to the attached protocol. The primer sequences used are shown in Table 2.

[0145]

[0146] The amplified products were quantified using a QuantStudio® 3 real-time PCR system (Thermo Fisher Scientific), and the relative expression level of the PKG gene was determined by the comparative CT method using the expression level of EF1α as an internal control.

[0147] (Results) The results are shown in Figure 2. Figure 2 reveals that the expression level of the PKG gene in the area-intensive-searching Orius strigiforme (selected strain) produced by a method based on behavioral characteristics is significantly higher than that in the wild-type Orius strigiforme (non-selected strain).

[0148] Example 2: Changes in behavioral characteristics of local-searching arthropods due to suppression of PKG gene expression (Objective) To verify changes in behavioral characteristics when the expression level of the PKG gene is suppressed in local-searching arthropods.

[0149] The results of Example 1 revealed that the expression level of the PKG gene was higher in the selected line compared to the non-selected line. Therefore, we used RNAi to confirm whether knocking down the PKG gene in the selected line would result in changes in the behavioral characteristics of the selected line. In the control group, we performed knockdown of the EGFP gene, which does not have a target gene in Orius strigiformes and does not affect behavioral characteristics.

[0150] (Method) dsRNA for RNAi was synthesized using T7 RiboMAX. TM The RNAi was performed using the Express RNAi System (Promega) according to the attached protocol. dsRNA was synthesized for the target PKG gene and the control EGFP gene. Primers with T7 promoter sequences attached to both ends of each gene were used to synthesize each dsRNA. The sequences of each primer are shown in Table 3.

[0151]

[0152] Each synthesized dsRNA was adjusted to 2 μg / μL in blue-stained RNase-free water and microinjected at 0.01 μL into final-instar larvae of the regionally concentrated searching Orius strigiforme (selected strain) used in Example 1. Three days after injection, individuals that had molted normally and emerged as adults were examined for their behavioral characteristics based on their locomotion activity.

[0153] The number of individuals for measuring locomotor activity was 35 males and 35 females for PKG dsRNA, and 30 and 29 males and 39 females for EGFP dsRNA. The basic method followed the method disclosed in Example 1 of JP 2014-207872 A. Specifically, the locomotor activity of Orius strigiformes was measured for 1 hour at 25°C using an insect behavior monitor (Drosophila Activity Monitor 2; TriKinetics).

[0154] (Results) The results are shown in Figure 3. As shown in this figure, the PKG gene knockdown population of the area-searching Orius strigiforme showed a significant increase in walking activity in both males and females. In contrast, the EGFP gene knockdown population in the control group maintained the area-searching behavioral trait in both males and females. This result suggests that increased PKG gene expression is associated with the area-searching behavioral trait.

[0155] Example 3: Correlation between PKG gene expression level and walking activity level (Objective) As disclosed in JP 2014-207872 A, in area-focused exploring arthropods, there is a negative correlation between area-focused exploring time and walking activity level. Since Example 2 suggested that there is a relationship between the expression level of the PKG gene and the behavioral characteristics of area-focused exploring, we investigated whether there is a similar negative correlation between the expression level of the PKG gene and walking activity level.

[0156] (Method) The basic method followed the method in Example 1. The walking activity of 24 male, area-searching Orius strigiformes was measured for each individual. Total RNA was then extracted from each individual, and PKG mRNA was quantified by quantitative RT-PCR. The correlation between walking activity and PKG gene expression level for each individual was plotted as a graph.

[0157] (Results) The results are shown in Figure 4. As this figure shows, it was revealed that the expression level of the PKG gene also had a negative correlation with the amount of walking activity. These results demonstrate that the PKG gene is a gene related to the behavioral trait of area-focused exploration, and that when the expression level of the PKG gene is significantly higher than that of the wild type, the individual will be area-focused exploration.

[0158] Example 4: Creation of local-exploratory arthropods (Objective) The results of Examples 1 to 3 revealed that there is a negative correlation between local-exploratory behavioral characteristics and the expression level of the PKG gene, and that the expression level of the PKG gene in local-exploratory arthropods is significantly higher than that in wild-type arthropods. Therefore, in this Example, we selected and phylogenetically characterized local-exploratory arthropods based on the expression level of the PKG gene.

[0159] (Method) Orius strigiformes were used as test individuals. To prepare the selection population, the walking activity of 24 male and 24 female Orius strigiformes was measured using the Drosophila activity monitor, an insect behavioral measurement device described above, and the individuals with the best performance, i.e., low walking activity, were used for the initial male-female pairing. This advance preparation based on behavioral characteristics ensured that each individual in the selection population would have a probabilistically higher expression level of the PKG gene compared to wild-type individuals.

[0160] Twenty-four pairs of males and females obtained through pre-preparation based on the behavioral characteristics described above were paired (G0), and each G0 female was allowed to lay eggs for one week. After egg laying, total RNA was extracted from each male and female individual for each G0 parent pair, and the expression levels of the PKG gene were measured. Six pairs of eggs laid by G0 parent pairs with high PKG expression were selected, and the progeny (G1) of each pair were reared in the same container. These G1 individuals were identified by sex upon emergence and then paired again in the same manner as G0. After allowing the G1 females to lay eggs, total RNA was extracted from each G1 parent pair, and the expression levels of the PDK gene were measured. Six pairs of eggs laid by G1 parents with high expression levels were again selected, and the progeny (G2) of each pair were reared in the same container. This was repeated for several generations to establish a lineage of the localized search-oriented Orius strifei bug.

[0161] Example 5: Land settlement rate of a local-concentrated-searching arthropod and its control effect (1) (Purpose) To verify the land settlement rate of the systematized local-concentrated-searching Orius strifebug, which was produced using the method for producing a local-concentrated-searching arthropod of the present invention described in Example 4, and the resulting pest control effect.

[0162] (Method) The local-concentration-searching arthropods used were the local-concentration-searching Orius strifenidis (PKG selection line) selected and systematized based on the expression level of the PKG gene in Example 4, and the local-concentration-searching Orius strifenidis (activity selection line) selected and systematized based on the amount of walking activity disclosed in JP 2014-207872 A.

[0163] Strawberry and eggplant fields were used for the field survey. In the strawberry field, 86 strawberry seedlings (variety: Berry Pop Haruhi) were grown in elevated stands in two 0.5a vinyl greenhouses under the usual cultivation conditions used by ordinary farmers. In mid-November, 40 individuals of each of the above strains of Orius strigiformes were released, and the number of Orius strigiformes present on all strawberries in each greenhouse was monitored over time. Note that no thrips, which serve as food for the bugs, were found in the strawberry field during the survey period.

[0164] Meanwhile, in the eggplant fields, eggplants (variety: Togenashi Senryo No. 2) were planted in two 0.5a vinyl greenhouses in October and grown under the usual cultivation conditions used by ordinary farmers. In early November, the natural emergence of onion thrips was confirmed on the flowers, young fruits, and stems and leaves of the plants. Then, in late November, 40 individuals of each of the above strains of Orius strigiformes were released into each greenhouse, and the number of Orius strigiformes present on all eggplants in each greenhouse and the number of thrips present on 20 plants in each greenhouse were monitored over time.

[0165] (Results) The results are shown in Figures 5 and 6. Figure 5 shows the change in the number of Orius strigiformes over time after release in the strawberry field (A) and eggplant field (B), respectively. Figure 6 shows the change in the number of thrips over time in the eggplant field.

[0166] The results in Figure 5 show that in both fields, the population over time of the PKG-selected lines, selected based on PKG gene expression levels, and the activity-selected lines, selected based on walking activity levels, clearly demonstrated higher soil colonization of the PKG-selected lines. In particular, in the strawberry field (A), the PKG-selected lines remained established over the long term, even though there was no occurrence of thrips, which serve as food for Orius strigiformes, during the survey period. These results suggest that the method for producing area-intensive searching arthropods of the present invention, based on PKG gene expression levels, is more efficient than the conventional method for producing area-intensive searching arthropods based on walking activity levels.

[0167] Furthermore, the results in Figure 6 indicate that the PKG-selected lines with high soil colonization have a higher thrips control effect than the activity-selected lines.

[0168] Example 6: Transition of PKG gene expression level in each generation by selection (Objective) The selection based on the expression level of the PKG gene performed in Example 4 is repeated for each generation to identify the generation in which the expression level reaches saturation.

[0169] (Method) Orius strigiformes were used as test individuals. The basic procedure followed the selection method described in Example 4. In Example 4, selection was repeated for several generations, but in this example, selection was further repeated over additional generations to examine the expression level of the PKG gene in each generation. Selection was stopped when it was determined that the expression level of the PKG gene had reached saturation.

[0170] (Results) The results are shown in Figure 7. Selection based on PKG gene expression levels in Orius strigiformes revealed that expression levels reached a nearly saturated state and equilibrium in both males and females in the seventh (G7) or eighth (G8) generation. This suggests that phylogenetic differentiation based on PKG gene expression levels in locally intensively searching arthropods can be stabilized by repeating the process for seven to eight generations.

[0171] Example 7: Land settlement rate of locally intensively searching arthropods and its control effect (2) (Objective) The land settlement rate and control effect against thrips of the PKG-selected line and the activity-selected line created in Example 6 were examined.

[0172] (Method) The basic procedure was the same as that described in Example 5. The PKG gene expression selection line used was the eighth generation Orius strigiformes (PKG selection line) selected and systematized based on the expression level of the PKG gene in Example 6. The activity level selection line used was the regionally concentrated search-type Orius strigiformes (activity level selection line) selected and systematized based on the walking activity level (phenotype) disclosed in JP 2014-207872 A, which was also used in Example 5.

[0173] The field survey was conducted in a strawberry field. In three 0.5a vinyl greenhouses, 108 strawberry seedlings (variety: Koiminori) were grown in elevated stands under the usual cultivation conditions used by ordinary farmers.

[0174] Sixteen PKG-selected strains of Orius strigiformes, 54 activity-selected strains, and 54 unselected strains were released into each greenhouse twice, once in late March and once a week later, and the number of Orius strigiformes present on all strawberries in each greenhouse, as well as the number of thrips present on 48 flowers and 48 young fruits, were counted over time every week during the survey period.

[0175] (Results) The results are shown in Figures 8 and 9. Figure 8 shows the change over time in the number of confirmed Orius strigiformes released in a greenhouse after release in a strawberry field. Figure 9 shows the change over time in the number of thrips present on flowers and young fruits in the strawberry field.

[0176] The results in Figure 8 indicate that the PKG-selected line after eight generations of selection has significantly higher soil colonization than the activity-selected line. Furthermore, while the activity-selected line had roughly the same number of individuals as the non-selected line 70 days after release, the PKG-selected line still had roughly twice as many individuals even 77 days after release, reaffirming the high soil colonization of the PKG-selected line in Example 5. Furthermore, in Example 5, there was no occurrence of thrips in the strawberry field, so the control effect could not be confirmed, but in this example, a high control effect against thrips was also confirmed.

[0177] The results in Figure 9 confirm that the PKG-selected line and the activity-selected line of Orius strigiformes tend to have a slightly stronger control effect on thrips than the PKG-selected line. It is noteworthy here that while the activity-selected and non-selected lines released in the field in this example had a total of 54 individuals, the number of PKG-selected lines was less than one-third of that, at 16 individuals. This suggests that the PKG-selected line after eight generations of selection has a control effect three times greater than the activity-selected line. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A method for producing a localized searching arthropod belonging to the class Insecta or Arachnida, comprising: a nucleic acid extraction step of extracting nucleic acid from each test individual in the homogeneous population; a measuring step of measuring the expression level of the cGMP-dependent protein kinase gene in the nucleic acid extracted from each test subject; and a selection step of selecting local concentrated search arthropods based on the expression level of the gene in the test individual measured in the measurement step. The method. 　 a mating step in which the local-concentrated searching arthropod produced according to claim 1 is used as at least one of the parent individuals; and The method of claim 1, further comprising a repeat step of repeating the nucleic acid extraction step, measurement step, and selection step of claim 1 on the hybrid population obtained in the mating step. 　 The method of claim 2 , further comprising a step of repeating the crossing and repeating steps multiple times to produce a line. 　 The method according to any one of claims 1 to 3, wherein the cGMP-dependent protein kinase gene is a polynucleotide consisting of any one of the following base sequences (a) to (c): (a) the base sequence shown in SEQ ID NO: 1; (b) a base sequence containing one or more base additions, deletions, and / or substitutions in the base sequence shown in SEQ ID NO: 1; and (c) A nucleotide sequence having 90% or more nucleotide identity with the nucleotide sequence shown in SEQ ID NO:

1. The method according to any one of claims 1 to 4, wherein the localized searching arthropod is a natural enemy organism for biological control. 　 The method for producing a stink bug according to claim 5, wherein the natural enemy belongs to any one of the group consisting of Anthocoridae, Miridae, Lygaeidae, Coccinellidae, and Phytoseiidae. 　 A kit for detecting arthropods in a localized area, comprising a probe capable of specifically binding to a polynucleotide or a fragment thereof derived from the mRNA of the cGMP-dependent protein kinase gene, which is a localized arthropod marker, and / or a primer that specifically recognizes and amplifies the same. 　 The probe and / or primer is a polynucleotide selected from the group consisting of any one of the following (a) to (e): (a) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1, or a fragment thereof containing 15 or more consecutive bases; (b) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1; (c) a polynucleotide consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1, or a fragment thereof containing 15 or more consecutive bases; (d) a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1; and (e) a polynucleotide that hybridizes under stringent conditions with any one of the polynucleotides (a) to (d); The kit of claim 7, wherein the polynucleotide is selected from the group consisting of: 　 The kit according to claim 7 or 8, wherein the localized searching arthropod is a natural enemy organism for biological control. A nucleic acid preparation for producing a localized search-type arthropod, comprising an expression vector capable of expressing a cGMP-dependent protein kinase gene or an active fragment thereof in a host cell. 　 The nucleic acid formulation according to claim 10, wherein the cGMP-dependent protein kinase gene is a polynucleotide consisting of any one of the following base sequences (a) to (c): (a) the base sequence shown in SEQ ID NO: 1; (b) a base sequence containing one or more base additions, deletions, and / or substitutions in the base sequence shown in SEQ ID NO: 1; and (c) A nucleotide sequence having 90% or more nucleotide identity with the nucleotide sequence shown in SEQ ID NO:

1. A localized searching arthropod obtained by the method of any one of claims 1 to 6, or comprising the nucleic acid formulation of claim 10 or 11. 　 The area-concentrated searching arthropod according to claim 12, wherein the area-concentrated searching arthropod is a natural enemy organism for biological control.

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