Method for supporting extermination of pests

Abstract

To deal with extermination by grasping the invasion state of even an insect pest which is difficult to be captured by trap investigation.SOLUTION: The pest extermination support method includes a step of selecting a primer set effective for DNA analysis of a pest which is an extermination candidate in an indoor target area from a plurality of primer sets, setting a plurality of sample collection positions in the target area, performing DNA analysis using the primer set for a sample collected at each of the sample collection positions, generating a distribution map 50 of the target area in which the sample collection position and a type of pest specified for each sample collection position based on the DNA analysis are associated with each other, and performing pest extermination measures based on the distribution map 50.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method for assisting in the extermination of pests. [Background technology]

[0002] For example, in food factories and the like, it is common to monitor the infestation of pests for hygiene management purposes. Generally, to monitor the infestation, trap surveys are conducted by placing traps in locations where pests are likely to pass through (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-049173 Summary of the Invention [Problem to be solved by the invention]

[0004] The advantage of trap surveys is that they allow for quick estimation of the presence or absence of pests simply by collecting samples. However, trap surveys are difficult to use to capture individual insects such as eggs, larvae, and pupae. Furthermore, traps are difficult to set up in narrow or dirty spaces, and adhesive tape loses its adhesive strength in humid locations. Therefore, there are limitations on sample collection locations, making it difficult to obtain a detailed understanding of pest distribution using trap surveys alone. Furthermore, identifying pests captured by traps requires workers who can identify pests visually, and securing such workers is difficult. [Means for solving the problem]

[0005] The present disclosure provides a pest extermination support method that includes selecting a primer set effective for DNA analysis of pests that are candidates for extermination in a target indoor area from among a plurality of primer sets, setting a plurality of sampling locations in the target area, performing DNA analysis using the primer set on samples collected at each of the sampling locations, and generating a distribution map of the target area that associates the sampling locations with the pest species identified for each sampling location based on the DNA analysis. [Effects of the Invention]

[0006] According to the present disclosure, it is possible to understand the infestation status of pests that are difficult to capture by trap surveys and take measures to exterminate them. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a conceptual diagram of a universal primer evaluation process according to one embodiment of a method for inspecting indoor pests. [Figure 2] FIG. 2 is a diagram illustrating a configuration of an information processing device used in the field survey process of the embodiment. [Figure 3] 10 is a flowchart showing the procedure of the extermination support method of the embodiment. [Figure 4] FIG. 2 is a diagram showing an example of a layout diagram of a target area according to the embodiment. [Figure 5] 10 is a flowchart showing detailed steps of the extermination support method of the embodiment. [Figure 6] FIG. 2 is a diagram showing an example of a distribution map of a target area according to the embodiment. [Figure 7] FIG. 10 is a diagram illustrating an example of evaluation information according to the embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0008] An embodiment of the extermination support method will be described below. In this method, a building such as a food factory is investigated, and the intrusion of indoor pests and their types are estimated. If pest intrusion is estimated, the distribution and movement routes of the pests are further estimated. In this case, the pests investigated are arthropods.

[0009] The extermination support method uses a detection method called environmental metabarcoding, which is metabarcoding of environmental DNA. Environmental metabarcoding is a method for comprehensively detecting species belonging to the taxonomic group being investigated. Environmental metabarcoding uses universal primers designed based on base sequence regions common to pest species. This analysis method uses a primer set that combines a universal forward primer and a universal reverse primer; hereafter, this primer set will be simply referred to as the universal primer.

[0010] In environmental metabarcoding, PCR products amplified using universal primers are sequenced using a next-generation sequencer, and the sequence is compared with sequences registered in a database to identify the type of pest.

[0011] The pest control support method includes a universal primer evaluation step and a field survey step using the evaluation results of the universal primer. <Universal primer evaluation process> The evaluation process will be outlined below. Experiments conducted by the inventors have revealed that universal primers have unique characteristics in DNA amplification. For example, even when the same sample is amplified, one universal primer may detect Blattodea but not Araneae, whereas another universal primer may not detect Blattodea but detects Araneae.

[0012] Therefore, in the evaluation step, as a preparatory step for conducting a field survey, the results of evaluating the properties of a plurality of universal primers targeted at arthropods are stored in advance in a database. Next, the evaluation process will be described in detail.

[0013] As shown in Figure 1, environmental metabarcoding of pests in buildings involves collecting a sample of sediment 100 from a designated sampling location within the building. The sediment 100 may contain environmental DNA, which is DNA derived from pest bodies or excrement. The "sediment" referred to here may be solid or liquid. Examples of sediments include dust accumulated on indoor floors, matter attached to walls, floors, or ceilings, biofilms formed in drains and gutters, and water accumulated indoors.

[0014] Methods for collecting the deposit 100 include sucking it up with a vacuum cleaner, wiping the walls, floors, or ceilings with a nonwoven fabric, and collecting water from a set cup. When vacuuming dust, components such as a tank and nozzle that collect the sucked dust are replaced for each sample collection position to prevent contamination. When collecting water from a cup, a cup with an opening and filled with water is set at the sample collection position in advance.

[0015] Once a sample is collected, it is purified and DNA is extracted using a soil sample DNA extraction kit or similar. The DNA extract 101 collected at the same location and on the same collection date and time is then divided into multiple extracts, and each of the divided extracts 101 is amplified using a different type of universal primer. The universal primers are designed for arthropods. For example, these universal primers amplify the 16S rRNA region or the mitochondrial CO1 region (mitochondrial cytochrome c oxidase subunit one). The sequence of each amplified sample is then determined using a next-generation sequencer to obtain analytical results. In other words, the only difference between the samples is the type of universal primer; the other analytical conditions are the same.

[0016] The characteristics of the universal primers are evaluated from the analysis results using each universal primer. The number of reads in each analysis result varies. In the example of Figure 1, for each analysis result using each universal primer, the total number of reads is set to 100%, and the vertical width of the graph indicates the percentage of reads for the detected pest species. The type of pest detected varies depending on the universal primer used. Also, for example, the analysis results using "Primer A" and "Primer B" both include "Battleroaches," but the ratio of pests to the total number of reads is different.

[0017] Next, the characteristics of the universal primer are evaluated based on these analysis results. That is, the types of pest insects that the universal primer can easily detect are evaluated. This evaluation may involve determining whether the proportion of the DNA reads of the pest insects is equal to or greater than a predetermined ratio. For example, if the analysis results using "Universal Primer A" show that the proportion of "Battleroaches" relative to the total number of reads is equal to or greater than a predetermined ratio, the universal primer is evaluated as being suitable for detecting "Battleroaches." Furthermore, if the analysis results using "Universal Primer B" show that the proportion of "Psocids" and "Collembola" relative to the total number of reads is equal to or greater than a predetermined ratio, the universal primer is evaluated as being suitable for detecting "Psocids" and "Collembola." As a result, the universal primer may be evaluated as being capable of specifically detecting one psocid or as being capable of balanced detection of multiple psocids. Furthermore, the results of trap surveys conducted at the same sample collection location and on the same collection date and time may also be taken into consideration.

[0018] Evaluation data 20 indicating the evaluation results of the universal primers is stored in the evaluation data storage unit 13. The evaluation data 20 is data that associates the identifiers of the universal primers with the types of pests for which the universal primers are suitable. The evaluation data 20 is updated by receiving feedback on the results of field surveys.

[0019] <Field survey process> The implementation survey process will be explained with reference to Figure 2. In the implementation survey, pests to be exterminated are selected based on environmental information such as the location and time of year, as well as interviews with building users.

[0020] First, samples are collected according to the pests to be exterminated (Step S1). For example, multiple sample collection locations are determined according to the pests within the area of ​​a building to be exterminated (hereinafter referred to as the target area). Samples are then collected at the sample collection locations. The method of collecting samples is the same as in the evaluation process. At this time, for example, deposits are collected from locations that are likely to be the movement path or habitat of the pest to be detected. If the target includes Blattonia, in addition to locations where Blattonia are expected to inhabit, possible routes that Blattonia may take from these habitats are estimated, and multiple locations on these routes are selected as sample collection locations. Furthermore, if the target is a flying insect such as Diptera or Hymenoptera, deposits may be collected not only from the floor but also from the wall or ceiling.

[0021] In addition, while collecting the sediments, traps may be placed at locations appropriate for the type of pest to be detected. The type of trap is not particularly limited, but examples include light traps that attract pests using light, and floor traps with adhesive tape that capture pests. In this collection method, the traps are replaced every month, and the presence or absence of pests caught in the traps is visually confirmed.

[0022] Next, a universal primer corresponding to the pest insect to be exterminated is selected from the universal primers stored in the evaluation data storage unit 13 (step S2). At this time, one or more universal primers may be selected.

[0023] Then, environmental DNA analysis is performed using the selected universal primers (step S3). The environmental DNA analysis is the same as the evaluation step. The analysis result data of the environmental DNA analysis is fed back to the evaluation data storage unit 13.

[0024] Once the DNA of the pest has been identified through environmental DNA analysis, pest control measures are formulated (step S4). In this process, a distribution map 50 of the pests in the target area is generated, and specific control measures are formulated based on the distribution map 50.

[0025] <Generate disinfection measures> The formulation of eradication measures will be specifically described with reference to Figures 3 to 6. The distribution map 50 is generated by the information processing device 10.

[0026] As shown in Fig. 3, the information processing device 10 is a personal computer, a tablet, or the like. The information processing device 10 includes a processor (processing circuit) such as a CPU, a communication device, and a storage device (all of which are not shown). The storage device stores programs for performing each process. When the processor executes the programs, the information processing device 10 functions as an analysis result acquisition unit 11 and a distribution estimation unit 12. The information processing device 10 is connected to an input device 16 such as a mouse and a keyboard, and an output device 17 such as a display (monitor).

[0027] The information processing device 10 includes the above-described evaluation data storage unit 13 as well as a building data storage unit 14. The building data storage unit 14 stores a layout diagram 30 (see FIG. 4) of the target area of ​​the building.

[0028] 4 is an example of a layout diagram 30. The layout diagram 30 shows the positions of entrances and exits 31. The layout diagram 30 may also show the positions of at least one of drainage channels 32 and drainage outlets 33 in the target area. Furthermore, the layout diagram 30 may also show the position of any one of air intake vents 35, air intake ducts 36, exhaust vents 37, and exhaust ducts 38. If the target area is a factory, the layout diagram 30 may also show the positions of indoor equipment 39 in each process, such as production equipment.

[0029] Furthermore, the layout diagram 30 may also show the movement lines of factory workers and hygiene levels (clean area, semi-clean area, contaminated area, etc.). The above-mentioned displays may also be switchable. 5, the distribution estimation unit 12 positions the sample collection position 40 on the layout diagram 30 (step S11). For example, the worker may use the input device 16 to specify an arbitrary position on the layout diagram 30, thereby positioning the sample collection position 40 on the layout diagram 30. Alternatively, the information processing device 10 may automatically position the sample collection position 40 on the layout diagram 30 based on position information (GNSS position information) transmitted from a terminal carried by the worker. The position information is transmitted by an operation of the worker who is at the sample collection position.

[0030] The analysis result acquisition unit 11 acquires analysis result data 41 obtained by environmental DNA analysis (step S12). The analysis result data 41 may be read from a storage medium or received from another information processing device connected to a sequencer or the like. The analysis result data 41 indicates the analysis results of each sample collected at multiple sample collection locations for each universal primer used in the analysis. For example, the analysis result data 41 includes the analysis results of "sample collection location 1" associated with "primer A," the analysis results of "sample collection location 2," etc., and the analysis results of "sample collection location 1" associated with "primer B," the analysis results of "sample collection location 2," etc.

[0031] The analysis result data 41 includes identifiers of sample collection locations and the number of reads for each sample collection location. The number of reads includes the total number of reads and the number of reads for each type of pest. Alternatively, instead of or in addition to the number of reads for each type of pest, the analysis result data 41 may include a composition ratio obtained by dividing the number of reads for each type of pest by the total number of reads.

[0032] The distribution estimation unit 12 generates a distribution map 50 by mapping the analysis results to the sampling locations 40 in the layout diagram 30 that are associated with the identifiers of the sampling locations included in the analysis result data 41 (step S13). Specifically, the distribution estimation unit 12 compares the identifiers of the sampling locations 40 determined in step S11 with the identifiers of the sampling locations 40 included in the analysis result data. Then, the distribution estimation unit 12 reads out the analysis results for the sampling locations 40 included in the analysis result data for each sampling location 40, and generates indices based on the analysis results. The distribution estimation unit 12 generates the distribution map 50 by displaying the generated indices at the sampling locations 40 in the layout diagram 30.

[0033] FIG. 6 is an example of a distribution map 50 generated based on the layout map 30 shown in FIG. 4. In the distribution map 50, an indicator 60 is displayed in association with each sample collection location 40. The size of the indicator 60 is set based on the total number of reads. The indicator 60 also includes a composition ratio 61. The composition ratio 61 indicates the appearance tendency of that type of pest. If pest DNA is not detected at the sample collection location 40, a display indicating that it was not detected may be output. In FIG. 6, if pest DNA is not detected, the indicator 60 is not superimposed on the sample collection location 40.

[0034] In the example of Figure 6, the indicator 60 is a pie chart, and the composition ratio 61 is a sector, semicircle, or the like, according to the ratio of the number of leads. The composition ratio 61 can be identified as representing which pest composition ratio it represents, depending on the display mode, such as its color or pattern. The size of the indicator 60 allows one to estimate the relative density between the sample collection locations 40.

[0035] For example, it is estimated that a relatively large index 60 on the distribution map 50 indicates a higher density of pests than a relatively small index 60. The indexes 60 are displayed overlaid on the layout map 30, so it is possible to grasp the distribution of pests and their positional relationship with the drain outlet 33, air intake 35, exhaust vent 37, entrance / exit 31, etc. Furthermore, the area (size) of the diagram of the composition ratio 61 allows the relative density of the pest species at the sampling location 40 to be estimated.

[0036] As shown in FIG. 6, the distribution map 50 may display a pest movement path 62. When sampling locations 40 where a specific pest species has been detected are adjacent to each other, the distribution estimation unit 12 assumes that these sampling locations 40 are on the pest's movement path and displays a movement path 62 extending from one sampling location 40 to the other sampling location 40. The display mode, such as the color or pattern, of the movement path 62 may be the same as the display mode of the composition ratio 61. This allows a worker or the like checking the distribution map 50 to confirm, at a glance, the relative density of, for example, "Battleroaches" and their movement path 62. Furthermore, when the area of ​​the composition ratio 61 is large, it is estimated that the sampling location 40 may be an intrusion point.

[0037] Once the distribution map 50 is generated in this manner, extermination measures are formulated or implemented for each pest based on the distribution map 50 (steps S14 to S16). At this time, the distribution estimation unit 12 may output an extermination method to the output device 17 using a database (not shown) in which extermination measures according to the type of pest are stored, or an operator may formulate the extermination measures. Examples of extermination measures include spraying chemicals along the movement route shown in the distribution map 50, and installing intrusion prevention devices or traps according to the size or characteristics of the pests at openings through which the pests are predicted to enter. Furthermore, extermination measures are primarily carried out at sampling locations 40 where the density is predicted to be high.

[0038] <Effects of this embodiment> As described above, according to the first embodiment, the following effects can be obtained. (1) In the above embodiment, for a target area, an effective universal primer (primer set) is selected according to the pest insects to be exterminated, and the type of pest is identified using DNA analysis. This makes it possible to grasp the infestation status of pest insects that are difficult to capture with traps. Furthermore, a pest distribution map 50 can be generated from DNA analysis, allowing effective extermination measures to be formulated.

[0039] (2) In the above embodiment, the distribution map 50 displays the indicators 60 indicating the types of multiple pests, so that the distribution map 50 allows the user to grasp the distribution of pests in detail. (3) In the above embodiment, the movement route 62 for each type of pest insect is estimated based on the distribution map 50, so that effective extermination measures can be taken.

[0040] <Example of change> The above-described embodiment can be modified as follows: Each embodiment and the following modifications can be combined with each other to the extent that no technical contradiction occurs.

[0041] In the above embodiment, the distribution estimation unit 12 generates the distribution map 50 by displaying circular indicators 60 at the sample collection positions 40 on the layout map 30, but the indicators 60 may take other forms. For example, the indicators 60 may be bar graphs or other graphs. Alternatively, the distribution estimation unit 12 may display a heat map for each type of pest.

[0042] In the above embodiment, the layout diagram 30 is a two-dimensional drawing. Alternatively, the layout diagram 30 may be a three-dimensional drawing showing the layout (arrangement) of the target area in the building in the width, depth, and height directions. This allows the height of the sample collection positions to be reflected in the distribution diagram 50. For example, when flying pests such as dipterans are candidates for extermination, samples may be collected by wiping the ceiling, walls, and floor with a nonwoven fabric. The distribution diagram 50 can show the difference in height between the sample collection positions 40 on the ceiling and walls and the sample collection positions 40 on the floor. This allows the distribution of pests to be grasped in the height direction of the target area, enabling effective extermination measures to be taken.

[0043] In the aspect of estimating the movement path 62, a common position of the movement paths 62 of a plurality of pests may be identified, and measures to exterminate the identified plurality of pests may be taken at the common position. In this case, pests can be exterminated efficiently.

[0044] The extermination response based on the distribution map 50 may be determined depending on the characteristics of the pest to be exterminated. For example, in the case of a pest with a wide range of movement, a pesticide may be sprayed at all sample collection locations 40 where the pest was detected and their surrounding areas, with the sample collection location 40 with the highest composition ratio 61 as the center. In contrast, in the case of a pest with a narrow range of movement, a pesticide may be sprayed in a narrower area centered on the sample collection location 40 with the highest composition ratio 61. The distribution map 50 may also show the illuminance, room temperature, and humidity at each indoor location. The extermination response may then take into account the environmental preferences of the pest.

[0045] The information processing device 10 may be part of a system (extermination support system) composed of multiple devices. For example, the extermination support system is a pest extermination support system that, when a processor executes a program, selects from multiple primer sets a primer set effective for DNA analysis of pests that are candidates for extermination in a target indoor area, sets multiple sample collection positions in the target area, performs DNA analysis using the primer set on samples collected at each sample collection position, generates a distribution map of the target area that associates the sample collection positions with the type of pest identified for each sample collection position based on the DNA analysis, and takes action to exterminate the pests based on the distribution map.

[0046] <Example> Examples of environmental DNA analysis are described below. The examples are not intended to limit the scope of the present disclosure. The scope of the present disclosure is limited only by the claims.

[0047] For the trap survey, one light trap (MP600, Benhur Fuyo Co., Ltd.) and one floor-standing sticky trap (Shimada Co., Ltd.) were set up. The sticky paper of the light trap and the floor-standing sticky trap were replaced every month, and the captured insects were identified to the eye level under a stereomicroscope.

[0048] Sediment (dust) samples were collected once a month from the vicinity of the trap installation. A nonwoven fabric sample collection bag was placed between the body and crevice nozzle of a vacuum cleaner (SV12FF, Dyson Technology Limited) equipped with a filter with performance equivalent to a HEPA (High Efficiency Particulate Air Filter), and dust was sucked from the sample collection location for approximately 1 minute. To prevent contamination, the crevice nozzle and vacuum cleaner attachment were changed for each sample collection location.

[0049] DNA was extracted from the samples using the "DNA Extrap Soil DNA Kit Plus ver.2" (manufactured by Nippon Steel Environment Co., Ltd.). Next, PCR was performed to amplify the target region using the extracted DNA as a template. The universal primers used for DNA amplification are listed in Table 1.

[0050] [Table 1] The PCR kit used was "KAPA3G Plant" (manufactured by KAPA BIOSYSTEMS), which is resistant to PCR inhibitors. The PCR conditions are as shown in Table 2. "Step 2" to "Step 4" in Table 2 were repeated 45 times.

[0051] [Table 2] The PCR product was purified using the "MonoFAS DNA Purification Kit" (GL Science), and the adapter sequence required for next-generation sequencing and the index sequence for sample identification were added according to the protocol of the "Nextera XT Index Kit" (Illumina).

[0052] The PCR products were purified using "NucleoMag NGS Clean-up and Size Select" (Takara Bio). The prepared samples were analyzed using the next-generation sequencer "Miseq" (Illumina) to decode the base sequence. The analysis was performed using the "Miseq Reagent Kit v3."

[0053] The sequence data decoded by the next-generation sequencer was analyzed using the analysis software "QIIME2." Low-quality gene sequences were removed from the obtained gene sequences, and primer and index sequences were deleted from the sequences. Then, clustering was performed based on 100% homology matches, and similar sequences were organized into OTUs (Operational Taxonomic Units), which are taxonomic units. The closest relative species of each OTU were identified using a BLAST search (NCBI nt database).

[0054] The detection results using multiple universal primers for one sample are shown in Figure 7. The table in Figure 7 shows the types of pest insects that are most prevalent for each primer. The trapping survey detected Myriapoda, Araneae, Collembola, Cardinalida, Coleoptera, Hymenoptera, Hemiptera, and Diptera. Diptera, Cardinalida (Psocoptera), Collembola, and Hymenoptera were the most frequently caught. No cockroaches were caught in the trapping survey.

[0055] On the other hand, in environmental DNA analysis, the types and composition of pests detected varied greatly depending on the primers. Many primers were able to amplify DNA from Diptera, Blattella, Coleoptera, and Cardiac orders. Few primers were able to amplify DNA from Hymenoptera and Lepidoptera. This may be due to issues with primer compatibility or the possibility that the DNA of those types of pests contained in the samples was extremely low.

[0056] Next, the technical ideas that can be understood from the above-described embodiment and other examples will be described below. [A] The extermination support method, wherein the distribution map maps the appearance trends of the identified pests in addition to the types of pests. [B] if a plurality of the pest insects are identified based on the DNA analysis, identifying a common position in the movement route of the plurality of the pest insects; An extermination support method, wherein extermination measures are taken against the identified plurality of pests at the common location. [C] In selecting the primer set, the extermination support method uses evaluation information that associates the primer set with the type of pest that matches the characteristics of the primer set to select the primer set that is suitable for the pest that is a candidate for extermination. [D] A pest extermination support system using one or more control circuits, wherein the control circuit selects a primer set effective for DNA analysis of pests that are candidates for extermination in a target area indoors from among the plurality of primer sets; setting a plurality of sample collection positions in the target area, and performing DNA analysis using the primer set on samples collected at each of the sample collection positions; A pest control support system characterized by generating a distribution map of a target area that associates the sample collection locations with the types of pests identified for each sample collection location based on the DNA analysis. [Explanation of symbols]

[0057] 10...information processing device, 11...analysis result acquisition unit, 12...distribution estimation unit, 13...evaluation information storage unit, 14...building data storage unit, 20...evaluation information, 30...layout map, 50...distribution map, 60...index, 61...composition ratio

Claims

1. selecting a primer set effective for DNA analysis of pests that are candidates for extermination in a target indoor area from among the plurality of primer sets; setting a plurality of sample collection positions in the target area, and performing the DNA analysis using the primer set on the samples collected at each of the sample collection positions; A pest extermination support method characterized by generating a distribution map of the target area that associates the sample collection locations with the types of pests identified for each sample collection location based on the DNA analysis.

2. The pest extermination support method according to claim 1 , further comprising displaying, on a layout map of the target area, indicators indicating the types of the plurality of pests identified based on the DNA analysis in association with the sample collection locations.

3. Predicting the migration route of each type of pest based on the distribution map; The pest extermination support method according to claim 1 or 2, wherein measures to exterminate the pest are taken in accordance with the movement route.

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