A method for identifying invertebrate species using cuticle proteins

Abstract

This invention provides a method for quickly and easily identifying invertebrate species. [Solution] A method for identifying the species of an invertebrate, comprising: a) a pretreatment step of contacting a sample containing the cuticle protein of the invertebrate with at least one pretreatment agent selected from the group consisting of acidifying agents, oxidizing agents, and mixtures thereof; b) a step of analyzing the peptide components contained in the sample after pretreatment; and c) a step of analyzing the species of the invertebrate based on the results of step b).

Description

[Technical Field]

[0001] The present invention relates to a method for identifying species of invertebrates by analyzing the cuticular proteins of the invertebrates. [Background Art]

[0002] Among butterflies and moths, some species carry plant viruses and the like and act as agricultural pests. Accurately grasping the trends of such agricultural pests and exterminating them is important for improving the productivity and quality of agricultural crops. Identification of these species is often performed based on morphological differences, particularly differences in wing patterns and the like. However, especially among moths, wing patterns are often very similar between different species, and it has often been difficult to quickly identify species by visual observation alone.

[0003] In protein analysis, it is common practice to first solubilize a sample, separate it by electrophoresis, chromatography or the like, and then examine its properties. However, since the scales of butterflies and moths repel water, solubilization has been difficult. Non-Patent Document 1 reports an example in which scales are shaken overnight in 8M urea, the precipitate is boiled for 15 minutes, then solubilized by sonication, further digested with trypsin for 20 hours, and then subjected to mass spectrometry. [Prior Art Literature] [Non-Patent Literature]

[0004] [Non-Patent Literature 1] Lie J. et al.,(2021) Lepidopteran wing scales contain abundant cross linked film-forming histidine-rich cuticular proteins. Commun. Biol. Vol. 4, pp. 491. [Summary of the Invention] [Problems to be Solved by the Invention]

[0005] The present inventor's objective is to provide a method for quickly and easily identifying invertebrate species. [Means for solving the problem]

[0006] To solve the above problems, the present inventors have conducted extensive research and have found that the species of invertebrate can be identified by treating the cuticle proteins of invertebrates with an acidifying agent, an oxidizing agent, or a mixture thereof, and then analyzing the results. This disclosure is based on this novel finding and specifically provides the following inventions. (1) A method for identifying the species of an invertebrate, a) A pretreatment step of contacting a sample containing the cuticle protein of the invertebrate with at least one pretreatment agent selected from the group consisting of acidifying agents, oxidizing agents, and mixtures thereof; b) A step of analyzing the peptide components contained in the sample after pretreatment; and c) A step of analyzing the species of the invertebrate based on the results of step b); Methods that include... (2) The method of (1), wherein step a) includes adding an organic solvent in addition to the pretreatment agent. (3) The method of (1) or (2), wherein step a) includes heating the pretreatment agent after it has been in contact with the sample. (4) Any method (1) to (3) wherein the pretreatment agent is selected from the group consisting of trifluoroacetic acid, formic acid, acetic acid, nitric acid, hydrochloric acid, difluoroacetic acid, monofluoroacetic acid, propionic acid, butyric acid, hypochlorite, hydrogen peroxide, hydrogen fluoride, bromoacetic acid, ozone, halogen, chlorine dioxide, manganese dioxide, or mixtures thereof. (5) In step b), the analysis is a mass spectrometry using an ionization means selected from the group consisting of APCI, CI, EI, ESI, FAB, FD, FI, LILBID, LSIMS, MALDI, PB, PD, SIMS, TSP, or a combination thereof, by any of (1) to (4). (6) The method of (5), wherein the ionization means is the ESI method or the MALDI method. (7) In step b), the analysis is performed by the method of (6), including MALDI-TOF-MS. (8) In step b), the analysis is performed by any of the methods (1) to (7), including separation by LC, MPLC, HPLC, or capillary electrophoresis. (9) Step c) is any of the methods (1) to (8) which utilize the analytical results obtained for a control group consisting of cuticle proteins of several known invertebrates. (10) The method of (9), wherein step c) utilizes a database in which analytical results obtained for a control group consisting of cuticle proteins of several known invertebrates are registered. (11) A system for identifying invertebrate species, comprising the following: - An analytical unit for analyzing peptide components contained in pre-treated samples obtained by treating samples containing cuticular proteins of invertebrates with at least one pre-treatment agent selected from the group consisting of acidifying agents, oxidizing agents, and mixtures thereof; and - An analysis unit that analyzes the species of invertebrates based on the analysis results output from the aforementioned analysis unit. (12) A pretreatment system of (11) further comprising: (12) a sample containing cuticular proteins of an invertebrate being treated with at least one pretreatment agent selected from the group consisting of acidifying agents, oxidizing agents, and mixtures thereof. (13) The system of (12), wherein the pre-processing unit is equipped with a heating means. (14) The analysis unit is any of the systems (11) to (13) that include a mass spectrometer. (15) The system described in (14), wherein the mass spectrometer is a MALDI-TOF-MS mass spectrometer. (16) The analysis unit is equipped with a database, the database being a database in which the obtained analysis results for a control group consisting of cuticle proteins of multiple invertebrates are registered in advance, one of the systems (11) to (15). [Effects of the Invention]

[0007] According to the method of the present invention, it is possible to easily identify invertebrate species in a short amount of time. [Brief explanation of the drawing]

[0008] [Figure 1] This is a flowchart illustrating the outline of the method of the present disclosure. The method of the present disclosure includes at least a sample preparation step, a sample pretreatment step (step a), an analysis step (step b), and an analysis step of the obtained results (step c). [Figure 2] This flowchart illustrates an example of how AI is used in the method of this disclosure. In the illustrated example, an AI is used that is constructed using a database of analysis results of known samples as training data. The analysis results of the target unknown sample, as well as the pre-processing and analysis conditions for the analysis, are input to the AI, and information such as the species, variety, and year of manufacture of the sample, which is determined by the AI, is output. [Figure 3] Figure 3A is a schematic diagram of an example of the system of the present invention. Figure 3A shows a schematic of the first embodiment of the system of the present invention. In the first embodiment of the system of the present invention, the test system 1 comprises an analysis unit 3 and an analysis unit 4. It also comprises an input unit 5 for inputting sample information and analysis conditions, an output unit 6 for outputting analysis results, and a control unit 7 for controlling each device within the test system 1. After a pre-treated sample S1 is set in a predetermined position within the test system 1 from an external source, the test system 1 performs analysis of the protein components in the sample and analyzes the analysis results. Figure 3B shows a second embodiment of the system of the present invention. In the second embodiment of the system of the present invention, the test system 1 comprises a pre-processing unit 2, an analysis unit 3, and an analysis unit 4. It also comprises an input unit 5 for inputting sample information and analysis conditions, an output unit 6 for outputting analysis results, and a control unit 7 for controlling each device within the test system 1. After an untreated sample S2 is set in a predetermined position within the test system 1 from an external source, the sample S2 is taken into the pre-processing unit 2 and pre-processing is performed. The sample preparation unit is equipped with a robotic arm, a reagent dispensing mechanism, etc., and can automatically perform sample preparation, transferring the prepared sample to the analysis unit 3. [Figure 4] These are mass spectra of silkworm and Japanese basket butterfly (Zizina otis) wings pre-treated at room temperature. Figure 4A shows the mass spectrum of silkworm, and Figure 4B shows the mass spectrum of Japanese basket butterfly wings. [Figure 5] These are mass spectra of samples obtained by pretreating silkworm and *Pseudozizeeria maha* wings under heating conditions. FIG. 5A is the mass spectrum of silkworm wings, and FIG. 5B is the mass spectrum of *Pseudozizeeria maha* wings. [Figure 6] These are mass spectra of samples obtained by pretreating wings of *Argynnis pandora*, *Ostrinia furnacalis*, *Pieris brassicae*, and *Ostrinia scapulalis* at room temperature. FIG. 6A is the mass spectrum of *Argynnis pandora*, FIG. 6B is the mass spectrum of *Ostrinia furnacalis*, FIG. 6C is the mass spectrum of *Pieris brassicae*, and FIG. 6D is the mass spectrum of *Ostrinia scapulalis*. [Figure 7] These are mass spectra of samples obtained by pretreating wings of *Argynnis pandora*, *Ostrinia furnacalis*, *Pieris brassicae*, and *Ostrinia scapulalis* under heating conditions. FIG. 7A is the mass spectrum of *Argynnis pandora*, FIG. 7B is the mass spectrum of *Ostrinia furnacalis*, FIG. 7C is the mass spectrum of *Pieris brassicae*, and FIG. 7D is the mass spectrum of *Ostrinia scapulalis*. [Figure 8] These are mass spectra of samples obtained by pretreating exuviae of *Graptopsaltria nigrofuscata*, *Meimuna opalifera*, and *Platypleura kaempferi* under heating conditions. FIG. 8A is the mass spectrum of *Graptopsaltria nigrofuscata*, FIG. 8B is the mass spectrum of *Meimuna opalifera*, and FIG. 8C is the mass spectrum of *Platypleura kaempferi*. [Figure 9] These are mass spectra of samples obtained by pretreating shells of *Pleoticus muelleri* and black tiger shrimp under heating conditions. FIG. 9A is the mass spectrum of *Pleoticus muelleri*, and FIG. 9B is the mass spectrum of black tiger shrimp. MODE FOR CARRYING OUT THE INVENTION

[0009] <Method for identifying invertebrate species> 1. Overview The method of the present disclosure is a method for identifying an invertebrate species, comprising: a) a pretreatment step of contacting a sample containing cuticular protein of said invertebrate with at least one pretreatment agent selected from the group consisting of an acidifying agent, an oxidizing agent, and a mixture thereof; b) a step of analyzing peptide components contained in the sample after pretreatment; and c) a step of analyzing the species of said invertebrate based on the result of step b).

[0010] The inventors have found that the cuticles of invertebrates, such as the wings and scales of butterflies and moths, contain fewer types of proteins compared to other body sites, and there is little variation depending on the collection position on the cuticle of the same site (e.g., a single wing), thus sample collection and protein analysis are relatively easy. On the other hand, since samples containing invertebrate cuticle proteins have water-repellent properties, it is known that these proteins are difficult to solubilize compared to those in ordinary samples. The inventors have found that bringing such cuticle proteins into contact with an acidifying agent and / or an oxidizing agent causes at least a portion of the proteins contained in the sample to form small molecules, that is, protein fragmentation occurs. Furthermore, the inventors have found that the fragmentation pattern of the proteins differs depending on the organism species from which the cuticle protein is derived. This demonstrates that the origin species can be identified by acidifying and / or oxidizing a cuticle protein under predetermined conditions and analyzing the obtained peptides. In the present disclosure, the term "peptide" refers to a degradation fragment of a biologically derived protein regardless of its molecular weight.

[0011] In the present disclosure, the term "analysis" refers to acquiring output data directly obtained from a sample, so-called raw data, and the term "evaluation" refers to acquiring output information determined based on the aforementioned output data.

[0012] A flow diagram of each step in the method of the present disclosure is shown in Figure 1. The method of the present disclosure includes at least sample preparation, a sample pretreatment step (step a)), an analysis step (step b)), and an analysis step of the obtained results (step c)). Each of these steps is described in detail below. In addition to these steps, the method of the present disclosure may further include other steps.

[0013] 2. Description of Each Step 2-1 Sample Preparation The "sample" to be tested in the method disclosed herein is a sample containing cuticle proteins derived from invertebrates. In this disclosure, "invertebrate" refers to an animal other than a vertebrate that possesses cuticle proteins. Examples of such animals include arthropods, particularly insects, crustaceans, and spiders. In this disclosure, "cuticle" is also called the keratinous layer and refers to a robust membrane produced when cells constituting the epidermis of an organism secrete it to the outside. In arthropods, the cuticle constitutes the exoskeleton, and in mollusks, it constitutes the shell or the surface of an egg. In arthropods, it is known to consist mainly of chitin, protein, and calcium salts (such as calcium carbonate). In crustaceans, it is also known to contain wax. In arthropods, the cuticle is known to exist not only on the body surface but also on shells, exuviae, feathers, scales, etc. In this disclosure, "cuticle protein" refers to a protein as a component of the cuticle. The above-mentioned sample is not particularly limited as long as it contains cuticular proteins from an invertebrate, but for example, it can be a piece of cuticular tissue from an arthropod. More specifically, it can include arthropod exoskeletons, molted exoskeletons, wings, scales, etc. If the arthropod is an insect, not only adult cuticular tissue but also larval cuticular tissue can be used as a sample.

[0014] When using arthropod-derived samples, the species of origin is not particularly limited, and any of insects, crustaceans, spiders, mites, myriapods, etc., may be selected. In the case of insect-derived samples, for example, the methods disclosed herein may be used to distinguish between pests and other species. Examples of pests for which differentiation from other species is useful include the beet armyworm, tobacco budworm, fall armyworm, cutworm, white-spotted armyworm, mallew, leaf roller, diamondback moth, cabbage looper, tobacco whitefly, greenhouse whitefly, onion moth, corn borer, diamondback moth, white-banded moth, white-backed underwing moth, melon looper, cabbage looper, tomato leaf roller, potato leaf roller, potato moth, sweet potato hawk moth, and striped hawk moth. Alternatively, in the case of crustacean-derived samples, the method disclosed herein may be used, for example, to distinguish between crustaceans with high commercial value as food and others. Examples of crustaceans for which differentiation from other species is useful include Pacific white shrimp, red shrimp, Japanese tiger prawn, kuruma prawn, spot prawn, northern red shrimp, Shiba shrimp, sakura shrimp, spiny lobster, European lobster, Atlantic snow crab, giant snow crab, red snow crab, mud crab, Dungeness crab, striped shore crab, red shore crab, Japanese swimming crab, Taiwanese swimming crab, swimming crab, Japanese spider crab, Japanese mitten crab, and sawtooth swimming crab.

[0015] In addition to the foregoing, the methods of this disclosure can be used, for example, as a means of identifying silkworm varieties that produce silk from silkworm exuviae, or as a means of identifying invertebrates that can be used as food (e.g., for insect consumption) from a part of their body surface.

[0016] In the method disclosed herein, sample preparation may include collecting samples containing cuticular proteins from invertebrates, such as shells, exuviae, feathers, scales, etc., and cutting them into small pieces (cuticular fragments) of a few millimeters to a few centimeters in size.

[0017] 2-2 a) Pretreatment process In the method of this disclosure, "pretreatment" refers to treating the cuticle proteins contained in the sample with a pretreatment agent to make them analyzable. Specifically, the "pretreatment agent" is an acidifying agent and / or an oxidizing agent. The pretreatment agent may be used as a single solution, or two or more solutions may be used simultaneously or sequentially. When heating (described later) is performed, it is preferable to use a solvent that can dissolve fragmented peptides, etc., and has a boiling point of 70°C or higher, particularly 85°C or higher, such as water, as the solvent constituting the pretreatment agent.

[0018] In this disclosure, "acidifying agent" refers to a chemical used to place a sample under acidic conditions, specifically at a pH of less than 7.0, more particularly at pH 5.0 or lower, and even more specifically at pH 1.0 to 3.0. The type of acidifying agent is not particularly limited as long as it can acidify the sample. Preferably, trifluoroacetic acid, difluoroacetic acid, monofluoroacetic acid, formic acid, acetic acid, nitric acid, hydrochloric acid, propionic acid, butyric acid, hydrogen fluoride, bromoacetic acid, or combinations thereof can be used. Furthermore, multiple of these acids can be used sequentially. When used as a pretreatment agent, it is preferable to use an acidifying agent with a concentration of 0.01 to 10.0 N (normal), particularly 0.1 to 2.0 N, and even more preferably 0.2 to 1.0 N.

[0019] In this disclosure, the term "oxidizing agent" is not particularly limited to any agent capable of oxidizing a sample, especially proteins. Preferably, hydrogen peroxide, ozone, hypochlorite, halogens, chlorine dioxide, manganese dioxide, or combinations thereof can be used. When using hydrogen peroxide solution as the oxidizing agent, it is preferable to use it at a concentration of 0.01 to 10.0 M, particularly 0.1 to 2.0 M, and even more preferably 0.2 to 1.0 M.

[0020] The means of bringing the pretreatment agent into contact with the sample are not particularly limited as long as the pretreatment agent is in even contact with the sample, but for example, immersion, coating, spraying, solid-phase-gas contact with vapor can be used. The time for contact between the pretreatment agent and the sample (reaction time) is preferably 0.5 to 60 minutes, particularly 1 to 10 minutes, as long as it is sufficient to fragment the proteins contained in the sample. If the reaction time is longer than 60 minutes, it is conceivable that further fragmentation of molecules will occur, making it impossible to obtain the characteristic signals of each sample. Therefore, it is not necessary to set the reaction time to a long period of time, for example, 2 hours, 3 hours, or 4 hours or more.

[0021] The pretreatment step may include adding an organic solvent to the sample in addition to the pretreatment agent. The organic solvent may be added before, at the same time as, or after the pretreatment agent. Alternatively, the pretreatment agent and the organic solvent may be mixed beforehand before being added to the sample. The organic solvent is not particularly limited, but one with a solubility parameter (SP value) of 10 to 15 is preferably used. Examples of such organic solvents include acetonitrile; alcohols such as methanol, ethanol, n-propanol, i-propanol, n-butanol, 2-butanol, isobutanol, and tert-butanol; ketones such as acetone, cyclohexanone, and cyclopentanone; amides such as N,N-dimethylformamide (DMF); and sulfides such as dimethyl sulfoxide (DMSO). Acetonitrile is particularly preferably used. The amount of organic solvent added is not particularly limited, but can be 10 to 200%, particularly 50 to 100%, and even more preferably 80 to 120% of the weight of the pretreatment agent. Samples containing cuticle proteins typically repel water, but their solubility can be improved by using organic solvents.

[0022] The pretreatment step may be performed at room temperature (5-35°C, particularly 15-25°C), but it may also be performed under heated conditions. When the sample is a cuticular fragment, heating may be performed with the pretreatment agent and the cuticular fragment or its residue present together, or it may be performed on the pretreatment agent after the cuticular fragment or its residue has been removed by centrifugation or the like, i.e., on the protein / peptide extract. The heating temperature is not particularly limited, but when analyzing the sample as a liquid phase, it is preferable to raise it to 60-100°C, particularly 70-98°C, and even further to 85-95°C. Methods for performing such heating include, for example, sealing the pretreatment agent in which the sample is immersed in a container and heating it with a Peltier element, a water bath, an oil bath, etc., or irradiating the sample with microwaves. Alternatively, when analyzing the sample as a gas phase, the sample temperature can be, for example, 100-120°C.

[0023] When heating with a Peltier element or water bath, the heating time can range from 3 minutes to 24 hours, particularly 10 minutes to 10 hours, and even further 30 minutes to 3 hours. When using microwave irradiation, the heating time can range from 1 minute to 15 minutes, particularly 3 minutes to 7 minutes.

[0024] It was confirmed that cuticular proteins can be fragmented even when in contact with a pretreatment agent at room temperature, and that analysis by mass spectrometry is possible. Furthermore, it was found that applying heat during the pretreatment of samples containing cuticular proteins further fragments the proteins, resulting in a wider variety of mass spectral patterns.

[0025] The sample after pretreatment may be used with the cuticle residue still present, or the supernatant obtained by centrifugation, filtration, etc., of the pretreatment agent after the reaction may be used. Furthermore, if mass spectrometry is performed in the subsequent analytical step, the sample used should preferably be around 0.01 to 100 mg, particularly 0.05 to 5.0 mg, as this system is suitable for trace analysis.

[0026] The pretreatment conditions, such as the components of the pretreatment agent and the reaction time, can be appropriately selected depending on the type of sample and the purpose of identification. It has been found that the signal obtained in the analytical step described later differs depending on the pretreatment conditions. By referring to known results, it is possible to select conditions that make the desired test easier. Furthermore, it is possible to pretreat the same sample under multiple conditions in parallel and analyze each one.

[0027] The sample may be treated with a reducing agent before or after treatment with an acidifying agent and / or an oxidizing agent, if necessary. Any reducing agent commonly used in protein analysis can be used, such as dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl(phosphine) hydrochloride (TCEP), tributylphosphine, cysteine ​​hydrochloride, or hydroxysulfite.

[0028] In the pretreatment step of the method disclosed herein, it is preferable not to use an enzymatic reaction. Conventionally, proteases such as trypsin have been used in protein fragmentation. However, although the use of enzymes is effective in fragmenting proteins, the reagents used are expensive compared to oxidizing agents / acidifying agents, and the reaction takes a considerable amount of time (for example, about 20 hours). The pretreatment agent used in the method disclosed herein can fragment cuticular proteins in a sample in a short time and at low cost.

[0029] 2-3 b) Analysis process The sample processed in the pretreatment step is subjected to the analysis step. In the analysis step, the proteins fragmented by the pretreatment step are analyzed. Any known method can be used for protein analysis, such as electrophoresis, amino acid sequencing, liquid chromatography (LC), mass spectrometry, amino acid composition analysis, X-ray diffraction, nuclear magnetic resonance spectroscopy (NMR), and Fourier transform infrared absorption spectroscopy (FTIR), but mass spectrometry, which can easily analyze the properties of polymeric substances, is particularly suitable. Mass spectrometry is a method that ionizes a trace amount of compound in a high vacuum chamber, separates the generated ions by mass, and detects them.

[0030] Methods for ionizing compounds include, for example, atmospheric pressure chemical ionization (APCI), chemical ionization (CI), electron ionization (EI), electrospray ionization (ESI), fast atom bombardment (FAB), field desorption (FD), field ionization (FI), liquid secondary ion mass spectrometry (LILBID), liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption ionization (MALDI), particle beam spectrometry (PB), plasma desorption (PD), and secondary ion mass spectrometry. Examples include spectrometry (SIMS), thermospray (TSP), or a combination of these ionization methods. In particular, the MALDI method or ESI method, which are widely used for the analysis of polymers, especially proteins, can be suitably used. Since it has been found that the mass spectrometry spectrum (hereinafter referred to as "mass spectrum") of pretreated cuticle proteins produces different patterns depending on the ionization method, conditions that are more likely to achieve the test objective can be selected by referring to the results of analysis of known samples, etc.

[0031] Any known method can be used as a means of separating ionized molecules by mass, but time-of-flight mass spectrometry (TOF-MS), which is particularly suitable for separating macromolecules, can be preferably used. MALDI-TOF-MS is preferably included in the analysis of protein components in this disclosure.

[0032] When using MALDI-TOF-MS as the analytical method, the sample after pretreatment may be analyzed either with the pretreatment agent or by placing the supernatant in a cuvette or similar container. Alternatively, the fibers remaining in the pretreatment agent can be attached to conductive carbon double-sided tape and analyzed after drying. Conductive carbon double-sided tape can usually be those available for scanning electron microscopes (SEM) (for example, manufactured by Nissin EM Co., Ltd.).

[0033] Before or in lieu of mass spectrometry, separation may be performed by liquid chromatography (LC), middle pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC), capillary electrophoresis, etc.

[0034] 2-4 c) Analysis process b) This step involves analyzing the species of invertebrates based on the results (raw data) obtained in the analysis step. Preferably, the analysis step is carried out using the obtained analysis results on a control group consisting of cuticle proteins from several known invertebrates.

[0035] It has been found that the data obtained in the analysis process, particularly the mass spectral patterns, for samples containing pre-treated cuticle proteins vary depending on parameters derived from the sample's origin species and the pre-treatment and analysis conditions. Therefore, for example, by obtaining numerous mass spectra under diverse pre-treatment and analysis conditions for a control group consisting of many cuticle proteins of known origin, and accumulating the obtained spectral patterns as fingerprints for each control, it is possible to construct a database useful for analyzing unknown samples. Alternatively, the database may be constructed by obtaining mass spectra for a large number of controls under predetermined pre-treatment and analysis conditions. However, when using this database, the pre-treatment and analysis processes for unknown samples may be limited to the predetermined pre-treatment and analysis conditions. The mass spectral data accumulated in the database may be image data of the mass spectra, or a collection of numerical data of the m / z and signal intensity of each detected peak.

[0036] In the method disclosed herein, the analysis step may be performed by comparing the analytical results obtained from the target unknown sample, particularly the mass spectrum, with a constructed database. If necessary, the pretreatment and analytical conditions used to obtain the analytical results may also be compared. Furthermore, the analytical results of the sample to be compared do not need to be one from a single unknown sample; for example, multiple analytical results, such as multiple mass spectra, may be obtained by changing the pretreatment and analytical conditions, and these may be combined and compared with the database.

[0037] Matching may be performed manually, but preferably using a computer. Matching is performed, for example, by inputting data such as the analysis results of the target sample, extracting the data closest to the input data from the database, and outputting the extracted data and its background information (particularly the species of origin of the sample). Matching may also be performed using artificial intelligence (AI). When using AI, the aforementioned database can be used as training data, and a trained model can be constructed through machine learning using a known neural network. Using this model, information on the species of origin of an unknown sample can be output from data input from that sample. Figure 2 shows a flowchart of an example of an embodiment in which AI is used in the method of this disclosure. In the example shown in Figure 2, an AI constructed using a database of accumulated analysis results of known samples as training data is used. The analysis results of the target unknown sample, as well as the pre-processing and analysis conditions for the analysis, are input to the AI, and information on the species of origin of the sample determined by the AI ​​is output. Note that the embodiment of AI is not limited to the example shown in Figure 2.

[0038] 2-5 Other processes Depending on the type of sample being used, additional steps may be added to the above process. For example, if the sample is an insect wing, exuvia, etc., additional steps may be added to remove surface dirt, etc., or to finely chop the sample.

[0039] The analytical and analytical steps in the method disclosed herein are not limited to the analytical results of a sample treated with the aforementioned pretreatment agent, but may also include the analytical results of the same sample treated separately with a reducing agent and a basicizing agent.

[0040] The system described herein is a system for identifying invertebrate species, comprising the following: - An analytical unit for analyzing peptide components contained in pre-treated samples obtained by treating samples containing cuticular proteins of invertebrates with at least one pre-treatment agent selected from the group consisting of acidifying agents, oxidizing agents, and mixtures thereof; and - An analysis unit that analyzes the species of invertebrates based on the analysis results output from the aforementioned analysis unit.

[0041] The systems described herein, unless otherwise specified, are systems for carrying out the methods described herein and have the same characteristics as those described in the preceding section <Methods for Identifying Invertebrate Species>.

[0042] The system disclosed herein includes, in addition to the analysis unit and the analysis unit, - A pretreatment method in which a sample containing cuticular proteins of an invertebrate is treated with at least one pretreatment agent selected from the group consisting of acidifying agents, oxidizing agents, and mixtures thereof; It may also include the following.

[0043] Figure 3 illustrates an overview of the system of this disclosure. Figure 3A shows a first embodiment of the system of this disclosure. In the first embodiment of the system of this disclosure, the test system 1 comprises an analysis unit 3 and an analysis unit 4. It also comprises an input unit 5 for inputting sample information and analysis conditions, an output unit 6 for outputting analysis results, and a control unit 7 for controlling each device within the test system 1. Preferably, the control unit 7 is a computer, and the input unit 5 and output unit 6 are user interfaces. After a pre-treated sample S1, which has been treated with a "pre-treatment agent" from an external source, is set in a predetermined position within the test system 1, the test system 1 performs analysis of the protein components in the sample and analyzes the analysis results.

[0044] Figure 3B shows a second embodiment of the system of the present disclosure. In this second embodiment of the system of the present disclosure, the test system 1 comprises a pre-processing unit 2, an analysis unit 3, and an analysis unit 4. It also comprises an input unit 5 for inputting sample information and analysis conditions, an output unit 6 for outputting analysis results, and a control unit 7 for controlling each device within the test system 1. Preferably, the control unit 7 is a computer, and the input unit 5 and output unit 6 are user interfaces. After an unprocessed sample S2 is set in a predetermined position within the test system 1 from an external source, the sample S2 is taken into the pre-processing unit 2 and pre-processed. The pre-processing unit may be equipped with heating means capable of heating the sample to 50-99°C. Furthermore, the pre-processing unit may be equipped with a robotic arm, a reagent dispensing mechanism, etc., to automatically pre-process the sample and transfer the processed sample to the analysis unit 3.

[0045] The following configuration is common to both the first embodiment (Figure 3A) and the second embodiment (Figure 3B). The analysis unit 3 is equipped with a device for analyzing the protein components of the pre-treated sample S1. Any device capable of analyzing the protein components of the pre-treated sample may be used, and any known means such as an electrophoresis tank, amino acid sequencer, liquid chromatography (LC) apparatus, or mass spectrometer can be used, but a mass spectrometer capable of easily analyzing the properties of polymeric substances is preferably used. In particular, the MALDI-TOF-MS mass spectrometry method, which is widely used for the analysis of polymers, especially proteins, is preferably used. The analysis unit may be equipped with multiple types of analytical devices.

[0046] The analysis unit 4 analyzes the species of invertebrates based on the analysis results obtained by the analysis unit 3. The analysis unit 4 includes a data collection unit 41 that aggregates input information from the input unit 5 and analysis results from the analysis unit 3, a matching unit 42, and a database 43. The database 43 is a database in which the obtained analysis results for a control group consisting of cuticle proteins of multiple invertebrates are pre-registered, and the matching unit 42 includes software that compares the data aggregated in the data collection unit 41 with the data in the database 43. As for the software, existing software such as MALDI biotyper (manufactured by Bruker Daltonics) can be used. The matching unit, for example, compares the data collected from the analysis unit with the database, extracts the closest data in the database, and outputs the extracted data and its background information (such as the species from which the sample originates). The matching unit 42 may also include AI. The AI ​​here can suitably use a trained model that has been machine-trained using the database as training data, and by inputting the collected data into the AI, it outputs information about the species from which the sample originates as a result.

[0047] Alternatively, the analysis unit 4 may, without using the database 43, directly use mass spectral data of specimens analyzed under the same conditions as the target sample at approximately the same time (for example, on the same day) to perform a comparison of the data aggregated in the data acquisition unit 41 in the matching unit 42.

[0048] The system of this disclosure may include an analysis unit and an interpretation unit, and may be applicable to the implementation of the method of this disclosure as described above, and should not be construed as being limited to the first and second embodiments described above. [Examples]

[0049] <Example 1: Analysis of butterfly and moth wings> 1. Sample pretreatment Circular pieces with a diameter of 5 mm were punched out from the wings (including scales) of silkworms (Bombyx mori), Japanese blue butterflies (Zizeeria maha), Indian fritillary butterflies (Argyreus hyperbius), corn earworms (Ostrinia furnacalis), yellow butterflies (Eurema mandarina), and white-banded earworms (Spoladea recurvalis), and placed in centrifuge tubes. 10 μL of 70% formic acid was added and the tubes were vortexed for 1 minute, then 10 μL of acetonitrile was added and the tubes were vortexed for another 5 minutes. After centrifugation, the supernatant was divided into two portions: 5 μL (pre-treated sample 1) and 15 μL (sample 2).

[0050] 292.5 μL of water was added to sample 2 and heated at 98°C for 30 minutes. 75 μL was taken out, dried, and then redissolved in 5 μL of 2.5% formic acid (pre-treated sample 2).

[0051] 2. Analysis using MALDI-TOF-MS One μL each of pre-treated sample 1 and pre-treated sample 2 were dropped onto conductive carbon double-sided tape (manufactured by Nisshin EM Co., Ltd.) and dried. After drying each solution, two μL of matrix solution was dropped on top and dried. As the matrix solution, a saturated solution (cinnamon acid derivative matrix solution) was used, prepared by dissolving α-cyano-4-hydroxycinnamonic acid (α-CHCA) in a 50% acetonitrile aqueous solution containing 2.5% trifluoroacetic acid. The samples were set on the sample stage of a MALDI-TOF-MS mass spectrometer (Autoflex III or Microflex, Bruker Daltonics) and dried. Mass spectra were acquired in linear positive mode at m / z 1000-20000.

[0052] Figure 4 shows the mass spectra of pre-treated samples 1 of silkworms and Japanese basket clover. Figure 4A shows the mass spectrum of the silkworm wing, and Figure 4B shows the mass spectrum of the Japanese basket clover wing. It was confirmed that the two species could be clearly distinguished in samples treated at room temperature. Figure 5 shows the mass spectrum of pre-treated sample 2. Figure 5A shows the mass spectrum of the silkworm, and Figure 5B shows the mass spectrum of the Japanese basket clover. Many peaks were observed in the heat-treated sample, indicating that more detailed distinction is possible.

[0053] Figure 6 shows the mass spectra of pre-treated sample 1 of the Common Leopard Butterfly, the European Corn borer, the Northern Clouded Yellow butterfly, and the White-banded Pyralid moth. Figure 6A is the mass spectrum of the Common Leopard Butterfly, Figure 6B is the mass spectrum of the European Corn borer, Figure 6C is the mass spectrum of the Northern Clouded Yellow butterfly, and Figure 6D is the mass spectrum of the White-banded Pyralid moth. Figure 7 shows the mass spectra of pre-treated sample 2 of the Common Leopard Butterfly, the European Corn borer, the Northern Clouded Yellow butterfly, and the White-banded Pyralid moth. Figure 7A is the mass spectrum of the Common Leopard Butterfly, Figure 7B is the mass spectrum of the European Corn borer, Figure 7C is the mass spectrum of the Northern Clouded Yellow butterfly, and Figure 7D is the mass spectrum of the White-banded Pyralid moth. In all cases, it was shown that species identification was possible with both room temperature treatment and heat treatment. In particular, it was shown that identification became clearer with the addition of heat treatment.

[0054] Similar tests were conducted on multiple locations on the feathers of the same individual, and on multiple other individuals of the same species. It was confirmed that, in the case of feathers of the same species, similar mass spectra were obtained regardless of the location or individual (including sex differences) (data not shown).

[0055] <Example 2: Analysis of cicada exoskeletons> 1. Sample pretreatment 5mm square sections were cut from the split dorsal portion of the exuviae of cicadas Graptopsaltria nigrofuscata, Meimuna opalifera, and Platypleura kaempferi, and placed in centrifuge tubes. 10 μL of 70% formic acid was added and vortexed for 1 minute, then 10 μL of acetonitrile was added and vortexed for another 5 minutes. After centrifugation, 292.5 μL of water was added to 15 μL of the supernatant and heated at 98°C for 30 minutes. 75 μL was separated, dried, and then redissolved in 5 μL of 2.5% formic acid (pre-treated sample).

[0056] 2. Analysis using MALDI-TOF-MS One μL of each pre-treated sample was dropped onto conductive carbon double-sided tape (manufactured by Nisshin EM Co., Ltd.) and dried. After drying each solution, two μL of matrix solution was dropped on top and dried. As the matrix solution, a saturated solution (cinnamon acid derivative matrix solution) was used, prepared by dissolving α-cyano-4-hydroxycinnamonic acid (α-CHCA) in a 50% acetonitrile aqueous solution containing 2.5% trifluoroacetic acid. The samples were set on the sample stage of a MALDI-TOF-MS mass spectrometer (Autoflex III or Microflex, Bruker Daltonics) and dried. Mass spectra were acquired in linear positive mode at m / z 1000-20000.

[0057] Figure 8 shows the mass spectra of the cicada species *Graptopsaltria nigrofuscata*, *Meimuna opalifera*, and *Platypleura kaempferi*. Figure 8A shows the mass spectrum of *Graptopsaltria nigrofuscata*, Figure 8B shows the mass spectrum of *Meimuna opalifera*, and Figure 8C shows the mass spectrum of *Platypleura kaempferi*. It was confirmed that the three cicada species can be clearly distinguished.

[0058] <Example 3: Analysis of shrimp shells> 1. Sample pretreatment Argentine red shrimp (Pleoticus muelleri), black tiger shrimp (Penaeus monodon) A 5mm diameter circular piece was punched out from the shell and placed in a centrifuge tube. Water was added, and the tube was vortexed for 1 minute, then the water was removed. This process was repeated 5 times. 10 μL of 70% formic acid was added and the tube was vortexed for 1 minute, then 10 μL of acetonitrile was added and the tube was vortexed for another 5 minutes. After centrifugation, 292.5 μL of water was added to 15 μL of the supernatant and heated at 98°C for 30 minutes. 75 μL was taken out, dried, and then redissolved in 5 μL of 2.5% formic acid (pre-treated sample).

[0059] 2. Analysis using MALDI-TOF-MS One μL of each pre-treated sample was dropped onto conductive carbon double-sided tape (manufactured by Nisshin EM Co., Ltd.) and dried. After drying each solution, two μL of matrix solution was dropped on top and dried. As the matrix solution, a saturated solution (cinnamon acid derivative matrix solution) was used, prepared by dissolving α-cyano-4-hydroxycinnamonic acid (α-CHCA) in a 50% acetonitrile aqueous solution containing 2.5% trifluoroacetic acid. The samples were set on the sample stage of a MALDI-TOF-MS mass spectrometer (Autoflex III or Microflex, Bruker Daltonics) and dried. Mass spectra were acquired in linear positive mode at m / z 1000-20000.

[0060] Figure 9 shows the mass spectra of Argentine red shrimp and black tiger shrimp. Figure 9A shows the mass spectrum of Argentine red shrimp, and Figure 9B shows the mass spectrum of black tiger shrimp. It was confirmed that the two shrimp species can be clearly distinguished. [Industrial applicability]

[0061] The method disclosed herein can be used for ecological surveys of agricultural pests and quality assurance of fishery products, and is particularly useful in agriculture, fisheries reporting, and the food industry. [Explanation of Symbols]

[0062] 1…Testing System 2…Pre-treatment section 3…Analysis Department 4…Analysis department 41…Data Collection Department 42...Verification section 43…Database 5...Input section 6…Output section 7…Control Unit

Claims

1. A method for identifying invertebrate species, a) A step of contacting a sample containing the cuticle protein of the invertebrate with at least one pretreatment agent selected from the group consisting of acidifying agents, oxidizing agents, and mixtures thereof; b) A step of analyzing the peptide components contained in the sample after pretreatment; and c) A step of analyzing the species of the invertebrate based on the results of step b); Methods that include...

2. The method according to claim 1, wherein step a) includes adding an organic solvent in addition to the pretreatment agent.

3. The method according to claim 1, wherein step a) includes heating the pretreatment agent after contact with the sample.

4. The method according to claim 1, wherein the pretreatment agent is selected from the group consisting of trifluoroacetic acid, formic acid, acetic acid, nitric acid, hydrochloric acid, difluoroacetic acid, monofluoroacetic acid, propionic acid, butyric acid, hypochlorite, hydrogen peroxide, hydrogen fluoride, bromoacetic acid, ozone, halogen, chlorine dioxide, manganese dioxide, or mixtures thereof.

5. The method according to claim 1, wherein in step b), the analysis is mass spectrometry using an ionization means selected from the group consisting of the APCI method, CI method, EI method, ESI method, FAB method, FD method, FI method, LILBID method, LSIMS method, MALDI method, PB method, PD method, SIMS method, TSP method, or a combination thereof.

6. The method according to claim 5, wherein the ionization means is the ESI method or the MALDI method.

7. The method according to claim 6, wherein in step b), the analysis includes MALDI-TOF-MS.

8. The method according to claim 1, wherein in step b), the analysis includes separation by LC, MPLC, HPLC, and capillary electrophoresis.

9. The method according to claim 1, wherein step c) utilizes analytical results obtained for a control group consisting of cuticle proteins of several known invertebrates.

10. The method according to claim 9, wherein step c) utilizes a database in which analytical results obtained for a control group consisting of cuticle proteins of several known invertebrates are registered.

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