Nematode-sealed capsule, method for producing nematode-sealed capsule, and use of nematode-sealed capsule

Abstract

A nematode-sealed capsule according to the present disclosure includes: a sealing composition containing nematodes; and at least one film containing the sealing composition. The film contains, as a main component, a water-soluble polymer which is gelled through a reaction with a cation.

Description

[Technical Field]

[0001] The present invention relates to a capsule containing a nematode (hereinafter sometimes referred to as a "nematode-encapsulated capsule"), a method for producing a nematode-encapsulated capsule, and uses of the nematode-encapsulated capsule. Specifically, the present invention relates to a nematode-encapsulated capsule, a method for producing a nematode-encapsulated capsule, a method for evaluating the response of a nematode, a cancer testing method, a kit for evaluating the response of a nematode, and a production kit for producing a nematode-encapsulated capsule. [Background technology]

[0002] As described in Patent Documents 1 and 2, cancer testing using nematodes in urine samples has been attracting attention in recent years. This interest has led to an increasing need for nematode culture in medical institutions or individuals. For example, as described in Non-Patent Document 1, nematodes are supplied between skilled researchers, such as nematode researchers, by transporting them at room temperature in a state where the nematodes are cultured on an agar medium coated with Escherichia coli, which serves as food for the nematodes. Researchers then collect the nematodes from the agar medium and culture them under conditions suitable for the intended test.

[0003] Furthermore, Non-Patent Document 2 describes an experimental method using nematodes, which uses a special pack that encloses the nematodes and food. This experimental method was developed for space experiments. Non-patent document 3 states that entomopathogenic nematodes (EPNs) have great potential for use as biological control agents for insects that feed on plant roots, but have the disadvantages of being relatively short in storage and vulnerable to drying and ultraviolet light. 2+ -Embedding in alginate hydrogel has been described (Abstract, Materials and Methods, Fig. 1). Non-patent document 4, cited as a reference in Non-patent document 3, reports that a new method has been developed for long-term storage of infective larvae (IJ) of the entomopathogenic nematode (EPN) Steinernema feltiae IS-6 strain at 23±0.3°C, and that a suspension of nematodes in distilled water containing 18% glycerol and 2% sodium alginate is maintained in the presence of 0.5% CaCO 3 ·2H 2 It is described that calcium alginate granules were formed when the solution was dropped into O (Abstract). Non-patent document 5, which is cited as a reference for non-patent document 3, describes that the osmotic response of Steinernema carpocapsae and Steinernema fersai was studied from the perspective of dehydration and rehydration in the presence of calcium alginate and formaldehyde, specifically, that alginate-formulated IJ showed significantly different survival rates depending on the state of dehydrated or non-dehydrated IJ and the presence of an antibacterial agent, and that the formulation was carried out using a modified method of non-patent document 4 (Summary, last paragraph of right column 226 - line 15 of left column on page 227). Non-Patent Document 6, which is cited as a reference for Non-Patent Document 3, describes that in a series of experiments, the dehydration and rehydration responses of IJs of entomopathogenic nematodes of the order Rhabditida were investigated at various glycerol concentrations, temperatures, and incubation periods, that dehydrated nematodes were formulated in alginate granules using an optimized process, and that the formulation was carried out using the method of Non-Patent Document 4 with some modifications (Abstract, and the last paragraph on page 3 to line 2 on page 4). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2018-61515 [Patent Document 2] International Publication No. 2020 / 218501 [Non-patent literature]

[0005] [Non-Patent Document 1] Stiernagle, T., Maintenance of C. elegans. (February 11, 2006), WormBook, ed. The C. elegans Research Community, WormBook. [Non-patent document 2] Higashibata, A., et al., Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans. npj Microgravity (2016) 2,15022, 1-8. DOI: https: / / doi.org / 10.1038 / npjmgrav.2015.22 [Non-patent document 3] Kim, J., et al., Calcium-alginate beads as a formulation for the application of entomopathogenic nematodes to control rootworms. Jornal of Pest Science (2021) 94, 1197-1208. DOI: https: / / doi.org / 10.1007 / s10340-021-01349-4 [Non-patent document 4] Chen, S. and Glazer, I., A novel method for long-term storage of the entomopathogenic nematode Steinernema feltiae at room temperature. Biological Control (2005) 32(1), 104-110. DOI: https: / / doi.org / 10.1016 / j.biocontrol.2004.08.006 [Non-patent document 5] Kary, NE, et al., Effect of temperature, time and glycerol concentration on the dehydration and rehydration process of Steinernema carpocapsae and S. feltiae in alginate granule formulation. Nematology (2017) 19(2), 225-235. DOI: https: / / doi.org / 10.1163 / 15685411-00003042 [Non-patent document 6] Kary, NE, et al., Effects of abiotic factors on the osmotic response of alginate-formulated entomopathogenic nematode, Heterorhabditis bacteriophora (Nematoda: Rhabditida). Biocontrol Science and Technology (2018) 28(7), 688-701. DOI: https: / / doi.org / 10.1080 / 09583157.2018.1479731 Summary of the Invention [Problem to be solved by the invention]

[0006] The nematode supply method described in Non-Patent Document 1 requires appropriate knowledge, skills, and equipment, and cannot be easily performed by anyone. Furthermore, the experimental method described in Non-Patent Document 2 requires special culture conditions, etc., and is not necessarily suitable for cancer testing or various basic biological experiments using nematodes. Therefore, there is a need for the development of technology that enables even those who are not so-called experts, such as researchers who routinely conduct experiments using nematodes and are familiar with nematode culture or experiments, to easily use nematodes in experiments or tests.

[0007] An object of one aspect of the present invention is to realize a technique that enables even non-experts to easily use nematodes. [Means for solving the problem]

[0008] After extensive research, the inventors discovered that by using capsules in which nematodes are encapsulated in a biocompatible artificial membrane, even non-experts can easily use nematodes, and thus completed the present invention.

[0009] A nematode encapsulation capsule according to one embodiment of the present invention comprises an encapsulation composition containing a nematode and at least one layer of coating that encompasses the encapsulation composition, wherein the coating contains, as its main component, a water-soluble polymer that gels upon reacting with a cation. [Effects of the Invention]

[0010] According to one aspect of the present invention, even an unskilled person can easily use nematodes. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a microscope image of a spherical capsule enclosing an adult nematode, taken from above, in Example 1. [Figure 2] 1 shows microscope images taken from above of spherical capsules of various sizes enclosing adult nematodes in Example 1. [Figure 3]1 is a microscope image taken from above of a capsule of any shape enclosing an adult nematode in Example 1. [Figure 4] 10 is a microscope image of a spherical capsule enclosing nematode eggs taken from above in Example 2. [Figure 5] 10 shows microscopic images of spherical capsules enclosing nematode eggs taken daily from above in Example 3. [Figure 6] 10 shows microscopic images of spherical capsules enclosing adult nematodes taken daily from above in Example 4. [Figure 7] 10 is a microscope image taken from above of adult and larval nematodes crawling out of a nematode-enclosing capsule in Example 4. [Figure 8] 10 shows microscopic images taken daily from above of spherical capsules enclosing adult irradiated nematodes in Example 5. [Figure 9] 10 is a microscope image taken from above of irradiated adult nematodes crawling out of a nematode-encapsulating capsule in Example 5. [Figure 10] 10 shows microscopic images taken daily from above of spherical capsules enclosing adult nematodes exposed to a hatching inhibitor in Example 6. [Figure 11] 10 is a microscope image taken from above of adult nematodes exposed to a hatching inhibitor crawling out of a nematode-encapsulated capsule in Example 6. [Figure 12] 10 shows microscopic images taken daily from above of spherical capsules containing adult nematodes cultured in the absence of any hatching inhibitor in Example 6. [Figure 13] FIG. 10 shows spherical capsules of two different sizes containing adult nematodes enclosed in plastic dishes in Example 7. [Figure 14] In Example 8, spherical capsules containing adult nematodes were spread on leaf mold spread in a plastic dish, and images were taken from above every day. [Figure 15]FIG. 10 shows the results of supplying capsules containing adult nematodes to nematode test plate A1 and observing the supply area over time in Example 9. [Figure 16] FIG. 10 shows the results of supplying capsules containing adult nematodes to nematode test plate A2 in Example 9, and then observing the supply site over time. [Figure 17] FIG. 1 shows the results of supplying nematodes to a nematode test plate A1 by a conventional method and observing the state of the supply area over time in Comparative Example 1. [Figure 18] FIG. 10 shows the results of supplying nematodes to the nematode test plate A2 in Comparative Example 1 and observing the state of the supply area over time. [Figure 19] FIG. 10 shows the results of a chemotaxis test of nematodes to volatile substances, in which capsules containing adult nematodes were placed on nematode test plate A3 in Example 10. [Figure 20] FIG. 10 shows the results of a chemotaxis test of nematodes to volatile substances in Comparative Example 2, in which nematodes were supplied to nematode test plate A3 by a conventional method. [Figure 21] 1 is a flowchart showing an example of a method for manufacturing a nematode-encapsulating capsule according to the present embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Nematode-enclosed capsule] The nematode encapsulation capsule according to this embodiment comprises an encapsulation composition containing a nematode and at least one membrane layer. As used herein, the term "capsule" refers to at least one membrane layer that contains the encapsulation composition inside.

[0013] Encapsulated Composition The encapsulated composition contains nematodes. As used herein, nematodes include nematodes at various developmental stages. Each developmental stage of nematodes includes nematode eggs (including fertilized eggs), larvae (first to fourth instars), and adults. Additionally, adult nematodes include nematodes of various genders. Each gender of nematodes includes male and hermaphrodite nematodes.

[0014] [Nematodes] In this specification, the term "nematode" refers to both animals belonging to the phylum Nematoda and animals belonging to the phylum Nematoda in biological taxonomy. There are no particular limitations on the animals included in these categories, as long as they are terrestrial or semi-terrestrial and capable of moving on a solid substrate.

[0015] Animals belonging to the phylum Nematoda include non-parasitic nematodes (or free-living nematodes), plant-parasitic nematodes, insect-loving nematodes (including pathogenic nematodes, parasitic nematodes, parasitic nematodes, and zoophilic nematodes), and various nematodes that are parasitic on mammals, etc.

[0016] Examples of non-parasitic nematodes include Caenorhabditis elegans (hereinafter sometimes referred to as "C. elegans"), Aphelenchus avenae, Caenorhabditis angaria, Caenorhabditis brenneri, Caenorhabditis briggsae, Caenorhabditis japonica, Caenorhabditis remanei, and Pristionchus pacificus.

[0017] Examples of plant-parasitic nematodes include Acreberoides nanus, Bastiania gracilis, Wilsonema othophorum, Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica, Meloidogyne hapla, Meloidogyne marytlandi, Meloidogyne mali, Heterodera glycines, Heterodera schachtii, and Heterodera mali. elachista), potato cyst nematode (Globodera rostochiensis), potato white cyst nematode (Globodera pallida), lotus root root nematode (Hirschmanniella diversa), Imamura root nematode (Hirschmanniella immamuri), northern root lesion nematode (Pratylenchus penetrans), southern root lesion nematode (Pratylenchus coffeae), walnut root lesion nematode (Pratylenchus vulnus), wheat root lesion nematode (Pratylenchus neglectus), channel root lesion nematode (Pratylenchus loosi Loof), chapin nematode (Pratylenchus curvitatus), Kumamoto root lesion nematode (Pratylenchus kumamotoensis), false root nematode (Pratylenchus pseudocoffeae), root nematode (Ditylenchus dipsaci), root nematode (Helicotylenchus dihystera), root nematode (Helicotylenchuserythrinae), Kanayasayawa nematode (Hemicriconemoides kanayaensis), Shiba-iridoku nematode (Paratrichodorus mirzai), Minor-iridoku nematode (Paratrichodorus minor (Colbran) Siddiqi or Trichodorus minor Colbran), Rib-leaf nematode (Trichodorus porosus), Sorghum root-leaf nematode (Pratylenchus zeae Graham), Sugarcane root-leaf nematode (Tylenchorhynchus nudus), Rice root-leaf nematode (Aphelenchoides besseyi), Wheat grain nematode (Anguina tritici), Cattle-leaf nematode (Ditylenchus destractor), Peel nematode (Aphelenchoides Targeted pests include the nematode Aphelenchoides fragariae, the long-horn nematode Longidorus spp., the grape sting nematode Xiphinema index, the strawberry nematode Aphelenchoides fragariae, the strawberry root nematode Nothotylenchus acris, the pineapple root-lesion nematode Pratylenchus brachyurus, the banana root-miner nematode Radopholus similis, the pineapple root-lesion nematode Caenorhabditis inopinata, and the coffee sting nematode Xiphinema brevicolle.

[0018] Examples of insect-parasitic nematodes include the bumblebee gall nematode (Sphaerularia bombi), the hornet gall nematode (Sphaerularia vespae), the leafhopper nematode (Agamermis unka), the leafhopper nematode (Amphimermis zuimushi), the Japanese stag beetle nematode (Hexamermis microamphidis), Steinernema carpocapsae, Steinernema kushidai, the oyster mushroom nematode (Iotonchium ungulatum), Iotonchium californicum, Iotonchium cateniforme, and Iotonchium lacariee. laccariae, Iotonchium russulae, Caenorhabditis auriculariae, and Bursaphelenchus tadamiensis, Contortylenchus sp., Contortylenchus genitalicola, Romanomermis culicivorax, etc.

[0019] Examples of zoophilic nematodes on insects include Caenorhabditis japonica, Pristionchus pacificus, Bursaphelenchus xylophilus, Bursaphelenchus mucronatus, Bursaphelenchus doui, Bursaphelenchus firmae, Bursaphelenchus conicaudatus, Bursaphelenchus luxuriosae, Bursaphelenchus hunti, Bursaphelenchus okinawaensis, Bursaphelenchus yongensis, Bursaphelenchus kiyoharai, and Bursaphelenchus Examples of target species include Bursaphelenchus cocophilus, Bursaphelenchus niphades, Bursaphelenchus sexdentati, Teratorhabditis synpapillata, Caenorhabditis briggsae, and Caenorhabditis remanei. Of these, Cenorhabditis japonica, Pristionchus pacificus, Cenorhabditis briggsae, and Cenorhabditis remanei are treated in the laboratory in the same way as free-living (non-parasitic) nematodes.

[0020] Examples of parasitic nematodes that can infect mammals include Strongyloides stercoralis, filariasis, roundworms (ascaris), anisakiasis, whipworms, hookworms, Gnathostoma spp., and Trichinella spiralis.

[0021] Strongyloides nematodes are classified into three types according to the taxonomic group of their main hosts: the parasitic Strongyloides pereira (Strongyloides pereira) of the order Anura (Amphibia). Hereinafter, the genus name of Strongyloides, "Strongyloides," will be abbreviated simply as "S."(S. carinii, S. amphibiophilus, S. bufonis, S. physali, S. spiralis, S. prokopici, S. mascomai, etc.), parasitic cephalopods of the order Tokage (S. cruzi, S. darevskyi, S. ophiusensis, etc.), parasitic cephalopods of the order Hebi (S. ophidiae, S. mirzai, S. gulae, S. serpentis, etc.), parasitic cephalopods of the order Kounotori (S. cubaensis, S. ardeae, S. herodiae, etc.), parasitic cephalopods of the order Kiji (S. avium, S. *S. oswaldoi, S. pavonis, etc.*, *S. minimum, etc.*, *S. turkmenica, etc.*, *S. quiscali Barus, etc.*, *S. thylacis, etc.*, *S. akbari, S. rostombekowi, etc.*, *S. stercoralis, S. fuelleborni, S. fuelleborni kellyi, S. cebus, etc.*, *S. cebus*, etc.* Parasitic nematodes of the order Squamata (S. leiperi, etc.), parasitic nematodes of the order Odontoidea (S. chapini, S. ratti, S. myopotami, S. venezuelensis, S. agoutii, S. robustus, S. sigmodontis, etc.), parasitic nematodes of the order Carnivora (S. nasua, S. felis, S. mustelorum, S. erschowi, S. planiceps, S. puttori, S. martis, S. vulpis, S. tumefasciens, S. lutrae, S. procyonis, etc.), parasitic nematodes of the order Proboscidea (S. elephantis, etc.), parasitic nematodes of the order Perissodactyla (S. westeri, etc.), parasitic fecal nematodes of Artiodactyla (S. papillosus, S. ransomi, etc.), etc.Among these, S. stercoralis, S. fuelleborni, and S. fuelleborni kellyi are known as human strongyloides, while S. procyonis, the raccoon worm, and S. ransomi, the pig worm, can also infect humans.

[0022] Heartworms include Parafilaria multipapillosa, Stephanofilaria okinawaensis, Wuchereria bancrofti, Brugia malayi, Onchocerca cervicalis, Onchocerca gibsoni, Onchocerca gutturosa, Onchocerca volvulus, Acanthocheilonema reconditum, Setaria digitata, Setaria equina, Setaria labiatopapillosa, Setaria marshalli, Dirofilaria immitis, and Loa Examples of targets include loa).

[0023] Targeted roundworms (crocodiles) include human roundworms (Ascaris lumbricoides), pig roundworms (Ascaris suum), bear roundworms (Baylisascaris transfuga), cervical abscess roundworms (Lagochilascaris minor), bovine roundworms (Toxocara vitulorum or Neoascaris vitulorum), equine roundworms (Parascaris equorum), raccoon roundworms (Baylisascaris procyonis), dog roundworms (Toxocara canis), cat roundworms (Toxocara cati), dog minor roundworms (Toxocara leonine), and raccoon roundworms (Toxocara tanuki).

[0024] Targeted anisakids include Anisakis pegreffii, Anisakis simplex sensu stricto, and Anisakis simplex C, commonly known as type I Anisakis, type II Anisakis (also known as sperm whale Anisakis), striped dolphin Anisakis (Anisakis typica), sea lion Psudoterranova decipiens, and seal Contracaecum osculatum.

[0025] Target whipworms include Trichuris discolor, Trichuris muris, Trichuris ovis, Trichuris suis, Trichuris trichiura, and Trichuris vulpis.

[0026] Hookworms include the Brazilian hookworm (Ancylostoma braziliense), dog hookworm (Ancylostoma caninum), Ceylon hookworm (Ancylostoma ceylanicum), Duodenale hookworm (Ancylostoma duodenale), Kusima raccoon dog hookworm (Ancylostoma kusimaense), Malaysian hookworm (Ancylostoma malayanum), Miyazaki raccoon dog hookworm (Arthrostoma miyazakiense), cat hookworm (Ancylostoma tubaeforme), stenocephala hookworm (Uncinaria stenocephala), bovine hookworm (Bunostomum phlebotomum), sheep hookworm (Bunostomum trigonocephalum), and pig hookworm (Globocepharus Examples of targets include Necator urosubulatus, and American hookworm (Necator americanus).

[0027] Target gnathostoma species include Gnathostoma nipponicum, Gnathostoma spinigerum, Gnathostoma hispidum, Gnathostoma doloresi, Gnathostoma procyonis, and Gnathostoma vietnamicum.

[0028] Target trichinella (trichinella) includes Trichinella britovi, Trichinella spiralis, Trichinella nativa, Trichinella nelsoni, and Trichinella pseudospiralis.

[0029] Other target mammalian parasitic nematodes include the Caucasian stomach worm (Abbreviata caucasica), the cat stomach worm (Physaloptera praeputialis), the oriental eye worm (Thelazia callipaeda), the Rhodesian eye worm (Thelazia rhodesi), the Scriabin's eye worm (Thelazia skrjabini), the small-mouthed horse stomach worm (Habronema microstoma), the fly-breed horse stomach worm (Habronema muscae), the large-mouthed horse stomach worm (Draschia megastoma), the red-spotted spiral worm (Crassicauda giliakiana), the beautiful esophagus worm (Gongylonema pulchrum), the round-necked pig stomach worm (Ascarops strongylina), and the six-winged pig stomach worm (Physocephalus sexalatus). Also, strongyles such as the donkey strongyle (Strongylus asini), the toothless strongyle (Strongylus edentates), the horse strongyle (Strongylus equinus), and the common strongyle (Strongylus vulgaris), as well as Eucoleus annulate, Eucoleus contorta, Pearsonema feliscati, Eucoleus perforans, Paracapillaria philippinensis, or Capillaria philippinensis, Calodium hepatica, or Capillaria aerophila, Aonchotheca bovis, Eucoleus aerophile, and Pearsonema Examples of targets include capillary nematodes (also called capillary nematodes) such as Capillary plica.Other examples of target insects include trichostrongylus worms such as Trichostrongylus axei, Trichostrongylus colubriformis, and Trichostrongylus orientalis, haemonchus contortus and Mecistocirrus digitatus, Dictyocaulus filaria, Dictyocaulus viviparus, Nematodirus filicollis, angiostrongylus worms such as Angiostrongylus cantonensis and Angiostrongylus costaricensis, Metastrongylus elongatus, and Filaroides hirthi. Further examples of pinworms that can be targeted include human pinworms (Enterobius vermicularis), rabbit pinworms (Passalurus ambiguous), rat cecal pinworms (Syphacia muris), mouse cecal pinworms (Syphacia obvelata), and horse pinworms (Oxyuris equi).

[0030] Furthermore, examples of animals belonging to the phylum Nematoda include wireworms, such as Gordius robustus, Gordius ogatai, Pseudogordius tanganykae, and Chordodes japonensis.

[0031] The nematode encapsulated in the nematode encapsulation capsule of this embodiment is preferably Caenorhabditis elegans or a closely related species thereof, such as Caenorhabditis brenneri, Caenorhabditis briggsae, Caenorhabditis japonica, Caenorhabditis remanei, and Caenorhabditis inopinata.

[0032] The encapsulation composition may contain components other than nematodes. Examples of other components include water-soluble polymers that gel when reacted with cations; liquids in which nematodes can survive, such as water and buffer solutions; nematode culture solutions; nematode food such as E. coli; E. coli culture solutions; E. coli food such as yeast; labeling substances such as dyes and fluorescent substances for making the encapsulation composition more visible; antibiotics for preventing contamination by bacteria or fungi; and hatching inhibitors (also called hatching inhibitors). Details of water-soluble polymers will be described later.

[0033] By adjusting the amount of food such as E. coli in the encapsulated composition, the nutritional state of the nematodes can be adjusted, and the developmental stage or growth rate of the nematodes can be controlled. Controlling the developmental stage or growth rate of the nematodes makes it easier to transport or store the nematode encapsulated capsules for medium to long periods of time.

[0034] When the nematodes encapsulated in the nematode encapsulation capsules are in the egg stage of development, if the amount of food, such as E. coli, in the encapsulation composition is low, development will cease at the first instar larva (L1 larva) stage. Nematodes in this stage of development are called resistant larvae. Resistant larvae can survive for about a month without nutrition. If environmental conditions, such as nutritional status, improve, the resistant larvae will return to the normal developmental stage and grow into adults. Examples of cases where the amount of food, such as E. coli, in the encapsulation composition is low include situations where, immediately after production of the nematode encapsulation capsules, the amount of food is 10% or less, or 5% or less, of the amount of eggs in the encapsulation composition, taken as 100% by mass.

[0035] When the nematodes to be encapsulated in the nematode encapsulation capsules are at the L1 larval stage, if the amount of food such as E. coli in the encapsulation composition is small, development will stop at the L1 larval stage and the larvae will become resistant. Examples of cases where the amount of food such as E. coli in the encapsulation composition is small include when, immediately after production of the nematode encapsulation capsules, the amount of food is 10% or less, or 5% or less, when the amount of L1 larvae in the encapsulation composition is 100% by mass.

[0036] Examples of hatching inhibitors include fluorodeoxyuridine (5-Fluoro-2'-deoxyuridine: FUdR, also known as fluxuridine), a DNA synthesis inhibitor, and S,S-Bis(1-methylpropyl)=O-ethyl-phosphorodithioate (also known as Cadusafos), an organophosphate insecticide that inhibits acetylcholinesterase activity. Hatching inhibitors can suppress the proliferation of nematodes after use of nematode-encapsulating capsules and prevent the spread of nematodes into the environment.

[0037] The number of nematodes in the encapsulated composition can be selected appropriately depending on the purpose and size of the nematode encapsulated capsule, and may be one (one in the case of an egg) or multiple (multiple in the case of an egg).

[0038] The nematode may be an irradiated nematode. By using irradiated nematodes, the proportion of eggs that hatch can be adjusted. Furthermore, by using irradiated larvae, development can be controlled (e.g., development can be stopped or delayed). Irradiation is described below.

[0039] Alternatively, the nematodes may be treated (typically exposed) to a hatching inhibitor. By using adult nematodes exposed to a hatching inhibitor for a certain period of time, the number of eggs laid by the adults or the proportion of eggs that hatch can be adjusted. Hatching inhibitor treatment will be described later.

[0040] Furthermore, the nematodes may be sterilized nematodes. By using sterilized nematodes, it is possible to suppress the proliferation of nematodes after use of the nematode encapsulation capsule and prevent the spread of nematodes into the environment. Sterilization treatment will be described later.

[0041] (coating) The coating of the nematode encapsulation capsule according to this embodiment is a membrane that forms the outer phase of the nematode encapsulation capsule, and this membrane encapsulates the encapsulation composition containing the nematode inside (hereinafter, this may be referred to as the "external phase membrane"). The coating contains, as a main component, a water-soluble polymer that gels upon reacting with cations. In this specification, "containing a water-soluble polymer as a main component" means that the coating contains 50% by mass or more of the water-soluble polymer when the components in the coating excluding the liquid component are taken as 100% by mass.

[0042] The coating is preferably made of a material that is not edible by nematodes, that does not have a negative effect on nematodes, and / or that is oxygen permeable.

[0043] [Water-soluble polymer] The water-soluble polymer that gels by reacting with a cation is preferably a divalent cation (e.g., Mg 2+ , Ca 2+) and more preferably, a water-soluble polymer (calcium-coagulating polymer compound) that gels upon reacting with calcium ions.

[0044] Examples of the water-soluble polymer include proteins and polysaccharides. Specific examples of water-soluble polymers include guar gum, casein, pectin, sodium cellulose glycolate, sodium alginate, sodium polyacrylate, agar, kappa carrageenan, iota carrageenan, lambda carrageenan, gluten, dextrin, xanthan gum, methylcellulose, starch, gelatin, locust bean gum, galactan, albumin, and keratin. Those that dissolve in water at room temperature or below are preferred. Among these, sodium alginate is more preferred. Even if a polymer dissolves in hot water but not in cold water, it may be soluble if converted into a sodium salt, as long as it can be prepared at low temperatures. Furthermore, the water-soluble polymer may be one type or two or more types.

[0045] The coating may contain components other than the water-soluble polymer. Examples of such components include dyes, fluorescent or other labeling substances, nematodes, liquid components in which nematodes can survive, such as water and buffer solutions, nematode culture medium components, nematode food such as E. coli, E. coli culture medium components, E. coli food such as yeast, and antibiotics for preventing contamination by bacteria or fungi. When the coating contains a dye or labeling substance, the external shape of the nematode-encapsulated capsule can be easily observed visually or under a microscope (e.g., under a fluorescent microscope).

[0046] The thickness and hardness of the coating can be appropriately selected depending on the application and size of the nematode encapsulating capsule, etc. For example, by making the coating thinner, the encapsulated nematodes can be induced to break the coating and escape to the outside of the nematode encapsulating capsule.

[0047] In the nematode encapsulation capsule according to this embodiment, the coating may be single-layered or multi-layered.

[0048] (Shape of nematode capsule) The shape of the nematode-encapsulating capsule according to this embodiment is not particularly limited as long as it can encapsulate the desired amount of nematodes. Examples of capsule shapes include spherical, oval, teardrop, and gourd shapes. The capsule may also have one or more flat surfaces.

[0049] The size of the nematode-encapsulating capsule according to this embodiment may be appropriately selected depending on the type and developmental stage (egg, larva, adult, etc.) of nematodes to be encapsulated, whether or not the nematodes have been sterilized prior to encapsulation, their sex, size, number, etc. The outer diameter of the capsule may be several μm, several tens of μm, or several hundred μm. The outer diameter of the capsule may be several mm, several tens of mm, or several hundred mm. The outer diameter of the capsule may be 10 cm or less, or 1 cm or less. The outer diameter of the capsule may be 10 μm or more, or 20 μm or more. In this specification, the outer diameter of the capsule refers to the maximum diameter of the cross section of the capsule.

[0050] The nematodes encapsulated in the nematode encapsulation capsules according to the present embodiment require oxygen for survival, but the oxygen-permeable membrane of the capsules allows them to survive for several days within the capsules. Furthermore, if the encapsulation composition of the nematode encapsulation capsules contains nematode food such as E. coli, the nematodes can be grown within the capsules.

[0051] Furthermore, the nematode-encapsulated capsules according to this embodiment can be stored in good condition for several days even when sealed in a container such as a plastic dish. In other words, the nematode-encapsulated capsules according to this embodiment can be transported, and the nematodes encapsulated in the capsules can be used at their destination. Furthermore, the nematodes encapsulated in the capsules can be removed by a simple operation such as puncturing the capsules. Therefore, even non-experts can use the capsules to conduct experiments using nematodes.

[0052] When storing the nematode-encapsulated capsule according to this embodiment, a liquid such as water may be poured into the storage container, which can reduce the impact of vibrations on the nematodes during transportation.

[0053] Furthermore, increasing the number of nematode-encapsulated capsules in the same storage container and storing them in a dense state can prevent the nematode-encapsulated capsules from rolling or breaking due to vibrations during transportation. Furthermore, if the nematode-encapsulated capsules are not spherical but have one or more flat surfaces, placing them in the storage container with that surface facing downwards can prevent the capsules from rolling due to vibrations, thereby reducing the impact of vibrations during transportation on the nematodes inside the capsules.

[0054] [Method for producing nematode-encapsulated capsules] Methods for producing nematode-encapsulating capsules according to this embodiment include (1) a method of encapsulating nematodes simultaneously with the preparation of a membrane that will become the outer membrane of the capsule (hereinafter, sometimes referred to as "Method (1)"), and (2) a method of injecting nematodes after the preparation of a membrane that will become the outer membrane of the capsule (hereinafter, sometimes referred to as "Method (2)"). These two methods will be explained below with reference to the flowchart in Figure 21.

[0055] [(S1) Determining the developmental stage of the nematodes to be encapsulated and other conditions, and culturing the nematodes] In this step, depending on the use of the nematode encapsulation capsule, the type of nematode to be encapsulated, its developmental stage (egg, larva, adult, etc.), whether or not the nematodes have been sterilized before encapsulation, the sex and number of nematodes to be encapsulated, or the amount (volume) of the liquid containing the nematodes (hereinafter sometimes referred to as "nematode liquid"), the amount of food, etc. may be determined. Nematodes may be cultured before encapsulation to obtain nematodes at the desired developmental stage.

[0056] [Nematode Sterilization Treatment Before Nematode Encapsulation] Sterilizing the nematodes to be enclosed in the nematode-encapsulating capsules suppresses the generation of the next generation and prevents the nematodes from multiplying after use in experiments, etc., or from spreading uncontrollably throughout the environment. Sterilization methods include irradiation or chemical treatment with hatching inhibitors, as well as the use of reproductively impaired mutants, and can be selected according to the purpose.

[0057] (Sterilization of nematodes by irradiation) Irradiating nematodes with radiation (X-rays, gamma rays, electron beams, heavy particle beams, proton beams, etc.) can suppress their reproductive function. The method of irradiating nematodes can be selected depending on the radiation irradiation facility, the type of nematode, the purpose of irradiation, etc. Examples of irradiating nematodes include irradiating them on a culture plate, irradiating them in a culture solution contained in a centrifuge tube, or irradiating them while they are sealed in a microchip for holding nematodes. Other examples of irradiating nematodes include irradiating the entire container containing the nematodes to irradiate the entire body of the nematode, and irradiating only the gonads of the nematode using a special irradiation device that can target radiation at specific sites (e.g., a heavy ion microbeam irradiator).

[0058] The dose required to make all fertilized eggs from the irradiated generation unable to hatch (hereinafter referred to as the "sterilization dose") varies depending on the type of nematode, developmental stage or sex, type of radiation (radiation quality) or dose rate, etc. maleIf the irradiated individual is male, we evaluate whether the next generation (fertilized eggs) fertilized by mating with a female or hermaphrodite that has not been sterilized after irradiation can hatch. If the irradiated individual is female, we evaluate whether the next generation (fertilized eggs) fertilized by mating with a male that has not been sterilized after irradiation can hatch. On the other hand, if the irradiated individual is hermaphrodite, we evaluate whether the next generation (fertilized eggs) that self-fertilized before or after irradiation, or the next generation (fertilized eggs) that fertilized by mating with a male after irradiation can hatch. For example, when irradiating adult hermaphrodites of C. elegans before egg-laying, the effective dose that reduces the hatching rate by 37% is ED 37 is about 184 Gy (gray: unit of absorbed dose) for X-rays or gamma rays, and about 184 Gy for carbon ions, a type of heavy particle beam. is about It has been reported that the threshold dose required for hatching is 41 Gy. The threshold dose (sterilization dose: ED0) for X-rays or gamma rays is approximately 500 Gy. By irradiating adult worms with a dose lower than the sterilization dose, it is possible to adjust the rate at which only a certain percentage of eggs hatch. Furthermore, by irradiating fertilized eggs or larvae with a dose lower than the sterilization dose, it is possible to stop or delay the development of nematodes. On the other hand, by irradiating fertilized eggs or larvae with a dose much higher than the sterilization dose, it is possible to temporarily suppress the movement of nematodes, which is advantageous when using nematode-encapsulated capsules for the purpose of observing nematodes.

[0059] (Nematode sterilization treatment by chemical treatment) As a method for suppressing (sterilizing) the reproductive function of nematodes, chemical treatments such as culturing nematodes on a medium containing fluorodeoxyuridine (hereinafter referred to as "FUdR"), a type of hatching inhibitor, or in a culture medium containing FUdR, can also be selected.

[0060] The dose of drug required to prevent all eggs laid by the drug-treated generation from hatching varies depending on the nematode species, developmental stage, or sex, the type of drug, and the treatment (exposure) time. For example, if FUdR solution is added to a culture plate and pre-oviposition hermaphrodite adult C. elegans is exposed for one day, the sterilizing dose required to reduce the hatching rate of the next generation to 0% is 0.5 mg / mL or higher. By exposing pre-oviposition adult worms to a lower dose of drug than the sterilizing dose, it is possible to adjust the dose so that only a certain percentage of eggs hatch.

[0061] (Hatching inhibitor added during packaging) If the nematodes to be encapsulated in the nematode encapsulation capsules are larvae or adults before egg laying or adults that have not been treated with a hatching inhibitor, an agent such as FUdR can be added to the encapsulation composition of the nematode encapsulation capsules, in which case the hatching of eggs in the nematode encapsulation capsules can be inhibited.

[0062] [(S2) Selection of water-soluble polymers and cation solutions, and determination and preparation of various conditions such as concentration] In this step, a water-soluble polymer such as a calcium-coagulating polymer compound and a cation such as a calcium salt aqueous solution are selected, and various conditions such as concentration are determined. An example of a calcium-coagulating polymer compound is sodium alginate, and suitable cations for its coagulation include calcium salt aqueous solutions such as calcium lactate and calcium chloride. After selecting a water-soluble polymer and its corresponding cation, a solution containing the water-soluble polymer and an aqueous cation solution are prepared. The various conditions, such as concentration, may be determined based on the conditions used in known methods for producing artificial granules. For example, the conditions described in JP-A-7-133209 may be referenced.

[0063] By adjusting the concentration of the water-soluble polymer in the solution containing the water-soluble polymer, it is possible to adjust the thickness and hardness of the coating of the nematode-encapsulating capsules to be produced.

[0064] [(S3) Encapsulating nematodes before preparing the outer membrane?] In this step, it is decided whether to produce the nematode encapsulation capsules by method (1) or method (2).

[0065] [(S4) Putting nematodes into a solution containing a water-soluble polymer] This step is performed when method (1) is selected in (S3). An encapsulation composition is prepared by injecting nematodes into the solution containing the water-soluble polymer prepared in (S2). The solution containing the water-soluble polymer prepared in (S2) is in a sol state, and a nematode-containing sol can be prepared by injecting a nematode solution. Alternatively, nematodes may be collected one by one from the culture plate using a platinum wire picker and transferred into the aqueous sol. The number of nematodes to be placed in the solution containing the water-soluble polymer or the amount (volume) of nematode solution can be appropriately selected depending on the purpose and size of the nematode encapsulation capsule.

[0066] The flowchart in FIG. 21 shows an example in which the nematode liquid is injected into a solution containing a water-soluble polymer, but the encapsulation composition may also be prepared by adding a water-soluble polymer to the nematode liquid.

[0067] [(S5) Preparation of capsule outer membrane] In this step, the encapsulation composition containing the nematodes prepared in (S4) is reacted with the solution containing the cations prepared in (S2) to produce a coating that will become the outer membrane of the nematode encapsulation capsule. For example, the solution containing the cations prepared in (S2) is dripped onto or sprayed onto the encapsulation composition containing the nematodes prepared in (S4), thereby producing a coating that will become the outer membrane of the nematode encapsulation capsule. Alternatively, the encapsulation composition prepared in (S4) is dripped onto or sprayed onto the solution containing the cations prepared in (S2), thereby producing a coating that will become the outer membrane of the nematode encapsulation capsule.

[0068] By adjusting the amount (volume) of the encapsulating composition that is reacted with the cation-containing solution, the size and shape of the nematode encapsulation capsules can be adjusted.

[0069] When the solution containing the cation prepared in (S2) is dripped onto the encapsulation composition containing the nematodes prepared in (S4), or when the encapsulation composition prepared in (S4) is dripped onto the solution containing the cation prepared in (S2), after the coating is formed, the dripped material is immersed in the receiving liquid until the desired coating thickness and hardness are achieved. Depending on the type of water-soluble biopolymer selected, for example, in the case of a calcium-coagulating compound, the longer the immersion time (e.g., about 5 minutes), the thicker and harder the coating will be. On the other hand, if the immersion time is short (e.g., less than 30 seconds), the coating will be thin and soft. If the coating is thin and soft, the nematodes encapsulated in the nematode encapsulation capsule can break the coating and escape from the nematode encapsulation capsule.

[0070] After immersing the dropped object in the receiving liquid, the dropped object is removed from the receiving liquid and washed with ultrapure water or the like to remove the receiving liquid components adhering to the outside of the coating so as to prevent the hardening of the coating from progressing.

[0071] From the viewpoint of ease of operation, it is preferable to add dropwise the solution containing the encapsulation composition prepared in (S4) to the solution containing the cation prepared in (S2).

[0072] When the solution containing the cation prepared in (S2) is added dropwise to the encapsulation composition prepared in (S4), Japanese Patent Application Laid-Open No. 5-92909, etc. can be referred to. When the encapsulation composition prepared in (S4) is added dropwise to the solution containing the cation prepared in (S2), Japanese Patent Application Laid-Open No. 7-133209, etc. can be referred to.

[0073] [(S6) Preparation of capsule outer membrane] This step is performed when method (2) is selected in (S3). A membrane that will become the capsule outer membrane is prepared by reacting the solution containing the water-soluble polymer prepared in (S2) with the solution containing the cation prepared in (S2). (S6) Preparation of the capsule outer membrane can be performed in the same manner as (S5) Preparation of the capsule outer membrane.

[0074] [(S7) Injecting nematodes into the capsule?] In this step, it is decided whether or not to inject nematodes into the capsule.

[0075] [(S8) Injection of nematodes into the capsule] This step is performed if you select to inject nematodes in (S7) "Inject nematodes into the capsule?". The nematodes are injected into the outer membrane of the capsule prepared in (S6). Methods for injecting nematodes include injection.

[0076] [(S9) Sealing of the nematode injection port in the outer membrane] In this step, after injecting the nematodes in (S8), the nematode injection port is sealed. Methods for sealing the nematode injection port opened when injecting the nematodes include dripping or spraying a water-soluble polymer compound solution onto the injection port, and then dripping or spraying an aqueous solution of a cation onto the injection port to form a film that seals the injection port.

[0077] [(S10) Are nematodes already enclosed inside the capsule?] This step is a decision step to be performed if the user chooses not to inject nematodes in (S7) "Inject nematodes into capsule?" If the capsule is multi-layered and a capsule with nematodes encapsulated inside has already been produced before the preparation of the capsule outer membrane in (S6), proceed to (S11). If the capsule consists of a single-layer outer membrane prepared immediately before in (S6), or if the capsule is multi-layered but does not encapsulate nematodes inside, return to (S2) to continue producing the nematode-encapsulated capsule.

[0078] [(S11) Created by stacking more layers of film?] In this step, if the capsule produced immediately before (S11) is a single layer, a decision is made as to whether to leave the nematode-encapsulated capsule with a single layer of coating or to add layers to create a multi-layered capsule. If the capsule is multi-layered, a decision is made as to whether to add another layer of coating. If it is decided to add another layer of coating, the process returns to (S2) and continues producing a multi-layered nematode-encapsulated capsule.

[0079] In a nematode encapsulation capsule having multiple membranes, the nematode may be encapsulated between the inner and outer layers or within the outer layer, rather than within the inner membrane or the encapsulation composition contained within the inner layer. Furthermore, the type, developmental stage, sex, etc. of the nematode may be varied depending on the encapsulation position.

[0080] In the method for producing a nematode encapsulation capsule according to this embodiment, a water-soluble polymer that gels upon reaction with a cation, such as calcium ion, is reacted with the cation to generate a gel. A coating is then formed on the surface of the gel. Because the encapsulation composition encapsulated in the coating is in a liquid or sol state, the nematode can be encapsulated in the nematode encapsulation capsule while maintaining its fluidity.

[0081] The method for producing nematode encapsulation capsules according to this embodiment does not induce gelation by heating, so it does not cause heat shock or other effects on the nematodes. Another advantage is that the production process and the components of the nematode encapsulation capsules do not chemically affect the nematodes. Furthermore, the coating formed by the method for producing nematode encapsulation capsules according to this embodiment is oxygen-permeable, so it can maintain the supply of oxygen essential for the survival of the nematodes encapsulated in the nematode encapsulation capsules.

[0082] [Irradiation of nematode capsules] After the nematode-encapsulating capsules are manufactured, they can be irradiated to inhibit the hatching of the encapsulated nematode eggs or the eggs laid by the adult nematodes within the capsules. In this case, the nematode-encapsulating capsules are placed in a container and the entire container is irradiated. As mentioned above, the sterilization dose varies depending on the type, developmental stage or sex of the nematode, the type or dose rate of radiation, etc. Because irradiation can decompose the membrane of the nematode-encapsulating capsules, it is desirable to avoid irradiation at a dose far exceeding the sterilization dose.

[0083] [Method for evaluating nematode response] The method for evaluating the response of nematodes according to this embodiment includes the steps of supplying nematode-encapsulated capsules to a nematode testing plate (hereinafter sometimes simply referred to as a "plate"), releasing nematodes from the nematode-encapsulated capsules, and evaluating the response of the nematodes.

[0084] (Step of supplying nematode-encapsulated capsules to nematode test plates) This step is a step of supplying nematode-encapsulated capsules to a nematode test plate to which a test substance has been partially supplied (hereinafter, this step may be referred to as the "supplying step").

[0085] There are no particular limitations on the nematode test plate, as long as it can be used in a nematode response evaluation test. As an example, a nematode trap plate (hereinafter sometimes referred to as a "nematode trap plate") having a drop tank described in Patent Document 2 (WO 2020 / 218501) may be used. When using a nematode trap plate, the nematode response evaluation method described in Patent Document 2 may be followed.

[0086] As another example, a nematode testing plate having a supply section for supplying nematodes, in which the supply section is treated to prevent the solution containing nematodes flowing out of the nematode-encapsulated capsules from spreading over the plate (hereinafter, this may be referred to as a "supply section-treated plate"). When a supply section-treated plate is used, nematodes can be supplied by supplying nematode-encapsulated capsules to the supply section. The supply section-treated plate will be described below.

[0087] (Supply section processing plate) The supply unit-processed plate is equipped with a supply unit on the solid phase that supplies nematode-containing capsules during the supply process. The supply unit is processed to suppress the spread of the nematode-containing solution in the nematode-containing capsule that flows out of the capsule during the process of releasing nematodes from the nematode-containing capsule, as described below. The processing to suppress spread allows the nematodes to remain within a predetermined range, and the starting position of the nematode's movement can be sufficiently controlled. Specific embodiments of the processing to suppress the spread of the nematode-containing solution flowing out of the nematode-containing capsule are not limited, but examples include making the supply unit a recess and making the bottom surface of the supply unit, i.e., the surface that comes into contact with the nematode-containing solution flowing out of the nematode-containing capsule, hydrophobic. Unless otherwise specified, the following description will be given for the case where the solid phase is a solid medium.

[0088] First, we will explain the use of a recess as the supply unit. By using a recess as the supply unit, the nematode-containing solution leaking out of the nematode-encapsulation capsule can be retained within the recess, thereby preventing the nematode-containing solution from spreading across the plate. Furthermore, if nematodes overlap and form a clump within the recess, the nematodes closest to the opening on the solid medium surface side of the recess will crawl out to the solid medium surface in order, allowing the nematode clump to dissolve when the nematodes are released. Therefore, by providing a recess, even if a clump of nematodes does form, the clump state can be prevented from persisting. Furthermore, since the nematode-encapsulation capsule can be retained within the recess, it is easy to supply the nematode-encapsulation capsule onto the plate.

[0089] The recesses are formed so as to extend from the surface of the solid medium toward the bottom of the solid medium. The depth of the recesses is not particularly limited as long as the effect of the recesses can be obtained. For example, the recesses may or may not reach the bottom surface of the solid medium.

[0090] The shape of the recess when viewed from the opening side is not particularly limited, but from the standpoint of ease of observation of the nematodes in the recess when observed with an upright microscope and ease of the nematodes crawling up to the surface of the solid medium after the solution in the nematode-encapsulating capsule has dried, it is preferable that the opening of the recess has the same shape as the bottom surface of the recess or a shape that is wider than the bottom surface.

[0091] The method for forming the recesses is not particularly limited, and examples thereof include a method of forming recesses by hollowing out a solid medium and a method of forming recesses by pouring a solid medium into a container that is previously provided with a structure for forming recesses.

[0092] The volume of the well is not particularly limited. For example, when the well is formed by hollowing out a solid medium, the volume of the well depends on the thickness of the solid medium.

[0093] The size, shape and material of the container in which the solid medium is formed are not particularly limited, and commercially available containers such as plastic dishes can be used.

[0094] As a supply section processing plate having a recess, for example, a bottom surface inner diameter of about 8.4 cm and a bottom surface area of ​​about 55 cm 2 A cylindrical depression with an inner diameter of 5 mm (bottom surface area approximately 0.2 cm) was placed in a circular plastic dish. 2 ) may be formed as a solid medium.

[0095] The recess may penetrate the solid medium, and the container may be exposed at the bottom surface of the recess. If the bottom surface of the container is hydrophobic, a portion of it may be exposed in the recess, and the solution containing nematodes that flows out of the supplied nematode encapsulation capsules may come into contact with the hydrophobic portion, thereby hastening the drying of the nematode liquid. In this specification, hydrophobicity refers to the property of repelling the liquid components of the solution containing nematodes that flows out of the nematode encapsulation capsules.

[0096] When the supply section is a recess, the hydrophobic bottom surface of the recess allows the nematode solution supplied (dropped) to the bottom surface of the recess to spread over the entire surface and then quickly move to the outer periphery of the bottom surface of the recess, with the center of the bottom surface of the recess first becoming dry. This allows the solution containing nematodes flowing out of the nematode encapsulation capsule to efficiently move to the outer periphery of the bottom surface of the recess, which is connected to the inner wall of the recess, along which the nematodes move as they crawl up to the surface of the solid medium. This allows the nematodes to be efficiently supplied to the surface of the solid medium.

[0097] When the container is exposed at the bottom surface of the well, the entire container may be made hydrophobic, thereby making the bottom surface of the well hydrophobic. The hydrophobic bottom surface of the well allows the liquid components of the nematode-containing solution flowing out of the nematode-encapsulated capsule onto the plate to be repelled by the hydrophobic portion, allowing the nematode-containing solution flowing out of the nematode-encapsulated capsule to move quickly from the center to the periphery of the bottom surface of the well. From this perspective, the container is preferably made of a material that does not absorb or transmit liquid. Examples of such materials include plastic, glass, silicone resins such as polydimethylsiloxane (PDMS), and metals such as aluminum.

[0098] The same effect can be achieved by applying a hydrophobic material such as a water-repellent polymer to the bottom surface of the recess, rather than exposing the hydrophobic container portion at the bottom surface of the recess. When applying a hydrophobic material such as a water-repellent polymer to the recess, it is not limited to the bottom surface, and it may also be applied to the inner wall (side surface) of the recess.

[0099] When a supply part other than a recess is provided, the surface of the supply part may be made hydrophobic by any method, for example, by placing a hydrophobic sheet of any material and shape on the nematode test plate and using the sheet as the supply part.

[0100] When the supply section is a recess, the inner wall of the recess connecting the bottom surface of the recess to the opening may be water-absorbent, which allows the liquid components of the nematode-containing solution flowing out of the nematode encapsulation capsule to be absorbed into the solid medium, thereby hastening the drying of the nematode solution in the recess.

[0101] In this embodiment, water absorption refers to the ability of a solid medium to absorb the liquid components of a solution (not limited to water) flowing out of a nematode-encapsulating capsule. For example, this refers to the solid medium absorbing 40% or more, preferably 50% or more, and more preferably 70% or more of the liquid components of the solution flowing out of a nematode-encapsulating capsule within about 30 minutes of the supply of the solution.

[0102] The bottom surface of the recess may be hydrophobic, and the inner wall of the recess may be water-absorbent. This configuration allows the nematode-containing solution flowing out of the nematode-encapsulating capsule to move to the outer periphery of the bottom surface of the recess. This allows the liquid components of the nematode-containing solution flowing out of the nematode-encapsulating capsule to be more efficiently absorbed by the inner wall of the recess, thereby shortening the drying time of the solution.

[0103] The method for making the inner wall of the recess water-absorbent is not particularly limited. For example, the inner wall may be made water-absorbent by hollowing out the solid medium to form a recess. By hollowing out the solid medium to form a recess, fine pores formed inside the solid medium as it solidifies are exposed on the inner wall of the recess, and the exposed pores act to make the inner wall of the recess water-absorbent.

[0104] Furthermore, compared to a container with a pre-defined structure for forming a recess, such as a container with a protruding member attached or inserted therein, in which a solid medium is poured into the container and allowed to solidify, the inner wall of a recess formed by hollowing out the solid medium has a rougher surface and a larger area. Thus, hollowing out the recess is presumed to increase the area available for absorbing the liquid components of the nematode-containing solution that flows out of the nematode-encapsulated capsule, as well as the number of micropores. For this reason, hollowing out the recess is preferred.

[0105] The area of ​​the inner wall of the recess is not particularly limited, but from the viewpoint of increasing the water absorption of the nematode solution on the inner wall of the recess, it is preferable to set the area to 0.1 cm 2 Over 4cm 2 Preferably less than 0.2 cm 2 More than 2cm 2 More preferably, it is:

[0106] Increasing the area of ​​the absorbent recess inner wall increases the absorbency. On the other hand, increasing the area of ​​the recess inner wall due to an increase in the amount of solid medium increases the humidity inside the supply unit processing plate and, ultimately, inside the recess, thereby inhibiting the drying of the liquid components of the nematode-containing solution flowing out of the nematode-encapsulation capsule. Furthermore, the time required for the liquid components of the nematode-containing solution flowing out of the nematode-encapsulation capsule to dry is affected by various conditions, such as the size and shape of the supply unit processing plate, the size, shape, and number of recesses, the amount (volume) of the nematode-containing solution flowing out of the nematode-encapsulation capsule, the amount (volume) of the solid medium, and the nematode population density. Therefore, the area of ​​the recess inner wall can be appropriately selected while taking into account the above-mentioned ranges and other conditions.

[0107] From the viewpoint of suitable release of nematodes, the depth of the recess is preferably 0.1 cm or more and 1 cm or less, and more preferably 0.14 cm or more and 0.6 cm or less. By ensuring that the depth of the recess is not below the lower limit, the area of ​​the inner wall of the recess can be kept within a sufficient range, thereby maintaining sufficient water absorption of the inner wall of the recess. Furthermore, by ensuring that the depth of the recess is not above the upper limit, the distance required for the nematodes to crawl out of the recess can be shortened, and the humidity within the recess can be controlled within a suitable range. This shortens the time until the nematodes are released.

[0108] The depth of the wells can be reduced by pouring a relatively large amount (volume) of solid medium into the feeder-processed plate, i.e., by making the thickness of the solid medium relatively thin. This shortens the distance from the bottom surface of the well to the surface of the solid medium, limiting the number of nematodes that accumulate along the inner wall from the bottom of the well toward the opening on the solid medium surface side, thereby suppressing the size of the nematode colony. Furthermore, the shorter distance along the inner wall of the well that the nematodes must climb facilitates nematode release. For example, when the well depth is 0.17 cm or more and 0.25 cm or less, nematode release can begin within 3 minutes of the release of the nematode from the nematode-encapsulation capsule.

[0109] The amount (volume) of solid medium may be varied as appropriate depending on the method for preparing the solid medium, the size of the container, and the conditions for the drying step described below. The amount of solid medium will be described below using the example of a solid medium in which the medium is poured into a container, solidified, and left overnight in a tightly sealed storage container at room temperature, with the supply section being a recess. Note that the moisture content of the solid medium changes depending on the length of storage time after solidification, so the conditions for the supply step and the drying step described below can be adjusted according to the state of the solid medium.

[0110] The amount (volume) of the solid medium is not particularly limited as long as it is within a range in which the test can be carried out suitably, with the basic rule being that the thickness should be such that the solution containing nematodes flowing out of the nematode-encapsulating capsule supplied to the recess does not overflow onto the surface of the solid medium. For example, from the viewpoint of suitably drying the liquid components of the solution containing nematodes flowing out of the nematode-encapsulating capsule, the amount of solid medium is preferably 40% or less of the volume of the container. There is no particular lower limit, but as long as it is 10% or more, the solution containing nematodes flowing out of the nematode-encapsulating capsule will not overflow onto the solid medium and will be within a range in which the test can be carried out. In order to achieve an amount of solid medium within this range, for example, a container with an inner diameter of the bottom surface of about 8.4 cm and a volume of about 72 cm is used. 3 When using a circular dish, the amount of solid medium should be about 10 mL to 30 mL, i.e., about 0.2 cm to 0.6 cm thick. The inner diameter of the bottom surface is about 5.2 cm, and the volume is about 21 cm. 3When using such a circular dish, the amount of solid medium may be about 3 mL to 10 mL, that is, the thickness may be about 0.14 cm to 0.5 cm.

[0111] When forming a recess by hollowing out a solid medium, the depth of the recess is the same as the thickness of the solid medium and depends on the amount (volume) of the solid medium, so the depth of the recess may be adjusted to be within the range described above.

[0112] The amount (volume) of the solid medium may be 20% or less of the capacity of the container. When other conditions, such as the amount of nematode-containing solution flowing out of the nematode-encapsulating capsule to be supplied, are the same, using a solid medium within this range will result in a faster drying rate of the liquid components of the nematode-containing solution flowing out of the nematode-encapsulating capsule to be supplied, compared to when a solid medium in an amount greater than this range is used. Therefore, a solid medium within this range can be suitably used when it is desired to relatively shorten the time required for the drying step described below. In this embodiment, "relatively short time required for the drying step" typically means that the time required for the drying step is 25 minutes or less. In order to obtain an amount of solid medium within this range, for example, a container with an inner diameter of the bottom surface of about 8.4 cm and a volume of about 82 cm is used. 3 When using a circular dish, the amount of solid medium should be about 10 mL, i.e., the thickness should be about 0.2 cm to 0.4 cm. The inner diameter of the bottom surface should be about 5.2 cm, and the volume should be about 25 cm. 3 When using a circular dish, the amount of solid medium should be about 3 mL, i.e., about 0.15 cm thick.

[0113] Depending on the test, the amount (volume) of solid medium may be 40% or more of the volume of the container. While there is no upper limit on the amount of solid medium, from the perspective of ease of testing, it is preferable that it be 50% or less of the volume of the container. When other conditions, such as the amount (volume) of the nematode-containing solution flowing out of the nematode-encapsulating capsule are the same, the drying rate of the liquid components of the nematode-containing solution flowing out of the nematode-encapsulating capsule is slower than when a solid medium less than this range is used. Therefore, solid media within this range are suitable for use when a relatively long drying time is desired. In this embodiment, "a relatively long drying time" typically means a drying time of 60 minutes to 180 minutes. Note that if a drying time exceeding 180 minutes is required, the nematodes used in the test may become starved, making the test unsuitable for normal testing. However, for tests using starving nematodes, a drying time exceeding 180 minutes may be selected.

[0114] There are no particular limitations on the number, shape, size, and location of the supply parts, and conditions can be changed appropriately depending on the type and number of nematodes used, the type and number of test subjects, the purpose of the test, etc. The number of supply parts can be, for example, 1, 2, 3, 4, 5, 6, or more. The shape of the supply part may be, for example, a circular, rectangular, polygonal, or other cross-sectional shape when the supply part is cut horizontally.

[0115] The size of the supply unit is preferably sufficiently small compared to the size of the supply unit-processed plate. For example, when the supply unit is a recess, the total area of ​​the openings of the recesses on the surface side of the plate may be approximately 2% or less of the surface of the solid medium, more preferably 0.4% or less. By keeping the area of ​​the openings of the recesses within the above range, the spread of the nematode-containing solution leaking from the nematode-encapsulated capsules on the plate can be suitably suppressed without interfering with the test. Furthermore, the position of the supply unit is typically near the center of the supply unit-processed plate, but is not particularly limited and can be determined depending on the purpose and content of the test.

[0116] The size of the container used in the supply unit processing plate is not particularly limited as long as it is suitable for the test. For example, in the case of a circular container, the inner diameter of the container bottom surface is preferably 3.0 cm to 15.0 cm, and the height (internal height) is preferably 0.5 cm to 3 cm. More preferably, the inner diameter is 5.0 cm to 10.0 cm, and the height (internal height) is 1 cm to 1.5 cm. Furthermore, in the case of a rectangular container, the inner dimensions are preferably 3 cm to 30 cm in length (typically short side), 3 cm to 30 cm in width (typically long side), and 0.5 cm to 3 cm in height. More preferably, the length is 5 cm to 15 cm, the width is 5 cm to 25 cm, and the height is 1 cm to 1.5 cm. By keeping the inner dimensions of the container bottom surface within this range, various gradients can be suitably formed within the supply unit processing plate, such as an odor gradient of the test substance in an olfactory chemotaxis test, a concentration gradient of salt or the like in a gustatory chemotaxis test, and a temperature gradient in a thermotaxis test. The volume of the container is 3.6 cm 3 More than 2700cm 3 Preferably less than 19.5cm 3 That's all, 565cm 3 It is preferable that:

[0117] (Step of releasing nematodes from nematode-encapsulating capsules) This step is a step of releasing nematodes from the nematode-encapsulating capsules supplied in the above-mentioned supply step (hereinafter, this step may be referred to as the "releasing step"). For example, nematodes may be released by making holes in the nematode-encapsulating capsules. After releasing the nematodes, the solution containing the nematodes may be pipetted or otherwise treated to maintain a constant nematode density.

[0118] When using a supply unit-processed plate, a drying step may be performed after the release step, in which the liquid components of the nematode-containing solution that has flowed out of the nematode-encapsulated capsule are dried to facilitate the movement of the nematodes on the plate. The drying of the liquid components of the nematode-containing solution that has flowed out of the nematode-encapsulated capsule may be performed by evaporating the solution. By performing the drying step, the liquid components of the nematode-containing solution that has flowed out of the nematode-encapsulated capsule dry, facilitating the movement of the nematodes, and the nematodes are released onto the supply unit-processed plate. The supply unit is processed to prevent the nematode-containing solution that has flowed out of the nematode-encapsulated capsule from spreading on the plate, thereby preventing the solution from spreading until the liquid components of the nematode-containing solution dry, and keeping the nematodes within a predetermined range. This allows for control of the timing of nematode release, particularly when using nematode-encapsulated capsules with an outer diameter of several centimeters that contain a large amount (volume) of encapsulated composition. In this specification, "release" refers to the drying of the liquid components of the nematode-containing solution that has flowed out of the nematode-encapsulating capsule, causing the nematodes to crawl out onto the supply processing plate. In this embodiment, "drying" refers to the removal of the liquid components of the nematode-containing solution that has flowed out of the nematode-encapsulating capsule to the extent that the nematodes can move freely on the supply processing plate, and typically refers to a state in which 80% or more of the liquid components have been removed.

[0119] (Step of assessing the response of nematodes) This step is a step of evaluating the response (for example, taxis) of the nematodes released in the above-mentioned releasing step to the test substance (hereinafter, sometimes referred to as a "response evaluation step").

[0120] C. elegans have chemotaxis (attraction to specific concentrations of sodium chloride (NaCl)), chemotaxis (avoidance from specific concentrations of diacetyl), chemotaxis (attraction to the urine of cancer patients), and chemotaxis (attraction to specific temperatures) (thermotaxis).

[0121] For example, if the test object is urine collected from a subject, the response evaluation step can determine the subject's likelihood of cancer. That is, the nematode response evaluation method according to this embodiment can be used in a cancer testing method. Cancer testing may include, for example, the steps of supplying a target test object to a portion of a nematode test plate (forming a concentration gradient of the target test object), supplying the nematode-encapsulated capsules to a predetermined position, releasing nematodes from the nematode-encapsulated capsules, and, after a certain period of time, measuring the number of nematodes attracted to or escaping from the test object, or a factor correlated with the number of nematodes. The nematode test plate is not particularly limited as long as it can be used in a test to evaluate the response of nematodes to cancer. For example, the nematode trap plate described above may be used. When using a nematode trap plate, the cancer testing method described in Patent Document 2 may be used. The number of nematodes attracted to the test object may be, for example, the number of nematodes present within a predetermined area of ​​the portion. Furthermore, the number of nematodes that have escaped from a portion of the nematode test plate may be the number of nematodes that exist outside a predetermined area from that portion.

[0122] Examples of test samples used in cancer testing methods include biological samples collected from subjects. Examples of such biological samples include body fluids such as urine, sweat, saliva, pleural effusion, ascites, stool, and blood (including individual components separated from blood, such as plasma and serum); cells such as biopsy cells, cancer tissues such as biopsy tissues and tissue sections; and preservation and culture solutions for biological tissues. The test sample is preferably a body fluid that can be easily collected from the mammal to be tested, and more preferably urine or blood.

[0123] The nematode response evaluation method according to this embodiment may also be used to conduct olfactory tests of nematodes on test objects that release odorants or taste tests of nematodes on test objects that have flavors. These chemotaxis tests may include the steps of supplying the target test object to a portion of the nematode test plate (forming a concentration gradient of the target test object), supplying the nematode-encapsulated capsules to a predetermined location, releasing nematodes from the nematode-encapsulated capsules, and, after a certain period of time, measuring the number of nematodes attracted to or escaping from the test object, or a factor correlated with the number of nematodes. For example, the olfactory test can be used to conduct a selection test for antagonistic or repellent organisms against plant-parasitic nematodes. There are no particular limitations on the nematode test plate, as long as it can be used in nematode chemotaxis tests. When using the nematode trap plate, the nematode response evaluation method described in Patent Document 2 may be used. The number of nematodes attracted to the test article may be, for example, the number of nematodes present within a predetermined area from the part, and the number of nematodes that have escaped from a part of the nematode test plate may be the number of nematodes present outside the predetermined area from the part.

[0124] Furthermore, the nematode response evaluation method according to this embodiment may be used to conduct a test to examine the nematode's response to temperature (thermotaxis). The thermotaxis test includes the steps of adjusting a portion of a nematode test plate to a desired temperature, supplying the nematode-containing capsule to a predetermined position, releasing nematodes from the nematode-containing capsule, and, after a certain period of time, counting the number of nematodes attracted to or escaping from the portion, or a factor correlated with the number of nematodes. The nematode test plate is not particularly limited as long as it can be used in a nematode thermotaxis test. When using the nematode trap plate, the test may be performed in accordance with the nematode response evaluation method described in Patent Document 2. The number of nematodes attracted to a portion of the nematode test plate may be, for example, the number of nematodes present within a predetermined area from the portion. Furthermore, the number of nematodes escaping from a portion of the nematode test plate may be the number of nematodes present outside the predetermined area from the portion.

[0125] [Nematode response evaluation kit] The nematode response evaluation kit according to this embodiment includes the nematode-encapsulated capsule. In addition to the capsule, it may also include a nematode test plate. The nematode test plate is not particularly limited as long as it can be used in nematode response evaluation tests, and examples include the above-described feeder-processed plate with a feeder formed thereon and a nematode trap plate. The evaluation kit may also include instructions describing procedures for evaluating nematode responses. These instructions may be written or printed on paper or other media, or may be attached to electronic media such as magnetic tape, computer-readable disks, or CD-ROMs. Instead of instructions, the kit may include an audio or video file explaining procedures for evaluating nematode responses. These instructions may be attached to electronic media such as magnetic tape, computer-readable disks, or CD-ROMs, or may be stored on an accessible cloud server or the like and accessed via an internet connection.

[0126] As described above, nematodes have the property of being attracted to the urine of cancer patients (a type of taxis), and therefore the nematode response evaluation kit according to this embodiment may be used as a cancer testing kit.

[0127] [Production kit for producing nematode-encapsulated capsules] The production kit for producing nematode encapsulation capsules according to this embodiment includes nematodes, a water-soluble polymer, and a cation salt. The nematodes may be nematodes that have been irradiated, exposed to a hatching inhibitor, or otherwise sterilized. Examples of the water-soluble polymer include alginates such as sodium alginate. Examples of the cation salt include calcium salts such as calcium chloride and calcium lactate. The production kit may also include a hatching inhibitor (e.g., powder or liquid) or a liquid containing the agent.

[0128] The production kit may also include instructions describing procedures for producing nematode-encapsulated capsules. These may be written or printed on paper or other media, or may be attached to electronic media such as magnetic tape, computer-readable disks, or CD-ROMs. Instead of instructions, the kit may include an audio file or video file explaining procedures for producing nematode-encapsulated capsules. These may be attached to electronic media such as magnetic tape, computer-readable disks, or CD-ROMs, or may be stored on an accessible cloud server or the like and be accessible via the Internet.

[0129] The nematode encapsulation capsules according to the present embodiment and the various techniques utilizing them described above allow even experimenters lacking the necessary nematode culturing skills and equipment to easily conduct experiments. Specifically, during an experiment, nematodes can be released from the capsule and delivered to nematode test plates by simply poking the capsule's membrane with a toothpick or chip, adding a chelating agent such as sodium citrate solution to dissolve the membrane, or contacting the capsule with a mixture of microorganisms and chemicals (typically by spreading the capsule on soil). For example, the use of the nematode encapsulation capsules according to the present embodiment is effective in space experiments, which require fewer experimenter operations and require less sample storage space. Furthermore, the use of the nematode encapsulation capsules according to the present embodiment is also effective in experimental situations where experimenters are unlikely to possess the necessary nematode culturing skills or equipment, such as in school education and at home. In other words, by obtaining the nematode encapsulation capsules according to the present embodiment, space experiments or experiments conducted by less experienced experimenters can easily obtain nematodes in good condition suitable for experiments. Furthermore, in situations where agricultural workers or pest nematode exterminators who are not skilled in nematode cultivation or the like release any nematode into the environment for the purpose of controlling pest nematodes, by obtaining the nematode-encapsulating capsule of this embodiment, any nematode can be used with simple operations.

[0130] Furthermore, nematode encapsulation capsules that enclose nematodes in good condition can be used to observe nematodes in their capsule state without breaking or dissolving the membrane, or to efficiently conduct experiments in which it is effective to keep nematodes in a fixed position (for example, ion microbeam irradiation experiments in which radiation is aimed at a range of several tens of micrometers within a specific tissue).

[0131] When nematode-containing capsules are used in experiments (experiences) at school or at home, it is preferable to suppress the proliferation of nematodes in the used nematode-containing capsules and prevent the spread of nematodes into the environment. In this regard, it is preferable to use nematode-containing capsules containing sterilized nematodes in experiments (experiences) at school or at home.

[0132] It is also envisioned that nematodes that cause severe damage to agricultural crops or trees can be controlled by scattering nematode-encapsulated capsules containing sterilized nematodes or predatory nematodes of the nematodes in the soil or injecting them into trees (sterile insect release). In this case, for example, the nematode-encapsulated capsules can be scattered, mixed, or buried in the soil or trees, and then left to slowly decompose the capsule membrane over several hours to several days due to microorganisms or chemicals contained in the soil or trees, releasing the nematodes. In order to prevent any nematodes from spreading more than necessary in the environment, it is preferable to use nematode-encapsulated capsules containing sterilized nematodes.

[0133] Furthermore, currently, cancer testing using nematodes is limited to specialized testing laboratories. To commercialize a "simple test kit" for use in mass screening at medical institutions, homes, workplaces, or local areas, a technology for supplying nematodes in good condition is essential. By enclosing and culturing nematodes in a nematode-encapsulating capsule according to one embodiment of the present invention, it is possible to supply nematodes in good condition at low cost. In cancer testing using nematodes or tests to evaluate the response of nematodes, the technology described in the published patent application by the present inventors regarding nematode test plates and nematode test methods (e.g., Patent Document 2) can also be used in combination.

[0134] Cancer testing using the nematode-encapsulated capsules according to this embodiment allows for simple and regular cancer testing, which enables early detection of risk factors that may be harmful to health and strengthens measures to mitigate and avoid risk factors, thereby extending healthy lifespan and contributing to the achievement of the Sustainable Development Goals (SDGs).

[0135] 〔summary〕 The nematode encapsulation capsule according to aspect 1 of the present invention comprises an encapsulation composition containing a nematode and at least one coating layer that encompasses the encapsulation composition, the coating containing as its main component a water-soluble polymer that gels upon reaction with a cation.

[0136] A nematode-encapsulating capsule according to a second aspect of the present invention may be the nematode-encapsulating capsule of the first aspect described above, having an outer diameter of 10 cm or less.

[0137] A nematode encapsulation capsule according to a third aspect of the present invention is the same as that of the first or second aspect, wherein the nematode is Caenorhabditis elegans or a closely related species thereof.

[0138] A nematode-encapsulating capsule according to a fourth aspect of the present invention may be in any one of the first to third aspects, wherein the water-soluble polymer is at least one water-soluble polymer selected from the group consisting of proteins and polysaccharides.

[0139] A nematode-encapsulating capsule according to a fifth aspect of the present invention may be one in which the nematode in any one of the first to fourth aspects is irradiated with radiation.

[0140] A nematode-encapsulating capsule according to a sixth aspect of the present invention may be one in which the nematode in any one of the first to fifth aspects has been treated with a hatching inhibitor.

[0141] A nematode-encapsulating capsule according to a seventh aspect of the present invention may be one in which the nematode in any one of the first to sixth aspects has been sterilized.

[0142] A nematode-encapsulating capsule according to an eighth aspect of the present invention is the encapsulated composition of any one of the first to seventh aspects, which may further contain a hatching inhibitor.

[0143] A method for producing a nematode encapsulation capsule according to aspect 9 of the present invention is a method for producing a nematode encapsulation capsule according to any one of aspects 1 to 8 above, and includes the steps of mixing the nematode and the water-soluble polymer to prepare an encapsulation composition, and reacting the encapsulation composition with a solution containing the cation to produce a nematode encapsulation capsule.

[0144] A method for producing a nematode encapsulated capsule according to aspect 10 of the present invention is a method for producing a nematode encapsulated capsule according to any one of aspects 1 to 8 above, and includes the steps of preparing a base for the coating of the nematode encapsulated capsule by reacting a solution containing the water-soluble polymer with a solution containing the cation, and producing the nematode encapsulated capsule by injecting the nematode into the base.

[0145] A method for producing a nematode-encapsulating capsule according to an eleventh aspect of the present invention may be the same as in the ninth aspect, and may further include a step of irradiating the nematode-encapsulating capsule with radiation.

[0146] A method for producing a nematode-encapsulating capsule according to a twelfth aspect of the present invention may be the same as in the tenth aspect, and may further include a step of irradiating the nematode-encapsulating capsule with radiation.

[0147] A method for evaluating the response of nematodes according to aspect 13 of the present invention includes the steps of supplying a test substance to a portion of a nematode test plate, supplying a nematode-encapsulated capsule according to any one of aspects 1 to 8 above to a predetermined position, releasing the nematode from the nematode-encapsulated capsule, and evaluating the response of the nematode to the test substance.

[0148] A method for evaluating the response of nematodes according to aspect 14 of the present invention may be such that, in aspect 13 above, the step of evaluating the response of the nematodes to the test substance includes a step of measuring, after a certain period of time, the number of nematodes attracted to or escaping from the part, or a factor correlated with the number of nematodes.

[0149] In the method for evaluating a response of a nematode according to aspect 15 of the present invention, in aspect 13 or 14 above, the test object may be a test object that releases an odorant or a test object that has a taste.

[0150] A cancer testing method according to aspect 16 of the present invention includes the steps of evaluating the response of a nematode to a biological sample collected from a subject using the response evaluation method according to any one of aspects 13 to 15 above, and determining the subject's likelihood of cancer on the basis of the evaluation.

[0151] A method for evaluating the response of nematodes to temperature according to aspect 17 of the present invention comprises the steps of adjusting a portion of a nematode test plate to a desired temperature, supplying a nematode-encapsulated capsule according to any one of aspects 1 to 8 above to a predetermined position, releasing the nematodes from the nematode-encapsulated capsule, and, after a certain period of time, measuring the number of nematodes that have been attracted to or escaped from the portion, or a factor correlated with the number of nematodes.

[0152] A kit for evaluating a nematode response according to an eighteenth aspect of the present invention includes the nematode-encapsulating capsule according to any one of the first to eighth aspects.

[0153] The kit for evaluating a response to a nematode according to Aspect 19 of the present invention may be used in Aspect 18 above for cancer testing.

[0154] A manufacturing kit for producing a nematode encapsulation capsule according to aspect 20 of the present invention is a manufacturing kit for producing a nematode encapsulation capsule according to any one of aspects 1 to 4 above, and comprises a nematode, a water-soluble polymer, and a salt of a cation.

[0155] A manufacturing kit for producing a nematode encapsulation capsule according to aspect 21 of the present invention is a manufacturing kit for producing a nematode encapsulation capsule according to aspect 5 above, and includes irradiated nematodes, a water-soluble polymer, and a salt of a cation.

[0156] A manufacturing kit for producing a nematode encapsulation capsule according to aspect 22 of the present invention is a manufacturing kit for producing a nematode encapsulation capsule according to aspect 6 above, and includes nematodes treated with a hatching inhibitor, a water-soluble polymer, and a salt of a cation.

[0157] A manufacturing kit for producing nematode encapsulation capsules according to aspect 23 of the present invention is a manufacturing kit for producing nematode encapsulation capsules according to aspect 7 above, and comprises sterilized nematodes, a water-soluble polymer, and a salt of a cation.

[0158] A manufacturing kit for producing a nematode encapsulation capsule according to aspect 24 of the present invention is a manufacturing kit for producing a nematode encapsulation capsule according to aspect 8 above, and comprises a nematode, a water-soluble polymer, a cationic salt, and a hatching inhibitor.

[0159] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents cited in this specification are incorporated by reference. [Example]

[0160] In each of the following examples and comparative examples, hermaphrodites of C. elegans were used as nematodes. C. elegans were cultured and raised at the optimum temperature of 20°C. Table 1 shows the manufacturing conditions for the nematode encapsulation capsules, which differ for each example, including the developmental stage of the nematodes to be encapsulated, whether or not prior sterilization treatment was performed, the method for recovering the nematodes and the method for adding them to the aqueous sol, the type of buffer used for the nematode washing solution and nematode solution, and the presence or absence of food in the nematode encapsulation capsules.

[0161] [Table 1]

[0162] Below, examples are given regarding the manufacturing process of nematode-encapsulating capsules and the cultivation of nematodes within the capsules.

[0163] Example 1: Production of capsules containing nematode individuals (adults) A typical method for producing nematode encapsulation capsules according to one embodiment of the present invention involves dropping an aqueous sol of a calcium-coagulating polymer compound encapsulating nematodes into an aqueous solution of calcium salt to form a gel. In this example, adult nematode encapsulation capsules were produced using this method. A wash buffer was used to wash the nematodes. Furthermore, a 20°C wash buffer containing washed nematodes (eggs or individuals) was used as the nematode solution for injection into the aqueous sol. The wash buffer was prepared by autoclaving 0.5 g of gelatin powder in 993 mL of ultrapure water, followed by adding 1 mL of 1 M MgSO4, 5 mL of 1 M potassium phosphate (pH 6.0), and 1 mL of 1 M CaCl2, and stirring. The nematode encapsulation capsules produced in this example did not contain E. coli as food.

[0164] (Preparation of aqueous sol and calcium salt solution) First, sodium alginate (2 g) was dissolved in 200 mL of S-basal buffer, a common solution used in nematode culture or various tests, and the resulting solution was stirred to prepare an aqueous sol that would serve as the base material for the nematode encapsulation capsules. The S-basal buffer was prepared by dissolving NaCl (5.9 g) in 949 mL of ultrapure water, adding 1 mL of ethanol containing 5 mg of cholesterol, and sterilizing the mixture under pressure. Then, 50 mL of 1 M potassium phosphate (pH 6.0) was added and the mixture was stirred. Next, calcium lactate (2 g) was dissolved in 200 mL of ultrapure water and stirred to prepare the calcium salt solution used in the manufacturing process to gel the aqueous sol.

[0165] (Manufacturing capsules containing adult nematodes) First, wash buffer containing adult nematodes 3 days after hatching was added dropwise to the above aqueous sol to prepare an aqueous sol containing nematodes (hereinafter sometimes referred to as "nematode sol"). Next, the nematode sol was extracted using a micropipette or electric pipettor and added dropwise to a beaker filled with calcium lactate solution, which was then immersed for approximately 5 minutes. This produced adult nematode capsules containing encapsulated adult nematodes. Finally, the adult nematode capsules were removed from the calcium lactate solution, and the outer membrane of the capsules was washed with ultrapure water.

[0166] (Results of Example 1) Figure 1 shows an image of a spherical capsule containing approximately 100 adult nematodes, captured from above using a digital camera attached to a microscope. Figure 2 shows examples of spherical capsules containing nematodes (adults) formed into various sizes by adjusting the diameter of the tip of the electric pipettor or micropipette tip and the amount of nematode sol dispensed. Figure 3 also shows examples of capsules containing nematodes (adults) formed into arbitrary shapes by reducing the amount of calcium lactate solution or by dispensing the nematode sol directly above the surface of the calcium lactate solution.

[0167] As shown in Figure 1, adult nematodes are encapsulated within the capsules. Furthermore, as shown in Figure 2, it is clear that nematode-encapsulated capsules of various sizes can be freely produced by adjusting the amount of nematode sol dripped. Furthermore, it is clear that nematode-encapsulated capsules of any shape, such as teardrop- or gourd-shaped, can be produced either in a controlled or accidental manner, not limited to spherical shapes, as shown in Figure 3.

[0168] From the above, it is clear that a nematode-encapsulated capsule containing nematodes inside can be produced by dropping a nematode sol, which is an aqueous sol into which a nematode liquid has been injected, into a calcium salt solution and immersing the nematode sol.

[0169] Example 2: Production of capsules containing nematode eggs and cultivation of nematodes under low nutritional conditions Nematode egg capsules were produced by dropping an aqueous sol of a calcium-coagulating polymer compound containing nematode eggs into an aqueous solution of calcium salt, causing gelation. The nematode solution used for injection into the aqueous sol was a 20°C wash buffer containing nematodes (eggs or individuals) that had been washed with wash buffer. The nematode egg capsules produced here were not supplemented with Escherichia coli, which serves as food, and the survival status of the nematode egg capsules under low-nutrient conditions was continuously observed.

[0170] (Manufacturing capsules containing nematode eggs) First, eggs were collected from adult nematodes using a conventional method. Next, a wash buffer containing eggs was added dropwise to the same aqueous sol prepared in Example 1 to prepare an aqueous sol containing nematode eggs (hereinafter sometimes referred to as "egg sol"). Next, the nematode sol was sucked out using a micropipette and added dropwise to a calcium lactate solution filled in a beaker, where it was immersed for approximately 5 minutes. In this way, nematode egg-encapsulated capsules containing nematode eggs were produced. Finally, the nematode egg-encapsulated capsules were removed from the calcium lactate solution, and the outer membrane of the capsules was washed with ultrapure water.

[0171] (Results of Example 2) Figure 4 shows an image of a spherical capsule containing approximately 10 nematode eggs, captured from above using a digital camera attached to a microscope. As shown in Figure 4, nematode eggs are clearly encapsulated within the capsule. Based on the results of Example 1, it is clear that nematode-encapsulated capsules can be produced using the same method for both eggs and individuals. Although not shown, the nematodes within the capsule hatched and became L1 larvae one day later. However, in this example, in which E. coli was not added to the nematode egg sol as food, the nematodes lacked nutrients for growth. Therefore, when starved, the nematodes transitioned to resistant larvae, which temporarily cease developmental stages, and survived within the capsule for at least one month. This demonstrates that regulating the nutritional status within the capsule can actively maintain nematodes in the resistant larval state for long periods of time.

[0172] Example 3: Production of capsules enclosing nematode eggs and culturing nematodes in the presence of E. coli as food Using the same method as in Example 2, an aqueous sol of a calcium-coagulating polymer compound encapsulating nematode eggs was dropped into an aqueous solution of calcium salt to gel, thereby producing nematode capsules encapsulating eggs. The nematode solution used for injection into the aqueous sol was a 20°C wash buffer containing nematodes (eggs or individuals) that had been washed with wash buffer. Escherichia coli, which serves as food, was added to the nematode egg capsules produced here, and the survival status of the nematodes inside the nematode egg capsules under nutrient-rich conditions was continuously observed.

[0173] (Manufacturing capsules containing nematode eggs) As in Example 2, eggs were collected from adult nematodes three days after hatching, and the wash buffer containing the eggs was added dropwise to the same aqueous sol prepared in Example 1 to prepare a nematode egg sol. A suspension of Escherichia coli (E. coli) was then added to the egg sol. The nematode sol was then drawn up using a micropipette and added dropwise to a calcium lactate solution in a beaker, where it was immersed for approximately five minutes. This resulted in the production of nematode egg-encapsulated capsules containing nematode eggs and E. coli (E. coli) as food. Finally, the nematode egg-encapsulated capsules were removed from the calcium lactate solution, and the outer membrane of the capsules was washed with ultrapure water.

[0174] (Results of Example 3) Immediately after production, spherical capsules containing approximately 100 nematode eggs were photographed from above every 24 hours (1 day) using a digital camera attached to a microscope. The results from immediately after production to 2 days later are shown in Figure 5.

[0175] As shown in Figure 5, manufacturing Immediately afterwards, it was clear that nematode eggs were enclosed within the nematode-enclosing capsule. Furthermore, after one day, the nematode eggs hatched within the capsule and became larvae (L1), which continued to grow gradually over the next two days. Furthermore, although not shown in the figure, it was confirmed that after three days, the nematodes became adults, and after four days, egg-laying of the next generation began. In contrast to the results of Example 2, in which resistant larvae were produced due to low nutritional conditions, growth at a normal rate was observed in this example, demonstrating that it is possible to grow (cultivate) nematodes appropriately by adjusting the nutritional conditions within the egg capsule.

[0176] Furthermore, although not shown in the figure, capsules containing nematode eggs were similarly produced using M9 buffer, which is commonly used in nematode culture or various tests, instead of the S-basal buffer used in Example 3, and the results of observing them for several days confirmed that nematodes grow well from eggs to adults even when M9 buffer was used as the solution. M9 buffer was prepared by autoclaving a liquid (approximately 100 mL) prepared by dissolving NaCl (50 g), KH2PO4 (30 g), and Na2HPO4 (60 g) in ultrapure water, followed by adding 1 mL of 1 M MgSO4 and stirring. This solution was then mixed with sterilized ultrapure water. Water The mixture was diluted 10 times and stirred to prepare the solution.

[0177] Example 4: Preparation of capsules containing adult nematodes and culturing of nematodes in the presence of E. coli as food Adult nematode capsules were produced by adding an aqueous sol of a calcium-coagulating polymer compound containing 3-day-old hatched adult nematodes to an aqueous solution of calcium salts, causing gelation. The nematode solution used for injection into the aqueous sol was 20°C M9 buffer containing nematodes (adults) recovered from a culture plate using M9 buffer.

[0178] (Manufacturing capsules containing adult nematodes) Eggs were collected from adult nematodes and cultured on plates coated with E. coli until three days after hatching. Adult nematodes three days after hatching were collected from the culture plate using M9 buffer and added dropwise to the same aqueous sol prepared in Example 1 to produce adult nematode sol. Next, the nematode sol was drawn up using a micropipette and added dropwise to a calcium lactate solution filled in a plastic dish, where it was immersed for approximately five minutes. This produced adult nematode capsules containing adult nematodes and their food, E. coli. Finally, the adult nematode capsules were removed from the calcium lactate solution, and the outer membrane of the capsules was washed with ultrapure water.

[0179] (Results of Example 4) Spherical capsules containing approximately 10 adult nematodes three days after hatching were photographed from above every 24 hours (days) using a digital camera attached to a microscope. Immediately after production, images were taken and observed from the top. The results from immediately after production to two days later are shown in Figure 6. As shown in Figure 6, it can be seen that the adult nematodes inside the capsules began laying eggs immediately after production of the adult nematode-containing capsules. Furthermore, one day later, the nematode eggs hatched inside the capsules, and more than 100 larvae were observed. Two days later, the number of larvae further increased, filling the capsules. The hatching rate of the eggs laid by the adults inside the capsules was no different from that observed in normal culture on medium, confirming normal development. These findings demonstrate that adult oviposition and egg hatching are possible inside the capsules.

[0180] Next, nematodes grown in the nematode-encapsulated capsules were removed and their locomotion was confirmed. A commercially available circular plastic dish with an 8.4 cm inner diameter at the bottom surface was used as the container for the nematode test plate. 10 mL of agar-based solid medium was poured into the container and used as plate A1 (described below). A 2-day-old nematode-encapsulated capsule was transferred onto plate A1 with tweezers and poked multiple times with a sterilized toothpick to expose the interior of the capsule. Figure 7 shows nematodes immediately crawling out onto plate A1. Figure 7 confirms that the eggs laid by the adult nematode (parent) encapsulated during the nematode-encapsulated capsule manufacturing process hatched, and the larvae crawled out of the capsule and moved around normally on plate A1. It was also revealed that when the number of adult nematodes (parent) in the nematode-encapsulated capsule is large relative to the capsule size, a large number of the next generation (offspring) emerges, resulting in the weakening or death of the parent generation.

[0181] Although not shown in the figure, when the nematode-enclosed capsule was poked multiple times with a sterilized toothpick one day after production to expose the inside of the capsule, the adult nematodes (parents) encapsulated in the capsule crawled out along with the next generation of larvae. This result demonstrated that the adult nematodes can be maintained normally within one day after egg laying, when the majority of the next generation has progressed from the egg to the L1 larval stage.

[0182] In light of the above, in experiments in which the parent generation of nematodes encapsulated in nematode capsules is removed from the capsules and used, it is effective to adjust the capsule manufacturing conditions, such as the number of individuals to be encapsulated, the capsule size, and the number of days for encapsulation, as well as the culture conditions, so that the parent generation survives along with the next generation. Furthermore, methods for inhibiting egg hatching, as described below, can also be selected.

[0183] Example 5: Preparation of capsules containing irradiated adult nematodes and culturing of nematodes in the presence of E. coli as food An experiment was conducted to confirm the effectiveness of nematode encapsulation capsules containing sterilized nematodes irradiated with cobalt 60 ( 60C) Irradiated nematode capsules were produced by dropping an aqueous sol of a calcium-coagulating polymer compound containing gamma-irradiated adult nematodes 3 days after hatching into an aqueous solution of calcium salt and allowing it to gel. The nematodes used for injection into the aqueous sol were adult nematodes irradiated with cobalt-60 gamma rays while swimming in a tube containing M9 buffer, and the nematode solution used was immediately after irradiation.

[0184] (irradiation of nematodes) As in Example 4, eggs were collected from adult nematodes and cultured on plates coated with E. coli until three days after hatching. Three-day-old adult nematodes were collected from the culture plates in M9 buffer and suspended in a tube. The nematode suspension was then irradiated with 500 Gy of cobalt-60 gamma rays, which corresponds to the sterilization dose for hermaphrodites.

[0185] (Production of capsules containing irradiated adult nematodes) Immediately after irradiation, the gamma-irradiated nematode solution was added dropwise to the same aqueous sol prepared in Example 1 to prepare a sterilized nematode adult sol. The nematode sol was then extracted using a micropipette and added dropwise to a calcium lactate solution in a plastic dish, which was then immersed for approximately 5 minutes. This resulted in the production of irradiated nematode capsules containing irradiated adult nematodes and E. coli as food. Finally, the sterilized nematode capsules were removed from the calcium lactate solution, and the outer membrane of the capsules was washed with ultrapure water.

[0186] (Results of Example 5) Spherical capsules containing approximately 10 adult nematodes three days after hatching, immediately after exposure to gamma rays, were photographed from above every 24 hours (days) using a digital camera attached to a microscope and observed. The results from immediately after production until two days later are shown in Figure 8. As shown in Figure 8, it can be seen that the adult nematodes inside the capsules began laying eggs immediately after production of the nematode-encapsulated capsules. One and two days later, the adult nematodes could be seen moving inside the capsules, but the eggs they laid did not hatch and no larvae were observed.

[0187] Example 5 was carried out under the same conditions and procedures as Example 4, except that adult nematodes were irradiated with gamma rays before the production of nematode-encapsulating capsules. Taking the results of Example 4 into consideration, it is clear that sterilization (hatching inhibition) treatment by irradiation inhibits the development of the next generation, even when adult nematodes are encapsulated three days after hatching, when they begin to lay eggs, and the capsules can be maintained in a state where only the parent generation is present at the time of production.

[0188] Next, nematodes grown in the nematode-encapsulated capsules were removed and their locomotion was confirmed. As in Example 4, irradiated nematode-encapsulated capsules two days after production were transferred with tweezers onto plate A1 and poked multiple times with a sterilized toothpick to expose the interior of the capsule. Immediately after this, nematodes crawled out onto the surface of plate A1, as shown in Figure 9. It has been known that nematodes irradiated with 500 Gy of cobalt-60 gamma rays exhibit a temporary decrease in locomotion, but this recovers after one day. In this example, too, no significant abnormalities in locomotion were observed in adult (parent) nematodes encapsulated immediately after irradiation and allowed to escape from the nematode-encapsulated capsule two days later. These results clearly demonstrate that encapsulating adult nematodes irradiated with a dose equivalent to a sterilizing dose for about two days does not result in problems with locomotion.

[0189] Example 6: Preparation of capsules containing adult nematodes exposed to a hatching inhibitor and culturing of nematodes in the presence of E. coli as food

[0190] Hatching inhibitor-treated nematode capsules were produced by adding an aqueous sol of a calcium-coagulating polymer compound containing 4-day-old hatched adult nematodes treated with a hatching inhibitor to an aqueous solution of calcium salt and allowing it to gel. In this example, adult nematodes were collected one by one from the culture plate using a platinum wire picker, without using nematode liquid, and transferred to the aqueous sol together with E. coli.

[0191] (Treatment of nematodes with hatching inhibitors) Nematodes were cultured on plates coated with E. coli until they reached adulthood three days after hatching (before egg laying began). FUdR solution was added to these nematode culture plates, and the nematodes were cultured for an additional day. FUdR solution was prepared by dissolving 5 mg of FUdR powder in 10 mL of sterilized ultrapure water and stirring. Although some adult nematodes laid eggs on the FUdR-added culture plates, the eggs did not hatch.

[0192] (Production of capsules containing adult nematodes exposed to hatching inhibitors) Using a platinum wire picker, adult nematodes exposed to the hatching inhibitor for one day were collected from the culture plate on the fourth day after hatching along with E. coli. These were then dropped into the same aqueous sol prepared in Example 1 to produce a sterilized nematode adult sol. The nematode sol was then drawn out using a micropipette and dropped into a calcium lactate solution filled in a plastic dish, where it was immersed for approximately five minutes. This resulted in the production of a hatching inhibitor-treated nematode encapsulation capsule containing the hatching inhibitor-exposed adult nematodes and the E. coli they served as food for. Finally, the sterilized nematode encapsulation capsule was removed from the calcium lactate solution, and the outer membrane of the capsule was washed with ultrapure water.

[0193] (Results of Example 6) Spherical capsules containing approximately 10 adult nematodes on the fourth day after hatching, whose hatching had been inhibited by exposure to FUdR, were observed by taking images from above every 24 hours (per day) starting immediately after production using a digital camera attached to a microscope. The results from immediately after production to two days later are shown in Figure 10. As shown in Figure 10, it can be seen that the adult nematodes inside the capsules began laying eggs immediately after production of the nematode-encapsulated capsules. One and two days later, adult nematodes could be seen moving inside the capsules, but the eggs they laid did not hatch and no larvae were observed.

[0194] Next, the nematodes grown in the nematode-encapsulated capsules were removed and their locomotion was confirmed. As in Example 4, a hatching inhibitor-treated nematode-encapsulated capsule two days after production was transferred with tweezers onto plate A1 and poked multiple times with a sterilized toothpick to expose the inside of the capsule. Figure 11 shows the nematodes crawling out onto the surface of plate A1 immediately after this. No significant abnormalities in locomotion were observed in the adult (parent) nematodes that had escaped from the nematode-encapsulated capsule two days after production. From these results, it is clear that there are no problems with locomotor function even if adult nematodes exposed to a hatching inhibitor are encapsulated in a capsule for about two days.

[0195] As shown in Figures 10 and 11, in capsules containing adult nematodes exposed to a hatching inhibitor on day 4 after hatching (corresponding to the egg-laying stage), the development of the next generation was suppressed, and it was confirmed that only the parent generation individuals present at the time of capsule production could be maintained for at least two days. To confirm that the absence of the next generation, excluding eggs, was due to the effect of exposure to the hatching inhibitor, similar nematode-encapsulated capsules were produced using nematodes cultured for one day on a culture plate supplemented with M9 buffer instead of FUdR solution (M9 buffer containing FUdR). The control results are shown in Figure 12. As shown in Figure 12, as in Example 4 using adult nematodes on day 3 after hatching, the adults laid eggs in the capsules, and two days later, the capsules were filled with hatched larvae. From this, it is clear that the fact that, although the adults laid eggs in the capsules containing adult nematodes exposed to a hatching inhibitor, the eggs did not hatch and no larvae emerged was due to the effect of the hatching inhibitor exposure treatment.

[0196] Furthermore, although not shown in the figure, in Example 6 described below, it was confirmed that similar results to those obtained when nematode-encapsulated capsules were produced using nematodes cultured for one day in a culture medium containing FUdR, instead of culturing them for one day on a culture plate supplemented with FUdR solution, were obtained.

[0197] From the results of Examples 1 to 3 and 4 to 6 relating to the manufacturing process of nematode-encapsulated capsules and the cultivation of nematodes within the capsules, it is clear that nematode-encapsulated capsules can be manufactured even if the conditions for capsule manufacturing and nematode cultivation are varied in various ways, such as the developmental stage of the nematodes to be encapsulated, whether or not sterilization treatment is performed, the method for recovering nematodes from the culture plate, the method for adding nematodes to the aqueous sol, the type of nematode solution used for washing or culturing the nematodes, whether or not food is added to the capsules, etc. It was also confirmed that by adjusting the manufacturing conditions for nematode-encapsulated capsules or the cultivation conditions for nematodes depending on the intended use of the nematodes, it is possible to suppress the generation of the next generation, control the nutritional status, control the population, etc.

[0198] Below are examples of the preservation and soil biodegradation of nematode encapsulated capsules.

[0199] Example 7. Preservation of nematode-encapsulated capsules (Manufacturing capsules containing adult nematodes) Nematode sol was prepared using the method of Example 1, and a suspension of Escherichia coli (E. coli) was added as food. Next, the nematode sol was aspirated using a micropipette, and the nematode sol was dripped into a beaker filled with calcium lactate solution and immersed for approximately 5 minutes. This produced spherical nematode encapsulation capsules with a diameter of approximately 3 mm containing adult nematodes. The nematode sol was also aspirated using an electric pipettor, and the nematode sol was dripped into a beaker filled with calcium lactate solution and immersed for approximately 5 minutes. This produced spherical nematode encapsulation capsules with a diameter of approximately 5 mm containing adult nematodes. Finally, the nematode encapsulation capsules were removed from the calcium lactate solution, and the outer membrane of the capsules was washed with ultrapure water.

[0200] (Storage of nematode capsules) Approximately 30 washed spherical nematode capsules, each approximately 3 mm in diameter, were transferred to a plastic dish with a bottom diameter of approximately 2.8 cm. Approximately 30 washed spherical nematode capsules, each approximately 5 mm in diameter, were transferred to a plastic dish with a bottom diameter of 5.4 cm. Each plastic dish was wiped clean with a paper wiper, then covered with a lid and sealed with paraffin film.

[0201] (Results of Example 7) Figure 13 shows two examples of plastic dishes storing spherical nematode capsules of two different sizes. In practice, the nematode capsules are sealed with paraffin film for storage, but Figure 13 shows the plastic dishes before wrapping them in paraffin film so that the contents can be clearly seen. The nematodes in the nematode capsules stored in the plastic dishes sealed with paraffin film were still viable in good condition after three days. This demonstrates that the storage method of this example does not adversely affect the survival of the nematodes in the nematode capsules.

[0202] Furthermore, it was found that by storing more nematode-encapsulated capsules in the same plastic dish than in the example shown in Figure 13 and storing them in a denser state, it was possible to prevent the capsules from rolling over due to vibrations during transportation.

[0203] Furthermore, as another method, the impact of vibrations on the nematodes during transportation can be reduced by using a nematode-encapsulating capsule that is not spherical but has one or more flat surfaces, or by filling the gaps with liquid.

[0204] Example 8. Biodegradation of nematode capsules in soil It is possible to gradually reduce the population of pest nematodes that cause severe damage to crops by sterilizing the pest nematodes or their antagonistic nematodes and then returning them to the soil. By encapsulating the target nematodes and spreading them, it is expected that the effects of improving work efficiency and slowing the rate at which nematodes migrate into the soil can be expected. In this example, the biodegradability of the nematode-encapsulated capsules in soil was examined to evaluate their environmental compatibility.

[0205] (Soil affinity evaluation test of nematode-encapsulated capsules) Spherical nematode capsules with a diameter of 2 to 3 mm containing adult nematodes 3 days after hatching were produced using the method of Example 4. A commercially available circular plastic dish with a bottom diameter of 5.4 cm was filled with commercially available leaf mold and moistened with water, after which approximately 50 nematode capsules were scattered over it, the dish was capped, and the dish was left to stand at 20°C.

[0206] (Results of Example 8) The condition of the nematode capsules on the leaf mold spread in a plastic dish was observed every 24 hours (1 day) starting immediately after spreading, with the lid removed. Figure 14 shows the results immediately after spreading, and after 1, 2, and 5 days. As shown in Figure 14, the nematode capsules spread on the leaf mold immediately after production (nematode encapsulation) were gradually assimilated into the leaf mold after 1 and 2 days. After 5 days, the biodegradation of the outer membrane had progressed to the point where the shape of the nematode capsules could no longer be seen. When a portion of the leaf mold was removed and observed under a microscope, multiple nematodes were observed moving around among the fragments of the nematode capsules mixed with the leaf mold.

[0207] From the above, it can be said that nematode-encapsulated capsules are a simple and efficient tool for transferring any nematode into soil. Furthermore, because the capsules are made of highly biodegradable materials, they do not place a burden on the environment, and therefore are clearly of great value in the agricultural, forestry, and fisheries sectors.

[0208] Below are examples and comparative examples relating to the nematode supply process and nematode response evaluation tests. In each example and comparative example, a commercially available circular plastic dish with an 8.4 cm inner diameter at the bottom surface was used as the container for the nematode test plate. 10 mL of agar-based solid medium was poured into the container and used as plate A1. Plate A2 was also used, in which one recess was created as the nematode supply area by hollowing out the solid medium of plate A1, and plate A3 was also used, in which two recesses were created on the left and right sides of the solid medium of plate A1 to form four recesses that served as nematode drop-in tanks. The recess that served as the nematode supply area was a cylindrical recess with an inner diameter of 5 mm centered on the center of the solid medium, and the recesses that served as nematode drop-in tanks were both cylindrical recesses with an inner diameter of 5 mm. Details of the nematode test plates used in the examples are shown in Table 2. Plate A3 is identical to plate A1 described in Patent Document 2.

[0209] [Table 2]

[0210] Example 9: Study of nematode supplying process The nematode supply process using a nematode-encapsulated capsule according to one embodiment of the present invention was evaluated using plate A1 into which 10 mL of solid medium was poured and plate A2, which similarly had a single depression in the center that served as a supply area for pouring solid medium and supplying nematodes.

[0211] First, a capsule containing adult nematodes prepared in the same manner as in Example 1 was clamped with tweezers and placed in the supply area of ​​each of plates A1 and A2, i.e., the center of plate A1 and the center recess of plate A2. In both plates, the nematode-encapsulated capsule was poked multiple times with a sterilized toothpick to expose the inside of the capsule. This caused nematodes to crawl out around the supply area of ​​plates A1 and A2. The liquid components contained in the capsule were sucked up with a micropipette. Immediately after the nematode-encapsulated capsule was added, images of an approximately 1.4 cm square area centered on the supply area of ​​plates A1 and A2 were taken from above using a digital camera attached to a microscope every minute to observe the nematode diffusion on the solid medium surface. The nematode diffusion on the solid medium surface was observed.

[0212] (Results of Example 9) The results of sequential observations of the areas around the supply area of ​​plates A1 and A2 are shown in Figures 15 and 16. Each figure shows, from left to right, images immediately after supplying the nematode-enclosed capsules and at the time when almost all of the nematodes had crawled out of the capsules onto the plate. The black dot in the center of the image marks the center of the supply area.

[0213] 15 and 16, when nematode-encapsulated capsules were supplied, nematodes crawled out within 12 minutes when plates A1 and A2 were used. Furthermore, when plate A2 was used, nematodes crawled out after 3 minutes, which demonstrated that the efficiency of nematode supply was increased by combining the method of supplying nematodes to the recesses (a nematode supply process using a supply-portion-processed plate) with the nematode-encapsulated capsules of the present invention.

[0214] Furthermore, when poking and breaking the nematode capsules with a toothpick, the nematode capsules on plate A2, which had been placed on the exposed bottom of the plate with a recess, were less likely to roll and easier to poke than the capsules on plate A1, which had been placed on the agar. Plate A2 also had the advantage that the solution containing nematodes that leaked out of the nematode capsules was confined within the recess, allowing the starting point of the nematode migration to be controlled within a certain range.

[0215] [Comparative Example 1. Supply of nematodes by conventional method] Plate A1 was filled with 10 mL of solid medium, and a well in the center was similarly filled with solid medium and provided with nematodes. Setting Using the bay plate A2 (a nematode feeding step using a feeding part-processed plate), a nematode feeding step according to a conventionally known method was evaluated.

[0216] First, the nematode solution containing washed adult nematodes was aspirated using a micropipette, and a droplet containing approximately 100 adult nematodes was dispensed into the supply area of ​​each nematode test plate. The liquid components of the nematode solution on plate A1 were removed using a conventional method, i.e., by wiping with a paper wiper. The liquid components of the nematode solution in the recesses of plate A2 were removed by suction using a micropipette. Immediately after dispensing the nematode solution, images of approximately 1.4 cm squares centered on the supply area of ​​plates A1 and A2 were taken from above every minute using a digital camera attached to a microscope to observe the spreading of nematodes on the solid medium surface.

[0217] (Results of Comparative Example 1) The results of sequential observations of the areas around the supply area of ​​plates A1 and A2 are shown in Figures 17 and 18. Each figure shows, from left to right, images taken immediately after supplying the nematode solution and at the time when almost all of the nematodes had crawled out onto the nematode test plate. The black dot in the center of the image marks the center of the supply area.

[0218] 17 and 18, when nematode liquid was supplied, the nematodes completed diffusion within 6 minutes when plates A1 and A2 were used. Comparing the results of nematode supply in Example 9 using the nematode-encapsulated capsules of the present invention with the results of this comparative example, there was no significant difference between the two, demonstrating that the nematode-encapsulated capsules of the present invention can be used as an alternative to conventional nematode supply methods.

[0219] Example 10: Test to evaluate the response of nematodes to volatile substances using nematode-encapsulated capsules A test to evaluate the response of nematodes to volatile substances was conducted using diacetyl (molar concentration 11.5 M at 15°C), a type of volatile substance, and dilutions of diacetyl diluted in two stages, 10-fold and 10 to the power of 5. The test method used was a method using a nematode trap plate.

[0220] First, plate A3 was prepared by placing two drop-in tanks side by side along the periphery on the left side of the solid medium of plate A1, the same as used in Example 1, and similarly placing two drop-in tanks in a control position on the right side. The left drop-in tank was filled with the liquid to be tested (hereinafter sometimes referred to as the "test liquid"), and the right drop-in tank was filled with a control buffer solution. As mentioned above, the test liquid in this example was a diluted solution of diacetyl, a type of volatile substance. A control buffer solution was used for dilution.

[0221] Next, several nematode-encapsulated capsules containing nematode eggs and grown to adulthood were poked with the tip of a micropipette to break the outer membrane, and the solution containing the nematodes was aspirated from the capsules and dropped onto the center of plate A3. Similarly, the same amount of solution containing nematodes aspirated from several of the same capsules was dropped onto plate A3, which was used for simultaneous experiments with different concentrations of test solution. The solution was then wiped off with a paper wiper, releasing the nematodes onto the plate. The plate was then covered with a lid and sealed with paraffin film and left to stand for at least 1 hour in a light-protected environment, after which the number of nematodes captured in the drop tank was counted.

[0222] (Results of Example 10) The total number of nematodes captured in each of the two drop-down tanks on the left filled with the test solution was defined as N1, and the total number of nematodes captured in each of the two drop-down tanks on the right filled with the control buffer solution was defined as N2.The value calculated by the following formula was defined as the chemotaxis index (CI) and the response was evaluated. Chemotaxis index (CI)=(N1-N2) / (N1+N2) A positive value indicates that the nematodes were attracted to the left drop tank, i.e., diacetyl, and a negative value indicates that the nematodes escaped from the drop tank. The test was conducted using three dilutions of diacetyl as the test solution, for a total of three concentrations. The average CI values ​​for two independent tests are shown in Figure 19.

[0223] As shown in Figure 19, the 10-fold dilution gave a negative value, indicating that the nematodes avoided diacetyl, while the 10^5 dilution gave a positive value, indicating that the nematodes were attracted to diacetyl.

[0224] Comparative Example 2: Test to evaluate the response of nematodes to volatile substances using a conventional nematode supply method As a comparative example, a nematode response evaluation was performed based on a conventionally known nematode supply method. The same plate A3 as used in Example 10, which had two drop tanks formed at symmetrical positions on the left and right, was used, and the test solution was a two-stage dilution of diacetyl, as in Example 10. A control buffer solution was used for dilution.

[0225] The nematode solution containing nematodes pre-washed with wash buffer was dropped onto the center of a nematode test plate, and the solution was wiped off with a paper wiper to release the nematodes onto the plate. The plate was then covered with a lid and sealed with paraffin film, and left to stand for at least 1 hour in a dark environment, after which the number of nematodes trapped in the drop tank was counted.

[0226] (Results of Comparative Example 2) The average CI values ​​for all three concentrations of the test solution, obtained from three independent tests, are shown in Figure 20. As shown in Figure 20, the 10-fold diluted solution produced negative values, indicating that the nematodes avoided diacetyl. On the other hand, the 10^5 diluted solution produced positive values, indicating that the nematodes were attracted to diacetyl.

[0227] The results of Example 10, in which a test was conducted using a nematode-encapsulated capsule supply method, and Comparative Example 2, in which a test was conducted using a conventional nematode supply method, were in good agreement. This demonstrates that nematode response evaluation tests can be conducted without problems using nematodes that have been encapsulated at the egg stage and grown to adulthood. [Industrial Applicability]

[0228] The present invention solves potential problems related to the preparation and supply of nematodes in various fields, including microbiology experiments, cell culture experiments, cancer testing using nematodes, space experiments, pest control, education, research, and industry. It is expected that the present invention will greatly contribute to improving the efficiency and reducing the cost of various biological experiments.

Claims

1. An encapsulated composition comprising a nematode (excluding only entomopathogenic nematodes (EPN)), and at least one coating layer containing the encapsulated composition; The nematode-encapsulating capsule has a coating containing, as a main component, a water-soluble polymer that gels upon reaction with a cation.

2. 2. The nematode-encapsulating capsule according to claim 1, having an outer diameter of 10 cm or less.

3. A nematode-encapsulated capsule as described in claim 1, wherein the nematode is a non-parasitic nematode.

4. The nematode encapsulation capsule of claim 1 , wherein the nematode is Caenorhabditis elegans or a closely related species thereof.

5. The nematode encapsulation capsule according to claim 1 , wherein the water-soluble polymer is at least one water-soluble polymer selected from the group consisting of proteins and polysaccharides.

6. A method for producing a nematode-containing encapsulated composition, comprising: an encapsulated composition containing a nematode; and at least one coating layer containing the encapsulated composition, the coating contains, as a main component, a water-soluble polymer that gels upon reaction with cations; A nematode encapsulation capsule, wherein the nematode is an irradiated nematode.

7. A method for producing a nematode-containing encapsulated composition, comprising: an encapsulated composition containing a nematode; and at least one coating layer containing the encapsulated composition, the coating contains, as a main component, a water-soluble polymer that gels upon reaction with cations; The nematode-encapsulating capsule is a nematode that has been treated with a hatching inhibitor, and the eggs laid by the treated generation of the nematode are in a state where they cannot hatch.

8. A method for producing a nematode-containing encapsulated composition, comprising: an encapsulated composition containing a nematode; and at least one coating layer containing the encapsulated composition, the coating contains, as a main component, a water-soluble polymer that gels upon reaction with cations; A nematode-encapsulating capsule, wherein the nematode is a nematode that has been sterilized, and the sterilization treatment is a treatment that suppresses the generation of the next generation.

9. A nematode-encapsulating capsule as described in claim 8, wherein the sterilized nematode is a sterilized disease-causing nematode or a sterilized predatory nematode of the disease-causing nematode.

10. The nematode encapsulation capsule of claim 1 , wherein the encapsulation composition further comprises a hatching inhibitor.

11. mixing the nematode and the water-soluble polymer to prepare an encapsulation composition; The method for producing a nematode encapsulation capsule according to any one of claims 1 to 10, comprising the step of reacting the encapsulation composition with a solution containing the cation to produce a nematode encapsulation capsule.

12. a step of reacting a solution containing the water-soluble polymer with a solution containing the cation to prepare a base for a coating of a nematode-encapsulating capsule; The method for producing a nematode encapsulation capsule according to any one of claims 3, 4 and 6 to 9, comprising the step of producing a nematode encapsulation capsule by injecting the nematode into the base.

13. The method of claim 12, further comprising: mixing the nematode and the water-soluble polymer to prepare an encapsulation composition; reacting the encapsulation composition with a solution containing the cation to produce a nematode encapsulation capsule; The method for producing a nematode encapsulating capsule according to any one of claims 3, 4 and 6 to 9, further comprising the step of irradiating the nematode encapsulating capsule with radiation.

14. The method for producing a nematode encapsulating capsule according to claim 12, further comprising the step of irradiating the nematode encapsulating capsule with radiation.

15. providing a test article to a portion of the nematode test plate; A step of supplying a nematode-encapsulating capsule according to any one of claims 3, 4, and 6 to 9 to a predetermined position; Releasing the nematodes from the nematode encapsulation capsule; and evaluating the response of the nematode to the test substance.

16. The response evaluation method of claim 15, wherein the step of evaluating the response of the nematodes to the test substance includes a step of measuring, after a certain period of time, the number of nematodes attracted to or escaping from the portion, or a factor correlated with the number of nematodes.

17. The response evaluation method according to claim 15, wherein the test object is an odorant-releasing test object or a taste-producing test object.

18. A step of evaluating the response of a nematode to a biological sample collected from a subject by the response evaluation method according to claim 15; and determining the subject's likelihood of cancer by said evaluation.

19. adjusting a portion of the nematode test plate to a desired temperature; A step of supplying a nematode-encapsulating capsule according to any one of claims 3, 4, and 6 to 9 to a predetermined position; Releasing the nematodes from the nematode encapsulation capsule; A method for evaluating the response of nematodes to temperature, comprising a step of measuring, after a certain period of time, the number of nematodes attracted to or escaping from the portion or a factor correlated with the number of nematodes.

20. A kit for evaluating nematode response, comprising the nematode-encapsulating capsule according to any one of claims 3, 4 and 6 to 9.

21. The kit for evaluating a nematode response according to claim 20, which is used for cancer testing.

22. A production kit for producing the nematode encapsulation capsule according to any one of claims 1 to 5, A manufacturing kit comprising a nematode, a water-soluble polymer, and a salt of a cation.

23. A production kit for producing the nematode encapsulation capsule according to claim 6, comprising: A manufacturing kit comprising an irradiated nematode, a water-soluble polymer, and a salt of a cation.

24. A production kit for producing the nematode encapsulation capsule according to claim 7, comprising: A manufacturing kit comprising nematodes treated with a hatching inhibitor, a water-soluble polymer, and a salt of a cation.

25. A production kit for producing the nematode encapsulation capsule according to claim 8 or 9, A manufacturing kit comprising sterilized nematodes, a water-soluble polymer, and a salt of a cation.

26. A production kit for producing the nematode encapsulation capsule according to claim 10, comprising: A manufacturing kit comprising a nematode, a water-soluble polymer, a cationic salt, and a hatching inhibitor.

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