Composition for culturing nematodes, culture kit, and screening method for candidate compound using nematodes

Abstract

To provide a novel culture composition and a novel culture kit that can culture nematodes, and to provide a novel screening method for a candidate compound using nematodes that can evaluate effects of the candidate compound.SOLUTION: The present invention provides a composition for culturing nematodes, comprising a nematode culture medium containing dead bacteria and / or dead fungi and formic acid. The present invention also provides a kit for culturing nematode, comprising dead bacteria and / or dead fungi, formic acid, and a nematode culture medium. The present invention also provides a screening method for a candidate compound using nematodes, the method comprising: (1) bringing a candidate compound into contact with nematodes in a nematode culture medium containing dead bacteria and / or dead fungi and formic acid, and culturing the nematodes; and (2) evaluating effects of the candidate compound on the nematodes using any phenotype of the nematodes cultured in the step (1) as an indicator.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition for culturing nematodes, a culture kit, and a method for screening candidate compounds using nematodes. [Background technology]

[0002] Nematodes are a general term for animals belonging to the phylum Nematoda, with thin, filamentous bodies that are essentially colorless and transparent and lack a segmented body structure. They live in soil and water, and although over 20,000 species have been identified to date, it is believed that many more species (e.g., over 1 million species) exist.

[0003] Nematodes are known to include both parasitic and free-living species. Caenorhabditis elegans (C. elegans) belongs to the free-living nematode family and is known as a model organism for multicellular organisms. The development and differentiation processes of all cells from fertilized eggs to adults have been elucidated as cell lineages. C. elegans is approximately 1 mm long and feeds on a variety of bacterial species found in the soil. C. elegans matures from fertilized eggs to adults in a short time (approximately 3 days) and is hermaphrodite, making genetic analysis easy and leading to its use in numerous research studies. The entire genome of C. elegans has also been determined, and because the types and number of genes contained therein are similar to those of humans in many respects, it is also being used to analyze genes related to human diseases (Non-Patent Document 1).

[0004] C. elegans are easy to culture and can be grown in large quantities on agar media, solid media on multi-well plates, or liquid media, allowing them to be used in a variety of assays. Methods using C. elegans to screen for compounds that can be used to treat human diseases have been attempted (Non-Patent Document 2). However, there are problems such as candidate compounds being taken up by the Escherichia coli bacteria used as food, resulting in the candidate compounds being metabolized by the bacteria or a decrease in the concentration of the candidate compounds, which may prevent the effects of the candidate compounds from being properly evaluated (Non-Patent Documents 3-5).

[0005] When nematodes were fed E. coli that had been killed by heat treatment to prevent the uptake and metabolism of candidate compounds by the E. coli that served as food, problems arose, such as the cessation of development at the early larval stage (L1-L2) (Non-Patent Documents 6-7). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Shohei Mitani, Comprehensive functional genomics using Caenorhabditis elegans as a model organism, Proc Jpn Acad Ser B Phys Biol Sci. 2017;93(8):561-577. [Non-patent document 2] Ai Ito, et al., Metolazone upregulates mitochondrial chaperones and extends lifespan in Caenorhabditis elegans, Biogerontology. 2021 Feb;22(1):119-131. [Non-patent document 3] Luxia Yao, et al., The antidiabetic drug metformin aids bacteria in hijacking vitamin B12 from the environment through RcdA, Commun Biol. 2023 Jan 24;6(1):96. [Non-patent document 4] Filipe Cabreiro, et al., Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism, Cell. 2013 Mar 28;153(1):228-39. [Non-patent document 5] Martina Klunemann, et al., Bioaccumulation of therapeutic drugs by human gut bacteria, Nature. 2021 Sep;597(7877):533-538. [Non-patent document 6] Bin Qi, et al., A vitamin-B2-sensing mechanism that regulates gut protease activity to impact animal's food behavior and growth, Elife. 2017 Jun 1:6:e26243. [Non-Patent Document 7] Bin Qi, et al., Microbial Siderophore Enterobactin Promotes Mitochondrial Iron Uptake and Development of the Host via Interaction with ATP Synthase, Cell. 2018 Oct 4;175(2):571-582.e11 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a novel culture composition and culture kit for culturing nematodes, and a novel screening method for candidate compounds using nematodes that allows for more accurate evaluation of the effects of candidate compounds. [Means for solving the problem]

[0008] The present inventors have conducted research and development in an effort to solve the above-mentioned problems, making extensive investigations from various angles. As a result, they have surprisingly found that when nematodes are cultured in a nematode rearing medium containing killed bacteria and / or killed fungi and formic acid, the nematodes do not stop developing at the early larval stage (L1-L2) but grow to adulthood, thereby completing the present invention. That is, the present invention encompasses the following inventions.

[0009] [1] A composition for culturing nematodes, comprising a nematode rearing medium containing dead bacteria and / or dead fungi and formic acid. [2] The composition for culturing nematodes according to item 1, further comprising glycerol. [3] The composition for culturing nematodes described in item 1 or 2, wherein the nematode is Caenorhabditis elegans (C. elegans). [4] The composition for culturing nematodes according to any one of items 1 to 3, wherein the dead bacteria and / or dead fungi are killed Escherichia coli. [5] The composition for culturing nematodes according to any one of items 1 to 4, wherein the dead bacteria and / or dead fungi are dead bacteria and / or dead fungi that have been killed by heat treatment and / or ultraviolet irradiation treatment. [6] A composition for culturing nematodes according to any one of items 1 to 5, for use in a method for screening for a candidate compound.

[0010] [7] A nematode culture kit, Dead bacteria and / or dead fungi Formic acid, and Nematode rearing medium A nematode culture kit including: [8] The nematode culture kit according to Item 7, further comprising glycerol. [9] The nematode culture kit according to item 7 or 8, wherein the nematode is Caenorhabditis elegans (C. elegans).

[10] The nematode culture kit according to any one of items 7 to 9, wherein the dead bacteria and / or dead fungi are killed Escherichia coli.

[11] The nematode culture kit according to any one of items 7 to 10, wherein the dead bacteria and / or dead fungi are killed by heat treatment and / or ultraviolet irradiation treatment.

[12] A nematode culture kit according to any one of items 7 to 11, for use in a method for screening a candidate compound.

[0011]

[13] A method for screening a candidate compound using a nematode, comprising: (1) contacting and culturing a candidate compound with nematodes in a nematode rearing medium containing killed bacteria and / or killed fungi and formic acid; and (2) evaluating the effect of the candidate compound on the nematode cultured in the step (1) using any phenotype of the nematode as an index; A method comprising:

[14] (3) The method according to Item 13, further comprising the step of selecting the candidate compound exhibiting a desired effect from the evaluation results of the step (2).

[15] The method according to Item 13 or 14, wherein the nematode is a nematode having a mutation in any gene.

[16] The method according to any one of items 13 to 15, wherein the nematode rearing medium in step (1) further contains glycerol.

[17] The method according to any one of items 13 to 16, wherein the nematode is Caenorhabditis elegans (C. elegans).

[18] The method according to any one of items 13 to 17, wherein the dead bacteria and / or dead fungi are killed Escherichia coli.

[19] The method according to any one of items 13 to 18, wherein the dead bacteria and / or dead fungi are dead bacteria and / or dead fungi that have been killed by heat treatment and / or ultraviolet irradiation treatment. [Effects of the Invention]

[0012] The present invention provides a composition for nematode culture and a nematode culture kit that can prevent the uptake and metabolism of a candidate compound by bacteria or fungi added as nematode food and allow the nematodes to grow to adulthood, thereby providing a new screening method using nematodes that can more accurately evaluate the effects of candidate compounds. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of Example 1, in which a compound was searched for that has the effect of enabling nematodes (C. elegans N2 strain), whose growth stops at the L1 larval stage when heat-killed Escherichia coli is added as food, to grow to adulthood. [Figure 2] FIG. 2 shows the results of Example 2, which confirmed the effect of adding formic acid on nematodes (C. elegans N2 strain) when they were cultured using as food Escherichia coli that had been killed by heating or ultraviolet (UV) irradiation. [Figure 3] FIG. 3 shows the results of Example 3, which confirmed the effect of adding formic acid and glycerol on nematodes (C. elegans N2 strain) when they were cultured using E. coli killed by heat treatment as food. [Figure 4] FIG. 4 shows the results of Example 3, which confirmed the effect of adding formic acid and glycerol on nematodes (C. elegans N2 strain) when they were cultured using E. coli killed by heat treatment as food. [Figure 5] FIG. 5 shows the results of Example 3, which investigated the effects of adding formic acid and tetrahydrofolic acid (THF) to nematodes (C. elegans N2 strain) whose growth stops at the L1 larval stage when heat-killed Escherichia coli is added as food. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, embodiments of the present invention will be described with reference to the contents of the examples and drawings as necessary. The configurations of the embodiments are illustrative, and the configurations of the present invention are not limited to the specific configurations of the embodiments. All documents cited in this specification are incorporated herein by reference in their entirety.

[0015] In one embodiment, the present invention provides a composition for culturing nematodes, comprising a nematode rearing medium containing killed bacteria and / or fungi and formic acid. The composition for culturing nematodes of the present invention may be used in a method for screening for candidate compounds. In another aspect, the composition for culturing nematodes of the present invention may further comprise glycerol.

[0016] In one embodiment, the present invention provides a nematode culture kit, comprising: Dead bacteria and / or dead fungi Formic acid, and Nematode rearing medium The present invention provides a culture kit comprising the above. For example, the kit may contain packaged killed bacteria and / or killed fungi, formic acid (or a salt thereof), and nematode rearing medium, and may further include an instruction manual for use with the kit. The kit may also further contain glycerol. The nematode culture kit of the present invention may be used in a method for screening for a candidate compound.

[0017] In one embodiment, the present invention provides a method for screening a candidate compound using a nematode, comprising: (1) contacting and culturing a candidate compound with nematodes in a nematode rearing medium containing killed bacteria and / or killed fungi and formic acid; and (2) evaluating the effect of the candidate compound on the nematode cultured in the step (1) using any phenotype of the nematode as an index; The present invention provides a method comprising:

[0018] In another embodiment, in the method of the present invention, the nematode rearing medium in the step (1) may further contain glycerol.

[0019] In yet another embodiment, the method of the present invention further comprises: (3) A step of selecting the candidate compound that exhibits the desired effect based on the evaluation results of the step (2).

[0020] Nematodes are a general term for animals belonging to the phylum Nematoda. They have a thin, filamentous body, are essentially colorless and transparent, and lack a segmented structure. Parasitic or free-living species are known. Caenorhabditis elegans (C. elegans) is preferred as a nematode applicable to the present invention, as it is used as a model organism for multicellular organisms. The entire genome of C. elegans has been determined, and the development and differentiation processes of all cells from fertilized eggs to adults have been elucidated as cell lineages. C. elegans has the advantage of being easy to conduct genetic analysis because it takes only about three days to mature from a fertilized egg to an adult and is hermaphrodite. Furthermore, genetic engineering methods for generating C. elegans mutants have been established, making it possible to obtain C. elegans mutants with mutations in desired genes, and these mutants can be applied to the present invention. In one embodiment, the phenotype of a C. elegans mutant can be used as an indicator to select the candidate compound exhibiting the desired effect.

[0021] The developmental process of C. elegans is broadly divided into the embryonic stage from fertilization to hatching, the larval stage from L1 to L4, and the adult stage where reproduction occurs. The larval stages from L1 to L4 are separated by molting. If C. elegans grows normally, a new generation will occur in about three days, but if there is a shortage of food during the L1 larval stage, the developmental pattern will change and the dauer larva will become dauer larvae, which can survive for about six months without food.

[0022] Hatched L1 larvae grow to adulthood through repeated molting when suitable food is available, but it has been reported that growth stops at the L1 stage when only heat-killed E. coli is available as food (Qi and Han, Cell, 2018 (Non-Patent Document 7)). After extensive research, the inventors of the present application discovered that adding formic acid to dead bacteria and / or dead fungi as food can cause the nematode C. elegans to grow from L1 larvae to adults.

[0023] In one embodiment of the present invention, the nematode may be a nematode having a mutation in any gene. The nematode having a mutation in any gene may be a nematode in which any gene has been modified by any genetic engineering technique, such as a transgenic nematode, a mutant knock-in nematode, or a gene knockout nematode. As used herein, "mutation" refers to an amino acid substitution, deletion, or insertion at any position in the amino acid sequence of a protein encoded by a gene. The mutated protein exhibits a different function from the wild-type protein, or does not exhibit that function, resulting in a phenotype different from that of the wild-type nematode (e.g., growth arrest at any larval stage (L1, L2, L3, or L4)). In one embodiment of the present invention, the nematode may be a nematode having a mutation (preferably a knockout mutation) in a gene orthologous to a human disease susceptibility gene. Screening of candidate compounds using nematodes having a mutation in such a gene results in a change in the nematode's phenotype (e.g., reversion to the wild-type phenotype), which can be used as an indicator to select candidate compounds effective for treating a disease.

[0024] A known nematode rearing medium can be used as the medium for rearing (culturing) nematodes. For example, a solid medium, such as NGM (Nematode Growth Medium) agar medium, or an agar-free liquid medium can be used. A standard NGM agar medium has the following composition, but the composition is not limited to the following, as long as it is capable of culturing nematodes.

[0025] <Standard NGM agar medium> 0.30% Sodium Chloride 0.25% Tryptone 1.7% Agar 5 μg / mL cholesterol 1mM Calcium Chloride 1mM Magnesium Sulfate 25mM Potassium Phosphate

[0026] NGM agar plates may be prepared according to known methods (e.g., Brenner (1974)), or may be purchased from, for example, Teknova or Genelinx International Inc. (trade name bioWORLD), and any plate capable of culturing nematodes may be used.

[0027] As used herein, "dead bacteria and / or dead fungi" refers to bacteria and / or fungi that can be used as food for nematodes and that have ceased to function. In the present invention, the use of dead bacteria and / or dead fungi prevents the candidate compound from being metabolized by the bacteria and / or fungi, or from being taken up by the bacteria and / or fungi, which would reduce the concentration of the candidate compound. This allows for accurate evaluation of the effect of the candidate compound on nematodes.

[0028] In one embodiment of the present invention, the "dead bacteria and / or dead fungi" may be bacteria and / or fungi that can be used as food for nematodes and are in a state where their vital activity has ceased. Applicable bacteria and / or fungi include, but are not limited to, bacteria of the genus Bacillus (e.g., Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis, etc.), bacteria of the genus Comamonas, Escherichia coli, Micrococcus luteus, bacteria of the genus Pseudomonas (e.g., Pantoea dispersa, etc.) (see Lesley MacNeil, et al., Worm 2:4, e26454; October / November / December 2013), Bacillus subtilis, lactic acid bacteria, fungi, etc. It is preferable to use Escherichia coli, which is particularly commonly used for culturing (raising) nematodes. There are no limitations on the strain of Escherichia coli that can be used, but for example, the OP50 strain may be used for culturing C. elegans.

[0029] The method for preparing "killed bacteria and / or fungi" from bacteria and / or fungi is not particularly limited, and may be, for example, heat treatment, ultraviolet irradiation treatment, radiation irradiation treatment (e.g., gamma ray irradiation, electron beam irradiation), gas sterilization treatment (e.g., ethylene oxide gas (EOG) sterilization), or disinfectant treatment (e.g., treatment with alcohol, sodium hypochlorite aqueous solution, etc.), with heat treatment, ultraviolet irradiation treatment, or radiation irradiation treatment being preferred. The duration or intensity of each treatment is not particularly limited as long as it is sufficient to stop (inactivate) the vital activity of the bacteria and / or fungi. For example, when heat treatment is applied, treatment may be performed at 70°C to 130°C for approximately 5 to 180 minutes. For example, when ultraviolet irradiation treatment is applied, treatment may be performed using an apparatus that irradiates with a light source having a wavelength of around 260 nm (e.g., approximately 240 nm to 290 nm), at which the absorption spectrum of DNA peaks.

[0030] In the present invention, the number of dead bacteria and / or dead fungi applied is not limited, as long as it is the amount necessary for the survival of nematodes. For example, when killed Escherichia coli are used as the dead bacteria and / or dead fungi, they may be inactivated after full growth in LB medium and applied to the nematode rearing medium. The dead bacteria and / or dead fungi may be applied to the nematode rearing medium, for example, by mixing them with the nematode rearing medium, or by applying a suspension containing the dead bacteria and / or dead fungi to the surface of an agar medium provided as a solid medium.

[0031] The inventors of the present application have discovered that the growth inhibition of nematodes caused by the addition of dead bacteria and / or dead fungi as food, or the growth delay or development caused by nutritional deficiency, can be restored by adding formic acid to the nematode rearing medium.

[0032] Furthermore, it was found that the addition of glycerol and / or tetrahydrofolic acid (THF) to the nematode rearing medium in addition to formic acid can improve the growth inhibition of nematodes caused by the addition of dead bacteria and / or dead fungi as food, or the growth retardation or development due to nutrient deficiency. More preferably, a combination of formic acid and glycerol is used, and adding these to the nematode rearing medium more effectively improves the growth inhibition of nematodes caused by the addition of dead bacteria and / or dead fungi as food, or the growth retardation or development due to nutrient deficiency.

[0033] Formic acid and tetrahydrofolic acid (THF) applicable to the present invention may be in the form of a pharmaceutically acceptable salt, and such pharmaceutically acceptable salts are included within the scope of the present invention. Examples of pharmaceutically acceptable salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, tartrate, citrate, fumarate, maleate, toluenesulfonate, and methanesulfonate; metal salts such as sodium salt, potassium salt, calcium salt, and aluminum salt; and salts with bases such as triethylamine salt, guanidine salt, ammonium salt, hydrazine salt, quinine salt, and cinchonine salt.

[0034] Formic acid, glycerol, and tetrahydrofolic acid (THF) applicable to the present invention may be added to a nematode rearing medium in an amount effective to prevent growth inhibition of nematodes caused by dead bacteria and / or dead fungi added as feed, or to recover growth retardation or development due to nutrient deficiency. Formic acid may be added at a concentration of, for example, 0.01 μM to 10 μM, preferably 0.05 μM to 5 μM, or 0.1 μM to 1 μM. Glycerol may be added at a concentration of, for example, 0.001 μM to 1 μM, preferably 0.01 μM to 0.5 μM, or 0.05 μM to 0.1 μM. Tetrahydrofolic acid (THF) may be added at a concentration of, for example, 0.01 μM to 1000 μM, preferably 0.1 μM to 100 μM. In addition to the additives described above, the nematode rearing medium of the present invention may also contain a component that promotes nematode growth.

[0035] As used herein, the term "candidate compound" may be, for example, an organic compound (a low molecular weight compound), a peptide, a protein, a nucleic acid (DNA, RNA), a tissue extract or cell culture supernatant of a mammal (e.g., mouse, rat, pig, cow, sheep, monkey, human, etc.), a compound or extract derived from a plant (e.g., a herbal extract, a compound derived from a herbal drug), and a compound or extract derived from a microorganism, or a culture product.

[0036] The step of evaluating the effect of a candidate compound on a nematode using any phenotype of the nematode as an indicator may be carried out by any known method, for example, by using an optical microscope to evaluate the morphological characteristics of the nematode (body length, width, etc.) as an indicator, or by using a system to detect the expression of a reporter gene (e.g., a gene expressing lacZ, green fluorescent protein, luciferase, acetohydroxyacid synthase, alkaline phosphatase, β-glucuronidase, chloramphenicol acetyltransferase, horseradish peroxidase, nopaline synthase, or octapine synthase) genetically engineered to correlate with the expression of a desired protein. [Example]

[0037] The present invention will now be described in more detail with reference to examples. The examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention can be modified, for example, by adding, deleting, or substituting constituent elements of the present invention, provided that the modifications do not depart from the spirit of the present invention.

[0038] In this example, we identified the nutrients necessary for further growth of L1 larvae by adding heat-treated E. coli and various compounds known to be involved in human metabolic pathways to the nematode culture medium.

[0039] [Experimental Method] In the examples, the experiments were basically carried out according to the following experimental method.

[0040] [1] C. elegans culture C. elegans were cultured according to known methods, for example, the methods described in WormBook - The Online Review of C. elegans Biology - (http: / / www.wormbook.org), edited by The C. elegans Research Community (see in particular the section "Maintenance of C. elegans (2006)" in WormBook).

[0041] C. elegans (wild-type N2 strain) was cultured at 20°C on Nematode Growth Medium (NGM) agar plates using E. coli OP50 as food, according to the method of Brenner (1974) (Genetics. 1974 May;77(1):71-94.). The alkaline bleach method was used to synchronize the nematodes. After culturing the nematodes to adulthood on NGM agar plates, they were washed with M9 buffer, collected, and suspended in an Eppendorf tube. The nematode suspension was mixed with an equal volume of bleach solution (a mixture of 4N NaOH, hypochlorous acid solution, and M9 buffer (2:3:5)). The mixture was centrifuged at 400xg and the supernatant was discarded. The nematodes were then ruptured, and only the fertilized eggs were collected. The collected fertilized eggs were inoculated onto NGM agar medium containing killed E. coli and the test compound, and cultured at 20° C. The day the eggs were collected was designated as day 0 of culture.

[0042] [2] Preparation of heat-killed E. coli A 20 ml suspension of E. coli OP50 strain grown to full growth in LB liquid medium was centrifuged, diluted 5-fold, and heat-killed in a hot water bath at 75°C for 90 minutes to prepare heat-killed E. coli. 40 μl of the heat-killed E. coli suspension was dropped onto a 35 mm diameter plate containing pre-prepared NGM agar medium. The plate surface was allowed to dry, and then a test compound was added and allowed to dry before use in nematode culture.

[0043] [3] Preparation of ultraviolet (UV) irradiation sterilization of E. coli UV irradiation of E. coli was performed according to the method described by Cabreiro et al. (Cell. 2013;153:228-239 (Non-Patent Document 4)). 20 ml of E. coli suspension, in which E. coli OP50 strain was grown to full growth in LB liquid medium, was centrifuged and diluted 5-fold to prepare a suspension. 40 μl of the E. coli suspension was dropped onto a 35 mm diameter plate containing pre-prepared NGM agar medium and cultured overnight at 20°C. The plate was then irradiated with UV light for 30 minutes using a UV Stratalinker 2400 (Stratagene) equipped with a bulb emitting light at a wavelength of 254 nm. Subsequently, a test compound was added, and the plate was allowed to dry before being used for nematode culture.

[0044] [Example 1] C. elegans N2 strain nematodes were cultured with heat-killed E. coli OP50 (heat-killed E. coli) and the test compounds formic acid and glycerol at final concentrations of 1 μM and 0.1 μM, respectively. The results are shown in Figure 1. When fed live E. coli, N2 nematodes grew to adulthood within 3–4 days (4 days of culture, Figure 1(A)). On the other hand, when fed only heat-killed E. coli, N2 nematodes stopped growing at the L1 stage (10 days of culture, Figure 1(B)). N2 nematodes fed heat-killed E. coli with formic acid and glycerol grew to adulthood within 12 ± 2 days of culture and remained fertile (10 days of culture, Figure 1(D)). The N2 strain of nematodes was able to grow to adulthood even when only formic acid was added to heat-killed E. coli (after 17 days of culture, Figure 1(C)).

[0045] [Example 2] Heat-killed and UV-irradiated E. coli strains were prepared using E. coli OP50 as described above. The effect of adding formic acid on the growth of the C. elegans N2 strain was examined, using culture with killed E. coli alone as a control. Whether killed by UV irradiation or heat, N2 strain worms reared on killed E. coli failed to develop into L2 larvae during the 17-day culture period (Figures 2(A) and (D) after 17 days of culture). In contrast, the addition of formic acid enabled N2 strain worms to progress beyond the L2 larval stage and reach adulthood (Figures 2(B), (C), (E), and (F)). Whether formic acid concentrations were 0.1 μM or 1 μM, N2 strain worms developed into adults and produced self-fertilized eggs over the 17-day culture period. The growth-promoting effect of formic acid on nematodes was not affected by the method of killing E. coli.

[0046] [Example 3] As shown in Example 2, adding formic acid to dead E. coli enabled the nematode C. elegans to develop beyond the L2 larval stage, but the growth rate was slower than when the nematode was raised on live E. coli. We searched for compounds that could increase the growth rate of the nematode C. elegans in combination with formic acid, which promotes development.

[0047] The growth of the N2 strain of nematodes was observed when glycerol was added to a combination of heat-killed E. coli and 1 μM formic acid (Figure 3). The combination of heat-killed E. coli and glycerol alone prevented larval development beyond the L2 stage and resulted in no growth to adulthood (Figure 3(B) and (C)). However, the addition of glycerol to the combination of heat-killed E. coli and formic acid was able to accelerate the growth rate of the N2 strain of nematodes. Figure 3 shows the results after 7 days of culture. In particular, the addition of glycerol at concentrations of 0.05 μM to 0.1 μM demonstrated a significant growth-promoting effect on the N2 strain of nematodes (Figure 3(E) and (F)).

[0048] The optimal concentration of formic acid was investigated for the combination of heat-killed E. coli, formic acid, and glycerol (Figure 4). When the formic acid concentration was varied under the condition of a glycerol concentration of 0.1 μM, the growth-promoting effect of the N2 strain of nematodes was observed across the entire range of formic acid concentrations tested. In particular, significant growth of the N2 strain of nematodes was observed at formic acid concentrations ranging from 0.1 to 1 μM (Figures 4(C) and (D)). Figure 4 shows the results after 7 days of culture.

[0049] We observed the growth of the N2 strain of C. elegans by adding tetrahydrofolic acid (THF) instead of glycerol to heat-killed E. coli alone or to a combination of heat-killed E. coli and formic acid (Figure 5). Adding THF (1 μM or 10 μM) to heat-killed E. coli alone did not allow C. elegans to develop beyond the L2 larval stage (Figures 5(B) and (C)). When THF was added to a combination of heat-killed E. coli and formic acid (1 μM), the N2 strain of C. elegans progressed beyond the L1 larval stage, but did not reach the fertile developmental stage. Figures 5(E) and (F), respectively, show C. elegans cultured for 7 days with heat-killed E. coli and formic acid plus 1 μM or 10 μM THF.

[0050] [Example 4] Screening compounds from a drug library using C. elegans reared on killed Escherichia coli

[0051] Eggs are collected from adult C. elegans N2 strain (wild-type) or mutant worms using the alkaline bleach method. The collected eggs are washed in M9 buffer and plated on NGM agar plates containing killed E. coli, formic acid (1 μM), and glycerol (0.1 μM). They are then cultured at 20°C until they reach a developmental stage appropriate for screening, from L2 larvae to adults. After recovering them in M9 buffer, they are plated at a density of approximately 10–15 eggs per well on 96-well NGM agar plates containing killed E. coli, formic acid, and glycerol. Candidate compounds from a drug library (e.g., the Validated Compound Library (Drug Discovery Institute, University of Tokyo) including the Prestwick Chemical Library (Prestwick Chemical)) and DMSO as a control are added to each well, and the desired phenotype is observed. Subsequently, candidate compounds added to worms exhibiting the desired phenotype are selected.

Claims

1. A composition for culturing nematodes, comprising a nematode rearing medium containing dead bacteria and / or dead fungi and formic acid.

2. The composition for culturing nematodes according to claim 1 , further comprising glycerol.

3. The composition for culturing nematodes according to claim 1 , wherein the nematode is Caenorhabditis elegans (C. elegans).

4. The composition for culturing nematodes according to claim 1 , wherein the dead bacteria and / or dead fungi are killed Escherichia coli.

5. 2. The composition for culturing nematodes according to claim 1, wherein the dead bacteria and / or dead fungi are killed by heat treatment and / or ultraviolet irradiation treatment.

6. The composition for culturing nematodes according to claim 1, which is used in a method for screening for a candidate compound.

7. A nematode culture kit, comprising: Dead bacteria and / or dead fungi Formic acid, and Nematode rearing medium A nematode culture kit including:

8. The nematode culture kit according to claim 7, further comprising glycerol.

9. The nematode culture kit according to claim 7, wherein the nematode is Caenorhabditis elegans (C. elegans).

10. The nematode culture kit according to claim 7 , wherein the dead bacteria and / or dead fungi are killed Escherichia coli.

11. 8. The nematode culture kit according to claim 7, wherein the dead bacteria and / or dead fungi are killed by heat treatment and / or ultraviolet irradiation treatment.

12. The nematode culture kit according to claim 7, for use in a method for screening a candidate compound.

13. A method for screening a candidate compound using a nematode, comprising: (1) contacting and culturing a candidate compound with nematodes in a nematode rearing medium containing killed bacteria and / or killed fungi and formic acid; and (2) evaluating the effect of the candidate compound on the nematode cultured in the step (1) using any phenotype of the nematode as an index; A method comprising:

14. (3) The method according to claim 13, further comprising the step of selecting the candidate compound that exhibits a desired effect from the evaluation results of the step (2).

15. The method according to claim 13 , wherein the nematode is a nematode having a mutation in any gene.

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