Parasitic lumbar flagellates belonging to amebic genus

Abstract

It is an object of the present invention to provide a parasitic lumbar flagellum belonging to the Amoeborphrya genus, which is capable of effectively preventing red tide derived from a lumbar flagellum belonging to the Karenia genus. The present invention provides an amebic parasitic lumbar flagellate having the ability to parasitize Karenia lumbar flagellate, a composition and a microbial pesticide each containing the amebic parasitic lumbar flagellate, a method for obtaining the amebic parasitic lumbar flagellate, a method for obtaining the amebic parasitic lumbar flagellate, and a method for obtaining the amebic parasitic lumbar flagellate. The invention also provides a method for inhibiting the proliferation of the Karenia lumbar flagellates.

Description

Technical Field

[0001] The present invention relates to Amoeba Amoebophrya ) is a parasitic dinoflagellate. Background Art

[0002] Red tides caused by excessive growth of plankton are known to cause hypoxia in seawater and cause great damage to aquaculture. One type of plankton that causes red tides is Karenia ( Karenia ) are dinoflagellates, and damage caused by plankton has been documented in coastal areas throughout the world.

[0003] NPL 1 reported that Flavobacterium spp. was isolated from Ise Bay. Flavobacterium sp.) has an algalytic effect on Karenia dinoflagellates. However, since Flavobacterium is a bacterium, it has not been used in practice as a red tide control agent due to its potential impact on organisms other than Karenia dinoflagellates. So far, no effective method has been found to eliminate Karenia dinoflagellates.

[0004] Reference List Non-patent literature NPL 1: Nippon Suisan Gakkaishi 70(4), 537-541 (2004) Summary of the invention

[0005] Technical issues One object of the present invention is to provide Amoeba ( Amoebophrya ) is a parasitic flagellate that is effective in preventing the spread of bacteria from Karenia ( Karenia ) is a red tide of the genus Dinoflagellate.

[0006] Solution To solve the above problems, the present inventors conducted extensive studies and found that specific parasitic dinoflagellates of the genus Amoeba have the ability to parasitize and kill dinoflagellates of the genus Karenia.

[0007] The present invention has been accomplished based on this discovery, and includes the broad aspects of the subject matter described below.

[0008] Item 1 A parasitic dinoflagellate of the genus Amoeba having the ability to parasitize dinoflagellates of the genus Karenia.

[0009] Item 2 A parasitic flagellate of the genus Amoeba, comprising one of the following nucleic acid molecules (1) to (12): (1) a nucleic acid molecule containing the base sequence of SEQ ID NO: 1, (2) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 1 are replaced, added or deleted, (3) a nucleic acid molecule comprising a base sequence having at least 90% identity with the base sequence of SEQ ID NO: 1, (4) a nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO: 1, (5) a nucleic acid molecule containing the base sequence of SEQ ID NO: 2, (6) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 2 are replaced, added or deleted, (7) a nucleic acid molecule comprising a base sequence that is at least 90% identical to the base sequence of SEQ ID NO: 2, (8) a nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO: 2, (9) a nucleic acid molecule containing the base sequence of SEQ ID NO: 3, (10) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 3 are replaced, added or deleted, (11) a nucleic acid molecule comprising a base sequence that is at least 90% identical to the base sequence of SEQ ID NO: 3, and (12) A nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO: 3.

[0010] Item 3 The parasitic dinoflagellate according to item 1, which has the ability to kill dinoflagellates of the genus Karenia.

[0011] Item 4 The parasitic dinoflagellate according to Item 1, which has the ability to inhibit the proliferation of dinoflagellates of the genus Karenia.

[0012] Item 5 A composition comprising the parasitic dinoflagellate of any one of items 1 to 4.

[0013] Item 6 The composition according to Item 5, which is used for preventing red tide.

[0014] Item 7 A microbial pesticide comprising the parasitic flagellate described in any one of items 1 to 4.

[0015] Item 8 A method for obtaining parasitic dinoflagellates of the genus Amoeba comprises the following steps: inoculating dinoflagellates of the genus Karenia infected with parasitic dinoflagellates of the genus Amoeba into a culture medium in which uninfected dinoflagellates of the genus Karenia are cultured.

[0016] Item 9 A parasitic dinoflagellate of the genus Amoeba obtained according to the method of item 8.

[0017] Item 10 A method for inhibiting the proliferation of Karenia algae comprises the following steps: making parasitic dinoflagellates of Amoeba parasitize on dinoflagellates of Karenia.

[0018] Beneficial Effects of the Invention The present invention provides a parasitic dinoflagellate of the genus Amoeba, which has the ability to parasitize the dinoflagellate of the genus Karenia, a microbial pesticide using the parasitic dinoflagellate and a method for preventing red tide using the parasitic dinoflagellate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The amoeba species ( Amoebophrya sp.) OSK2020 (a parasitic flagellate of the genus Amoeba) parasitizes Karenia mikimotoi ( Karenia mikimotoi ) (a dinoflagellate of the genus Karenia). Figure 1 The scale bars in the figure represent 20 μm. Figure 1 A: Image of uninfected Karenia mikimotoi observed using a light microscope. Figure 1 B: Image of uninfected Karenia mikimotoi observed using a fluorescence microscope. Figure 1 C: Image of Karenia mikimotoi at an early stage of infection observed using an optical microscope. Figure 1 D: Image of Karenia mikimotoi at the early stage of infection observed using a fluorescence microscope. Figure 1 E: Image of Karenia mikimotoi at mid-stage of infection observed using a light microscope. Figure 1 F: Image of Karenia mikimotoi at the middle stage of infection observed using a fluorescence microscope. Figure 1 G: Image of Karenia mikimotoi at a late stage of infection observed using an optical microscope. Figure 1 H: Image of Karenia mikimotoi at the late stage of infection observed using a fluorescence microscope. Figure 1 I: Images observed using an optical microscope when Karenia mikimotoi was destroyed and OSK2020 was released. Figure 1 J: Images observed using a fluorescence microscope when Karenia mikimotoi was destroyed and OSK2020 was released.

[0020] Figure 2A The results of an experiment in which the amoeba species OSK2020 in Example 1 was used to infect Karenina mikimotoi are shown. Figure 2A The graph shows the change in cell density of Karenia mikimotoi over time when the amoeba species OSK2020 was added to the culture medium of Karenia mikimotoi from 0 hours after the start of culture (treatment group) and when the amoeba species OSK2020 was not added (control group).

[0021] Figure 2B The results of an experiment in which the amoeba species OSK2020 in Example 1 was used to infect Karenina mikimotoi are shown. Figure 2B The infection rate (line graph) and infection status (bar graph) of Karenia mikimotoi when the amoeba species OSK2020 was added to the culture medium of Karenia mikimotoi from 0 hours after the start of culture are shown.

[0022] Figure 2C The results of an experiment in which the amoeba species OSK2020 in Example 1 was used to infect Karenia mikimotoi are shown. Figure 2C The graph shows the change in cell density of the amoeba species OSK2020 over time when the amoeba species OSK2020 was added to the culture medium of Karenia mikimotoi from 0 hours after the start of culture.

[0023] Figure 3 Results of molecular phylogenetic analysis of parasitic organisms by using nuclear 18S rDNA are shown.

[0024] Figure 4 Results of molecular phylogenetic analysis of parasitic organisms by using nuclear 28S rDNA are shown.

[0025] Figure 5 The number of days that the host survived when the host infected with the parasite was stored at low temperatures (14°C, 12°C and 10°C) in Example 7 (control group: 20°C). When the infected host was stored at an optimal temperature of about 20°C, the number of infected host cells decreased over the days, and the infected host cells completely disappeared after a period of time (10 to 20 days). However, when the infected host was stored at a low temperature, the number of days before its demise was extended.

[0026] Figure 6 In Example 8, it was shown that when the host infected with the parasite was exposed to low light intensity (20 μmol photons m -2 s -1 and 5 μmol photons m -2 s -1 ) (control group: 100 μmol photons m -2 s -1). When the infected host is exposed to about 100 μmol photons m -2 s -1 When stored under low light intensity, the number of infected host cells decreased over the days, and the infected host cells completely disappeared after a period of time (10 to 20 days). However, when the infected hosts were stored under low light intensity, the number of days before their demise was extended to a maximum of 90 days. DETAILED DESCRIPTION

[0027] In the present specification, singular nouns (having "a", "an", "the", etc.) include both the singular and the plural, unless otherwise clearly stated in the present specification or if an obvious contradiction is confirmed in the context. In the specification, the term "comprising" is a concept encompassing essentially consisting of and consisting of.

[0028] In one embodiment, the present invention provides Amoeba ( Amoebophrya ) is a parasitic flagellate of the genus Amoeba. In this embodiment, the parasitic flagellate of the genus Amoeba of the present invention has a parasitic Karenia ) is the ability of dinoflagellates.

[0029] Examples of Karenia dinoflagellates include Karenia mikimotoi ( Karenia mikimotoi )、 Karenia papilionacea and Karenia selliformis , preferably Karenia mikimotoi and Karenia papilionacea .

[0030] As used in this specification, "having the ability to parasitize" means that parasitism is confirmed in at least one cell of the Karenia dinoflagellates in the co-culture of the parasitic dinoflagellates of the genus Amoeba and the dinoflagellates of the genus Karenia. For example, according to the method described in the Examples given below, it can be confirmed whether the Karenia dinoflagellates are infected (parasitized) by the dinoflagellates of the genus Amoeba.

[0031] The conditions for co-cultivation are not particularly limited. For example, a common culture medium for culturing marine microalgae is used for the culture medium. A specific example is Daigo's IMK medium (manufactured by Nippon Pharmaceutical Co., Ltd.). The culture temperature is, for example, but not limited to, 15° C. to 30° C., preferably 20° C. to 25° C. The light intensity is, for example, but not particularly limited to, 10 μmol photons m -2 s -1 Up to 500 μmol photons m -2 s -1 , preferably 50 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1The light-dark cycle is, for example, but not particularly limited to, the following: 20 hours of light and 4 hours of darkness to 4 hours of light and 20 hours of darkness; preferably, the following: 12 hours of light and 12 hours of darkness to 14 hours of light and 10 hours of darkness. The concentration of the Karenia dinoflagellates at the start of co-culture is also not particularly limited; the concentration of the Karenia dinoflagellates is, for example, 500 cells mL -1 Up to 3000 cells mL -1 , preferably 1000 cells mL -1 Up to 2000 cells mL -1 The concentration of parasitic dinoflagellates of the genus Amoeba is also not particularly limited. For example, the concentration of parasitic dinoflagellates of the genus Amoeba may be 5000 cells mL -1 Up to 30,000 cells mL -1 , preferably 10000 cells mL -1 Up to 20,000 cells mL -1 .

[0032] When the parasitic dinoflagellates of the genus Amoeba of the present invention are co-cultured with the dinoflagellates of the genus Karenia (for example, co-cultured for 12 hours or longer according to the method described in Example 1), the infection rate of the dinoflagellates of the genus Karenia is not particularly limited as long as it exceeds 0%; however, the infection rate is preferably 10% or higher, more preferably 50% or higher, and even more preferably 90% or higher.

[0033] The parasitic dinoflagellate of the genus Amoeba of the present invention has the ability to inhibit the proliferation of dinoflagellate of the genus Karenia when co-cultured with dinoflagellate of the genus Karenia. This is because the parasitic dinoflagellate of the genus Amoeba of the present invention has the ability to kill the dinoflagellate of the genus Karenia after parasitizing the dinoflagellate of the genus Karenia. In the co-culture of the parasitic dinoflagellate of the genus Amoeba of the present invention and the dinoflagellate of the genus Karenia (for example, co-cultured for 12 hours or more according to the method described in Example 1), the parasitic dinoflagellate of the genus Amoeba of the present invention is preferably reduced by 10% or more, more preferably 50% or more, and even more preferably 90% or more of the dinoflagellate of the genus Karenia compared to before the co-culture.

[0034] The parasitic dinoflagellates of the genus Amoeba of the present invention have the ability to kill the dinoflagellates of the genus Karenia after parasitizing the dinoflagellates of the genus Karenia. In the co-cultivation of the parasitic dinoflagellates of the genus Amoeba of the present invention and the dinoflagellates of the genus Karenia (for example, co-cultivation for 12 hours or more according to the method described in Example 1), the parasitic dinoflagellates of the genus Amoeba of the present invention preferably kill 10% or more, more preferably 50% or more, and even more preferably 90% or more of the dinoflagellates of the genus Karenia.

[0035] Furthermore, when the parasitic dinoflagellates of the genus Amoeba of the present invention are sprayed onto the sea surface or seawater, for example as a microbial pesticide, it is preferred that the parasitic dinoflagellates of the genus Amoeba of the present invention have the ability to specifically parasitize dinoflagellates of the genus Karenia without worrying about infecting an unknown number of organisms, and have a high degree of safety. Specifically, when the parasitic dinoflagellates of the genus Amoeba of the present invention are co-cultured with an organism other than dinoflagellates of the genus Karenia, it is preferred that no infection is confirmed in the organism.

[0036] The parasitic flagellate of the genus Amoeba of the present invention preferably has one of the following nucleic acid molecules (1) to (12): (1) a nucleic acid molecule containing the base sequence of SEQ ID NO: 1, (2) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 1 are replaced, added or deleted, (3) a nucleic acid molecule comprising a base sequence having at least 90% identity with the base sequence of SEQ ID NO: 1, (4) a nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO: 1, (5) a nucleic acid molecule containing the base sequence of SEQ ID NO: 2, (6) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 2 are replaced, added or deleted, (7) a nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence that is complementary to the base sequence of SEQ ID NO: 2, (8) containing a base sequence that hybridizes under stringent conditions to a base sequence that is complementary to the base sequence of SEQ ID NO: 2, (9) a nucleic acid molecule containing the base sequence of SEQ ID NO: 3, (10) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 3 are replaced, added or deleted, (11) a nucleic acid molecule comprising a base sequence that is at least 90% identical to the base sequence of SEQ ID NO: 3, and (12) A nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO: 3.

[0037] In the present specification, the phrases "containing the base sequence" and "containing the base sequence" include the aspect in which the base sequence is included as a part of the full-length base sequence to the extent that the ability to parasitize Karenia dinoflagellates is not impaired, and the aspect in which the full-length base sequence is substantially composed of the base sequence.

[0038] In the above (1), the nucleic acid molecule includes a nucleic acid molecule containing the base sequence of SEQ ID NO: 1, so that the parasitic flagellate of the genus Amoeba having the nucleic acid molecule can have the ability to parasitize the flagellate of the genus Karenia.

[0039] In the above (2), the number of one or more bases to be replaced, deleted, added or inserted is not particularly limited, as long as the number is an integer greater than 1. For example, the number of the one or more bases may be 1 to about several tens, preferably about 1 to 30, more preferably about 1 to 15, even more preferably about 1 to 10, and particularly preferably about 1 to 5. The nucleic acid molecule includes a nucleic acid molecule containing a base sequence in which one or more bases in the base sequence of SEQ ID NO: 1 are replaced, added or deleted, so that the parasitic flagellate of the genus Amoeba having the nucleic acid molecule can have the ability to parasitize the flagellate of the genus Karenia.

[0040] In one aspect, the number of one or more bases to be replaced, added or deleted can be about 1 to 174, so that the identity of the base sequence to the base sequence of SEQ ID NO: 1 is at least 90%, preferably about 1 to 87, so that the identity is at least 95%, more preferably about 1 to 35, so that the identity is at least 98%, and particularly preferably about 1 to 17, so that the identity is at least 99%.

[0041] In the above (3), the identity of the base sequence may be at least 90%, preferably at least 95%, more preferably at least 98%, and particularly preferably at least 99%. The homology or identity of the base sequence may be less than 100%. The homology or identity between base sequences can be determined using a known algorithm, such as BLAST. The nucleic acid molecule includes a nucleic acid molecule containing a base sequence having at least 90% identity with the base sequence of SEQ ID NO: 1, so that the parasitic flagellate of the genus Amoeba having the nucleic acid molecule can have the ability to parasitize the flagellate of the genus Karenia.

[0042] In (4), (8) and (12) above, the phrase "stringent conditions" refers to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. An example of such conditions is hybridization in 1×SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6×SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA∙2Na, pH 7.4) at 42°C, followed by washing with 0.5×SSC at 42°C. However, stringent conditions are not limited to this example. For example, such conditions are described in, for example, MR Green et al., Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press (2012).

[0043] In the above (5), the nucleic acid molecule includes a nucleic acid molecule containing the base sequence of SEQ ID NO: 2, so that the parasitic flagellate of the genus Amoeba having the nucleic acid molecule can have the ability to parasitize the flagellate of the genus Karenia.

[0044] In the above (6), the number of one or more bases to be replaced, deleted, added or inserted is not particularly limited, as long as the number is an integer greater than 1. For example, the number of the one or more bases may be about 1 to several tens, preferably about 1 to 30, more preferably about 1 to 15, even more preferably about 1 to 10, and particularly preferably about 1 to 5. The nucleic acid molecule includes a nucleic acid molecule containing a base sequence in which one or more bases in the base sequence of SEQ ID NO: 2 are replaced, added or deleted, so that the parasitic flagellate of the genus Amoeba having the nucleic acid molecule can have the ability to parasitize the flagellate of the genus Karenia.

[0045] In one aspect, the number of one or more bases to be replaced, added or deleted can be about 1 to 76, so that the identity of the base sequence to the base sequence of SEQ ID NO: 2 is at least 90%, preferably about 1 to 38, so that the identity is at least 95%, more preferably about 1 to 15, so that the identity is at least 98%, and particularly preferably about 1 to 8, so that the identity is at least 99%.

[0046] In the above (7), the identity of the base sequence may be at least 90%, preferably at least 95%, more preferably at least 98%, and particularly preferably at least 99%. The homology or identity between the base sequences may be less than 100%. The homology or identity between the base sequences can be determined using a known algorithm, such as BLAST. The nucleic acid molecule includes a nucleic acid molecule containing a base sequence having at least 90% identity with the base sequence of SEQ ID NO: 2, so that the Amoeba parasitic flagellate having the nucleic acid molecule can have the ability to parasitize the Karenia flagellate.

[0047] In the above (9), the nucleic acid molecule includes a nucleic acid molecule containing the base sequence of SEQ ID NO: 3, so that the parasitic flagellate of the genus Amoeba having the nucleic acid molecule can have the ability to parasitize the flagellate of the genus Karenia.

[0048] In the above (10), the number of one or more bases to be replaced, deleted, added or inserted is not particularly limited, as long as the number is an integer greater than 1. For example, the number of the one or more bases may be about 1 to several tens, preferably about 1 to 30, more preferably about 1 to 15, even more preferably about 1 to 10, and particularly preferably about 1 to 5. The nucleic acid molecule includes a nucleic acid molecule containing a base sequence in which one or more bases in the base sequence of SEQ ID NO: 3 are replaced, added or deleted, so that the parasitic flagellate of the genus Amoeba having the nucleic acid molecule can have the ability to parasitize the flagellate of the genus Karenia.

[0049] In one aspect, the number of one or more bases to be replaced, added or deleted can be about 1 to 76, so that the identity of the base sequence to the base sequence of SEQ ID NO: 3 is at least 90%, preferably about 1 to 38, so that the identity is at least 95%, more preferably about 1 to 15, so that the identity is at least 98%, and particularly preferably about 1 to 8, so that the identity is at least 99%.

[0050] In the above (11), the identity of the base sequence may be at least 90%, preferably at least 95%, more preferably at least 98%, and particularly preferably at least 99%. The homology or identity between the base sequences may be less than 100%. The homology or identity between the base sequences can be determined using a known algorithm, such as BLAST. The nucleic acid molecule includes a nucleic acid molecule containing a base sequence having at least 90% identity with the base sequence of SEQ ID NO: 3, so that the parasitic flagellate of the genus Amoeba having the nucleic acid molecule can have the ability to parasitize the flagellate of the genus Karenia.

[0051] In the parasitic flagellate of the genus Amoeba having any one of the nucleic acid molecules (1) to (4) of the present invention, the nucleic acid molecule preferably also satisfies any one of (5) to (8).

[0052] Examples of such parasitic dinoflagellates of the genus Amoeba include Amoeba species ( Amoebophrya sp.) OSK2020 and amoeba species ( Amoebophrya sp.) HMN2020. The amoeba species OSK2020 has a nucleic acid molecule containing the base sequence of SEQ ID NO: 1 (18S rDNA, 1742 bases) and the base sequence of SEQ ID NO: 2 (28S rDNA, 759 bases). The amoeba species HMN2020 has a nucleic acid molecule containing the base sequence of SEQ ID NO: 3 (28S rDNA, 760 bases).

[0053] The cultivation of the parasitic flagellates of the genus Amoeba of the present invention can be carried out by, but not limited to, for example, co-cultivation with flagellates of the genus Karenia.

[0054] When the method for culturing the parasitic dinoflagellate of the genus Amoeba of the present invention includes the step of co-culturing with the dinoflagellate of the genus Karenia, the parasitic dinoflagellate of the genus Amoeba can be cultured, for example, according to the following steps.

[0055] (a) The parasitic dinoflagellate of the genus Amoeba of the present invention is inoculated into a culture medium in which dinoflagellates of the genus Karenia that are not infected (uninfected) by the parasitic dinoflagellate of the genus Amoeba are cultured.

[0056] (b) co-culturing the parasitic flagellate of the genus Amoeba and the flagellate of the genus Karenia.

[0057] (c) Two or more days after inoculation (after confirmation of spread of infection by microscopy), a portion of the culture medium containing infected hosts and / or individual parasitic flagellates of the species Amoeba from step (b) is inoculated into fresh medium containing uninfected flagellates of the species Karenia.

[0058] (d) Repeat steps (b) to (c).

[0059] In steps (a) to (d), the culture medium used for co-cultivation is preferably a common culture medium for marine microalgae. A specific example is Daigo's IMK medium (manufactured by Nippon Pharmaceutical Co., Ltd.).

[0060] In step (a), the concentration of Karenia dinoflagellates is preferably 1000 cells mL -1 Up to 2000 cells mL -1and the concentration of parasitic flagellates of the genus Amoeba was 10,000 cells mL -1 Up to 20,000 cells mL -1 The inoculation is performed so that the ratio of the concentration of the parasitic dinoflagellates of the genus Amoeba to the concentration of the dinoflagellates of the genus Karenia is in the range of 1:1 to 1:10.

[0061] In step (b), the co-cultivation is preferably carried out under the following conditions: a temperature of 16°C to 25°C (preferably 18°C ​​to 24°C), a light intensity of 50 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1 , the light-dark cycle ranged from 12 h of light and 12 h of darkness to 14 h of light and 10 h of darkness.

[0062] In step (c), replanting of the parasitic dinoflagellates of the amoeba genus in the culture medium is preferably performed 2 to 10 days after inoculation, more preferably 3 to 4 days after inoculation. Replanting within 2 to 10 days after inoculation is easy and preferred because the parasitic dinoflagellates of the amoeba genus can proliferate in the dinoflagellates of the Karenia genus (host), then destroy the host and escape. If the parasitic dinoflagellates of the amoeba genus of the present invention fail to parasitize the host, they will die within about 3 to 7 days. Therefore, if the operation of step (c) is not performed for 3 to 7 days or longer in a state where there are few new host cells after the parasitic dinoflagellates of the amoeba genus destroy the host and escape from the host cells, the parasitic dinoflagellates of the amoeba genus may not be able to parasitize the host and die.

[0063] In step (b) above, at a temperature of 16°C to 25°C and 50 μmol photons m -2 s -1 Up to 200 μmol photons m - 2 s -1After confirming the infection of the host by the parasitic dinoflagellates of the genus Amoeba, and before the host is destroyed and escapes (for example, 48 to 120 hours after inoculation), the temperature is lowered, preferably to 10°C to 15°C, more preferably to 12°C to 15°C, under a light intensity of 12 hours of light and 12 hours of darkness to a light-dark cycle of 14 hours of light and 10 hours of darkness. Compared with incubation at a temperature of 16°C to 25°C, storing the co-culture at a lower temperature can delay the infection rate of the parasitic dinoflagellates of the genus Amoeba to the dinoflagellates of the genus Karenia or prevent its infection. Storage within the above temperature range is preferred because the parasitic dinoflagellates of the genus Karenia can be stored for a longer period of time in a state where the dinoflagellates of the genus Amoeba are infected by the parasitic dinoflagellates of the genus Amoeba. This may be because creating a low temperature environment allows the parasites to proliferate in the host cells, temporarily delaying or ending the infection cycle, and maintaining the state in which the host keeps the parasites in its cells.

[0064] In this case, infection with uninfected Karenia dinoflagellates can be confirmed by storing the co-culture at a temperature of 10°C to 15°C, preferably for 15 to 40 days, more preferably for 20 to 40 days, and particularly preferably for 25 to 35 days, then returning the temperature to 16°C to 25°C, and then performing step (C).

[0065] Furthermore, in the above step (b), at a temperature of 16°C to 25°C and 50 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1 After confirming the infection of the host by the parasitic flagellates of the genus Amoeba and before the host is destroyed and escapes (e.g., 48 to 120 hours after inoculation), the light intensity is reduced, preferably to 5 μmol photons m -2 s -1 Up to 40 μmol photons m -2 s -1 , more preferably up to 5 μmol photons m -2 s -1 Up to 20 μmol photons m -2 s -1 , and particularly preferably up to 5 μmol photons m -2 s -1 Up to 10 μmol photons m -2 s -1 . Compared with 5 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s-1 Compared with culturing under a light intensity of 100,000, storing the co-culture under a lower light intensity can delay the infection rate of the Amoeba parasitic dinoflagellates to the Karenia dinoflagellates or prevent their infection. Storage within the above light intensity range is preferred because the Amoeba parasitic dinoflagellates can be stored for a longer period of time in a state where the Karenia dinoflagellates are infected by the Amoeba parasitic dinoflagellates. This may be because creating an environment with a low light intensity allows the parasite to proliferate in the host cell, reducing the activity of the parasite and making it impossible for the parasite to mature in the host cell.

[0066] In this case, the co-cultures were incubated at 5 μmol photons m -2 s -1 Up to 40 μmol photons m -2 s -1 The cells are preferably stored at a light intensity of 50 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1 , and then proceeding to step (C), infection with uninfected Karenia dinoflagellates can be confirmed.

[0067] Furthermore, in the above step (b), at a temperature of 16°C to 25°C and 50 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1 After confirming the infection of the host by the parasitic flagellates of the genus Amoeba and before the host is destroyed and escapes (e.g., 48 to 120 hours after inoculation), the co-culture can be stored by reducing the temperature and light intensity in combination with the above-mentioned low temperature condition and low light condition, with a light-dark cycle of 12 hours of light and 12 hours of darkness to 14 hours of light and 10 hours of darkness under a light intensity of 1.5 to 2.0 hours.

[0068] In this case, infection of Karenia dinoflagellates with parasitic Amoeba dinoflagellates was significantly increased at temperatures between 16°C and 25°C and 50 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1 This can slow the infection rate or end infection compared to culturing under higher light intensities.

[0069] The culture of Karenia dinoflagellates is preferably carried out under the following conditions: a temperature of 20°C to 25°C, a light intensity of 50 μmol photons m -2 s-1 Up to 100 μmol photons m -2 s -1 , the light-dark cycle was 14 hours of light and 10 hours of darkness, whether the culture was before or after infection with the parasitic dinoflagellate of the genus Amoeba of the present invention.

[0070] Examples of the parasitic dinoflagellates of the genus Amoeba of the present invention include Amoeba species ( Amoebophrya sp.) OSK2020. Amoeba species OSK2020 is deposited and maintained by the present applicant, who guarantees that the Amoeba species OSK2020 will be distributed in accordance with Article 27-3 of the Enforcement Regulations of the Patent Law of Japan, subject to compliance with the law.

[0071] Amoeba species OSK2020 is a parasitic flagellate of the genus Amoeba. Karenia mikimotoi ( Kareniamikimotoi ) (Karenia dinoflagellates). The nucleic acid molecule of the amoeba species OSK2020 contains the base sequence of SEQ ID NO: 1 (18S rDNA, 1742 bases) and the base sequence of SEQ ID NO: 2 (28S rDNA, 759 bases). The method for obtaining the amoeba species OSK2020 is, for example, as follows.

[0072] (i) A large number of Karenia mikimotoi growing in a seawater were observed using a fluorescence microscope. Karenia mikimotoi infected with an amoeba species had strong green fluorescence (derived from the amoeba) in its cells, so the infected Karenia mikimotoi was removed and inoculated into a culture medium containing uninfected Karenia mikimotoi that had been cultured in advance.

[0073] (ii) After inoculation, the cells were incubated for about several days to 1 week (temperature: 24°C, light-dark cycle: 14 h light and 10 h dark (light period: 100 μmol photons m -2 s -1 white fluorescent light)).

[0074] (iii) Confirmation of the spread of infection by amoeba species using fluorescence microscopy.

[0075] Furthermore, the method for culturing the amoeba species OSK2020 is, for example, as follows. According to the following culturing method, the amoeba species OSK2020 can be stably maintained.

[0076] (iv) Inoculating a portion of the culture medium containing Karenia mikimotoi cells infected with the amoeba species OSK2020 into a culture medium containing Karenia mikimotoi cells that have not been infected.

[0077] (v) After inoculation, the culture is carried out for 3 to 5 days, preferably 3 to 4 days (temperature: 16°C to 25°C (preferably 18°C ​​to 24°C), light-dark cycle: 14 hours of light and 10 hours of darkness (light period: 100 μmol photons m -2 s -1 white fluorescent light)).

[0078] (vi) Confirmation of the spread of infection with the amoeba species OSK2020 using fluorescence microscopy.

[0079] (vii) Repeat steps (iv) to (vi).

[0080] Figure 1 Shown is how the amoeba species OSK2020 parasitizes Karenia mikimotoi, a Karenia dinoflagellate (scale bars represent 20 μm). Figure 1 A and 1B show uninfected Karenia mikimotoi. Figure 1 C and 1D show Karenia mikimotoi at an early stage of infection. The amoeba species OSK2020 grows to several dozen cells in the host (K. mikimotoi). Figure 1 E and 1F show the intermediate stages of infection. Figure 1 G and 1H show the later stages of infection. The amoeba species OSK2020 grows to several hundred cells within the host (K. mikimotoi) and eventually penetrates and leaves the cell ( Figure 1 I and 1J). After that, hundreds of amoeba species OSK2020 are dispersed into the water and parasitize new hosts. This series of cycles is completed within 3 to 5 days, preferably 3 to 4 days.

[0081] In step (v) above, at a temperature between 16°C and 25°C and 100 μmol photons m -2 s -1 After confirming the infection of Karenia mikimotoi by the amoeba species OSK2020 under a light intensity of 14 hours of light and 10 hours of darkness, and before the Karenia mikimotoi is destroyed and escapes (for example, 48 to 120 hours after inoculation), the temperature is lowered, preferably to 10°C to 15°C, more preferably to 12°C to 15°C. Storing the co-culture at a lower temperature can delay the infection rate of Karenia mikimotoi by the amoeba species OSK2020 or prevent the infection, compared with incubation at a temperature of 16°C to 25°C. Storage within the above temperature range is preferred because the amoeba species OSK2020 can be stored for a longer period of time in a state where Karenia mikimotoi is infected by the amoeba species OSK2020.

[0082] In this case, the infection of uninfected Karenia mikimotoi can be confirmed by storing the co-culture at a temperature of 10°C to 15°C, preferably for 15 to 40 days, more preferably for 20 to 40 days, and particularly preferably for 25 to 35 days, then returning the temperature to 16°C to 25°C, and then performing step (iv) in step (vii).

[0083] Furthermore, in step (v) above, at a temperature of 16°C to 25°C and 100 μmol photons m -2 s -1 After confirming infection of the amoeba OSK2020 with K. mikimotoi under a light intensity of 14 h light and 10 h dark cycle and before the K. mikimotoi is destroyed and escapes (e.g., 48 to 120 h after inoculation), the light intensity is reduced, preferably to 5 μmol photons m -2 s -1 Up to 40 μmol photons m -2 s -1 , more preferably up to 5 μmol photons m -2 s -1 Up to 20 μmol photons m -2 s -1 , and particularly preferably up to 5 μmol photons m -2 s -1 Up to 10 μmol photons m -2 s -1 . Compared with 5 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1 Storing the co-culture under a lower light intensity can delay the infection rate of Karenia mikimoto by the amoeba species OSK2020 or prevent its infection compared to culturing under a light intensity of 1.4. Storage within the above light intensity range is preferred because the amoeba species OSK2020 can be stored for a longer period of time in a state where Karenia mikimoto is infected by the amoeba species OSK2020.

[0084] In this case, the co-cultures were incubated at 5 μmol photons m -2 s -1 Up to 40 μmol photons m -2 s -1 The cells are preferably stored at a light intensity of 50 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1, and then performing step (iv) in step (vii) to confirm the infection of uninfected Karenia mikimotoi.

[0085] Alternatively, in step (v) above, at a temperature of 16°C to 25°C and 100 μmol photons m -2 s -1 After confirming the infection of Karenia mikimotoi by the amoeba species OSK2020, and before the Karenia mikimotoi is destroyed and escapes (e.g., 48 to 120 hours after inoculation), the co-culture can be stored by reducing the temperature and light intensity by combining the above-mentioned low temperature conditions and low light conditions. Also in this case, for the infection of Karenia mikimotoi by the amoeba species OSK2020, the temperature and light intensity are reduced in accordance with the conditions at 16°C to 25°C and 50 μmol photons m -2 s -1 Up to 200 μmol photons m -2 s -1 This can slow the infection rate or end infection compared to culturing under higher light intensities.

[0086] In another aspect, the present invention provides a composition containing the parasitic flagellate of the genus Amoeba of the present invention. The composition can be used for the prevention of red tides. More preferably, the composition can be used for red tides derived from the growth of flagellate of the genus Karenia.

[0087] In another aspect, the present invention also provides a microbial pesticide, which contains the parasitic flagellate of the genus Amoeba of the present invention. The microbial pesticide is suitable for preventing red tides, and is particularly suitable for preventing red tides derived from the growth of flagellate of the genus Karenia.

[0088] For the composition or microbial pesticide of the present invention, the culture solution containing the parasitic flagellate of the genus Amoeba, in-situ seawater or in-situ seabed mud can be used as it is. However, the composition or microbial pesticide may also contain additives such as stabilizers and glycerol to the extent that the effect of the present invention is not impaired.

[0089] The composition or microbial pesticide of the present invention can be produced according to, for example, a method comprising the step of culturing the parasitic dinoflagellate of the genus Amoeba of the present invention. The step of culturing the parasitic dinoflagellate of the genus Amoeba of the present invention can be the above-mentioned method for co-culturing the parasitic dinoflagellate of the genus Amoeba and the dinoflagellate of the genus Karenia.

[0090] On the other hand, the present invention also provides a method for preventing red tides. The composition or microbial pesticide of the present invention is sprayed onto the sea surface or in the sea so that the parasitic flagellates of the genus Amoeba contained in the composition or microbial pesticide of the present invention infect the flagellates of the genus Karenia present in the sea surface or in the sea. This kills the flagellates of the genus Karenia present in the sea surface or in the sea, inhibits the growth of the flagellates of the genus Karenia, and prevents red tides caused by the growth of the flagellates of the genus Karenia. The amount of the composition or microbial pesticide of the present invention sprayed on the sea surface or in the sea is not particularly limited and is appropriately determined according to the area of ​​the sea surface to be sprayed, the depth of the sea, etc.

[0091] In a preferred embodiment, the parasitic dinoflagellates of the genus Amoeba of the present invention are expected to have a small impact on the ecosystem and be highly safe, because if the parasitic dinoflagellates cannot parasitize the host due to the lack of the host (i.e., the dinoflagellates of the genus Karenia), it will die within about 7 days. In addition, if the parasitic dinoflagellates of the genus Amoeba of the present invention specifically infect the dinoflagellates of the genus Karenia, there is almost no need to worry about nonspecific infection of many organisms. Therefore, the parasitic dinoflagellates of the genus Amoeba are expected to be highly safe.

[0092] Example The present invention is described in more detail below with reference to Examples. However, the present invention is not limited to these Examples.

[0093] Obtained amoeba species OSK2020 (parasitic flagellate of the genus Amoeba) On October 5, 2020, samples of seawater (water temperature: 24°C, salinity: 30) collected from Osaka Bay were infected with amoeba species ( Amoebophrya sp.) (a parasitic flagellate of the genus Amoeba) Karenia mikimotoi ) (a dinoflagellate of the genus Karenia). The infected Karenia mikimotoi individuals were inoculated into a medium containing uninfected Karenia mikimotoi (a strain collected from Ago Bay in 2013 and stored in Daigo'IMK medium with a salt content of 30). After inoculation, the culture was carried out for 4 days (temperature: 24°C, light-dark cycle: 14 h of light and 10 h of darkness (light period: 100 μmol photons m -2 s -1After the infection of the amoeba species spreads to the uninfected Karenia mikimotoi, about 1 / 10 to 1 / 20 of the culture medium (including the infected host) is aspirated with a micropipette and inoculated into a fresh culture medium (including the uninfected host), thereby obtaining the amoeba species OSK2020 (hereinafter sometimes referred to as "OSK2020"). According to this method, strains other than OSK2020 can also be obtained by performing similar operations in other water bodies where Karenia mikimotoi grows.

[0094] The stock culture of OSK2020 was carried out by inoculating a predetermined amount of Karenia mikimotoi infected with OSK2020 into a medium cultured with uninfected Karenia mikimotoi every 4 days. The stock culture was carried out under the following conditions: temperature was 24°C, and the light-dark cycle was 14 hours of light and 10 hours of darkness (light period: 100 μmol photons m -2 s -1 Using this culture method, OSK2020 was successfully and stably maintained for more than one year.

[0095] Example 1: Experiment of infecting Karenia mikimotoi with OSK2020 Infection experiments were conducted at the laboratory level to confirm the relationship between the cell density of Karenia mikimotoi, the host of OSK2020, and the parasitic effect of OSK2020. The culture medium of OSK2020 was passed through a filter with a pore size of 8 μm (Nuclepore track-etched membrane manufactured by Whatman) to remove host cells and isolate the migratory cells of OSK2020. 20 mL of migratory cells within 12 hours from the emergence of the host were plated at 16,000 cells mL -1 The initial concentration was inoculated into Karenia mikimotoi, which was in the logarithmic growth phase for the third time (initial cell concentration: 1600 cells mL -1 ) and cultured under the above conditions. Figure 2 shows the results.

[0096] Figure 2A The graph shows the changes in cell density of Karenia mikimotoi over time when OSK2020 was added to the culture medium of Karenia mikimotoi from 0 hours after the start of culture (treatment group) and when OSK2020 was not added (control group). Figure 2B The infection rate and infection status of Karenia mikimotoi over time when OSK2020 was added to the culture medium of Karenia mikimotoi from 0 hours after the start of culture are shown. Figure 2C The cell density of OSK2020 changes over time when OSK2020 is added to the culture medium of Karenia mikimotoi from 0 hours after the start of culture. From these results, it is clear that when OSK2020 is added, Karenia mikimotoi is infected and killed by OSK2020.

[0097] Example 2: Experiments on infection of other microorganisms with OSK2020 In order to confirm which can be the host of OSK2020, a cross-infection experiment was performed. The following 16 microorganisms were examined. The experiment was performed according to Kim, J. Phycol. 2006, 42, 1170-1173; Chambouvet et al., Protist 2011,162, 637-649; Chen et al., J. Eukaryot. Microbiol. 2018, 65, 448-457; and Nishitani et al., Harmful Algae 2021, 110, 102123. The operation of isolating OSK2020 wandering cells was performed in the same manner as in Example 1. The microorganism under study was cultured to obtain a suitable cell density, and then the culture medium containing the monomeric microorganism under study was added to a 24-well plate in an amount of 2 mL. Thereafter, OSK2020 migratory cells were added to make the ratio of migratory cells to host microorganisms (migratory cells: host microorganisms) reach 20:1, and the mixture was cultured at 20°C with a light-dark cycle of 14:10 (light period: 100 μmol photons m -2 s -1 White fluorescent lamp -2 s -1 The green autofluorescence of OSK2020 and the red autofluorescence of the chloroplasts of the host microorganisms were observed using a fluorescence microscope (IX71, manufactured by Olympus Corporation) equipped with a U-MNBV2 cube fluorescence unit (excitation filter: 420-440 nm, absorption filter: 475 nm, manufactured by Olympus Corporation) to confirm parasitism.

[0098] Microorganisms studied Red tide algae ( Akashiwo sanguinea ) A. catenella (Group I) A. pacificum Polycyclic algae ( Cochlodinium polykrikoides ) Gonyaula polytricha Gonyaulax polygramma ) Gonyaula spinulosa Gonyaulax spinifera ) Gymnodinium yum Gymnodinium impudicum ) Heterocarpus rotundus Heterocapsa circularisquama ) Karenia mikimotoi Karenia mikimotoi ) Karenia papilionacea Levanella serrata Levanderina fissa ) Prorocentrum koreanum Microdinoflagellates ( Prorocentrum minimum ) Prorocentrum mexicana Prorocentrum mexicanum ) Scripps cones Scrippsiella trochoidea ) Takayama xiamenensis The results are shown in Table 1. OSK2020 only infected Karenia mikimotoi and Karenia papilionacea This suggests that OSK2020 only parasitizes Karenia dinoflagellates and does not affect other organisms.

[0099] Table 1

[0100] Example 3: Extraction of DNA from amoeba species 2020Oct5-1 to 5 from seawater samples To confirm the genetic diversity of parasitic flagellates of the genus Amoeba that have the ability to parasitize Karenia mikimotoi, five species of infected Karenia mikimotoi (2020Oct5-1, 2020Oct5-2, 2020Oct5-3, 2020Oct5-4, and 2020Oct5-5) were collected from the seawater sample where OSK2020 was found according to the micropipette aspiration technique. Each cell was washed at least three times with filter-sterilized seawater (pore size: 0.1 μm) and then placed in a 0.2 mL PCR tube with 5% Chelex® suspension (Bio-Rad Laboratories, Hercules). DNA of the amoeba species 2020Oct5-1 to 2020Oct5-5 was extracted according to the method described by Richlen and Barber Mol.Ecol. Notes 2005, 5, 688-691.

[0101] Example 4: Extraction of DNA of amoeba species HMN2020 from seawater samples Karenia mikimotoi, which was confirmed to be parasitized by amoeba, was collected from lake water samples collected from Hamana Lake on December 10, 2020, using the micropipette aspiration technique. The cells were washed at least three times with seawater sterilized by a filter (pore size: 0.1 μm) and then placed in a 0.2 mL PCR tube with a 5% Chelex® suspension (Bio-Rad Laboratories, Hercules). DNA of the amoeba species HMN2020 was extracted according to the method described by Richlen and Barber Mol. Ecol. Notes 2005, 5, 688-691.

[0102] Example 5: Sequence determination PCR was performed on the nuclear 18S rDNA and nuclear 28S rDNA of OSK2020 and the nuclear 28S rRNA obtained in Examples 3 and 4. The sequence of 18S rDNA was determined according to the method described in Nishitani et al., Appl Environ Microbiol., 2010, 76, 2791-2798, and the sequence of nuclear 28S rDNA was determined according to the method described in Nishitani et al., Harmful Algae 2021, 110, 102123. Specifically, TOYOBO KOD plusver.2 enzyme and primers were used to amplify 18S rDNA and 28S rDNA in the extracted DNA of the genus Amoeba. The products obtained after the completion of PCR were confirmed by performing 1.5% TAE agarose gel electrophoresis, purified with Exo SAP IT (78200, Amphitheater, California), and then directly sequenced to determine the sequence.

[0103] The sequence of the nuclear 18S rDNA of the amoeba species OSK2020 obtained is represented by SEQ ID NO: 1. The sequence of the nuclear 28S rDNA of the amoeba species OSK2020 obtained is represented by SEQ ID NO: 2. The sequence of the nuclear 28S rDNA of the amoeba species 2020Oct5-1 obtained is represented by SEQ ID NO: 4. The sequences of the nuclear 28S rDNA of the amoeba species 2020Oct5-2, the amoeba species 2020Oct5-3, the amoeba species 2020Oct5-4 and the amoeba species 2020Oct5-5 obtained are represented by SEQ ID NO: 2. The sequence of the nuclear 28S rDNA of the amoeba species HMN2020 obtained is represented by SEQ ID NO: 3.

[0104] Example 6: Physical genetic analysis The sequences of the genus Amoeba obtained in Example 5 were confirmed using BLAST from GenBank, and a phylogenetic tree of nuclear 18S rDNA and nuclear 28S rDNA was created using MEGA ver.X software (Kumar et al., Mol. Biol. Evol. 2018, 35, 1547-1549) according to the maximum likelihood method. The topology of the phylogenetic tree was evaluated by a bootstrap test with 100 replicates. Figure 3 and 4 The results are shown.

[0105] Figure 3 Results of molecular phylogenetic analysis of parasitic organisms based on nuclear 18S rDNA are shown. Figure 3 The sequence of the amoeba species OSK2020 in the middle rectangular frame is represented by SEQ ID NO: 1. Figure 4 Results of molecular phylogenetic analysis of parasitic organisms based on nuclear 28S rDNA are shown. Amoebophrya sp.) OSK2020ex. Karenia mikimotoi ( Karenia mikimotoi ) and amoeba species ( Amoebophrya sp.) 2020Oct5-2 to 5-5 are represented by SEQ ID NO: 2. Amoeba species ( Amoebophrya sp.)HMN2020 ex. Karenia mikimotoi( Karenia mikimotoi ) is represented by SEQ ID NO: 3. Figure 4 The sequence of the amoeba species 2020Oct5-1 in is represented by SEQ ID NO: 4. The sequence of the nuclear 28S rDNA of the amoeba species 2020Oct5-1 differs from the sequence of SEQ ID NO: 2 by only one base.

[0106] These genetic analyses showed that the DNA sequence of the amoeba species isolated in the present invention did not match any existing sequence registered in the database. In other words, this suggests that this amoeba species may be a completely new species that has never been isolated anywhere in the world before. Currently, environmental DNA, including dinoflagellates, is being vigorously investigated in seas around the world, but the amoeba species did not match any of them. This suggests that the new species of the present invention, amoeba species OSK2020, is limited to water bodies in or near Osaka Bay, Japan.

[0107] Example 7: Experiment on cryogenic storage of infected host cells Daigo's IMK medium, host (Karenia mikimotoi) (2000 cells / mL), and parasite (Amoeba species OSK2020) (20000 cells / mL) were added to the wells of a 12-well microplate and incubated at 20°C (four microplates were prepared). When the infection rate reached about 70% (determined as day 0), the microplate was transferred to 20°C (control), 14°C, 12°C, and 10°C, and the number of days the infected host was maintained in the refrigerator (until complete demise) was recorded (days) Figure 5 ). The light intensity was set to 100 μmol photons m -2 s -1 .

[0108] Thereafter, it was confirmed whether the stopped infection cycle had resumed. The infected hosts stored at low temperature were replenished with uninfected hosts, and the water temperature was restored to 20° C. Whether the stopped infection cycle resumed and led to reinfection was investigated weekly.

[0109] In the control experimental group at 20°C (optimal temperature), the infected hosts survived for 19 days. However, in the experimental groups where the infection state was temporarily suspended at low temperatures of 14°C and 12°C, the survival days of the infected hosts in each group was extended to 28 days. In the experimental group placed at 10°C, the survival days of the infected hosts were extended to 36 days. It is believed that when placed at low temperatures, the activity of the parasite is reduced and it is unable to mature inside the host cells. In addition, attempts to resume the temporarily suspended infection cycle allowed the infected hosts to be successfully reinfected after being stored at 14°C for 19 days, 12°C for 28 days, and 10°C for 36 days, compared to the control group stored at 20°C for 7 days. Although the current passage interval of the host (Karenia mikimotoi) and the parasite (amoeba species OSK2020) is short at about 3 to 5 days, it was found that storing the infected hosts at 10°C was able to extend this time to about one month. Since host cells cannot withstand cold at temperatures below 10°C (e.g., 8°C), at which both the host and the parasite would be completely killed, the optimal storage temperature is expected to be 10°C to 12°C. Detailed consideration of storage conditions may enable long-term storage of one month or more. These results are considered key insights not only for reducing costs and labor, but also for producing parasitic organism preparations.

[0110] Example 8: Low light intensity storage experiment on infected host cells In the same manner as in the above experiment, Daigo's IMK medium, 2000 cells / mL of the host (Karenia mikimotoi), and 20,000 cells / mL of the parasite (Amoeba species OSK2020) were added to the wells of a 12-well microplate and incubated at 20°C in water (three microplates were prepared). The light intensity was set to 100 μmol photons m -2 s -1 When the infection rate reached about 70% (determined as day 0), the microplate was transferred to a light intensity of 100 μmol photons m -2 s -1 (control), 20 μmol photons m -2 s -1 and 5 μmol photons m -2 s -1 environment, and then recorded the number of days the infected hosts survived under low light intensity (until they died completely) ( Figure 6 ). The culture temperature during storage was set at 20°C.

[0111] Afterwards, confirm whether the stopped infection cycle has resumed. Replenish the infected hosts stored under low light intensity with uninfected hosts and restore the light intensity to 100 μmol photons m -2 s -1 The stopped infection cycles were investigated weekly to see whether they resumed and led to reinfection.

[0112] At a light intensity of 100 μmol photons m -2 s -1 In the control group (normal culture light intensity), the infected hosts survived for 18 days. However, in the -2 s -1 In the experimental group where the infection was temporarily suspended in a low light intensity environment, the survival days of the infected host were extended to 35 days. -2 s -1 The survival of the infected host group under low light intensities was extended to 90 days. It is believed that when placed under low light intensities, the activity of the parasite is reduced and it is unable to mature inside the host cells. In addition, attempts to resume the temporarily suspended infection cycle allowed the successful reinfection of hosts that had been stored for 90 days. Although the current subculture intervals of the host (Karenia mikimotoi) and the parasite (amoeba species OSK2020) are short at about 3 to 5 days, it was found that storing the infected hosts under low light intensities was able to extend this time to about three months. At less than 5 μmol photons m -2 s -1At a light intensity of 5 μmol photons m, the photosynthesizing host cells will not be able to tolerate the low light intensity conditions, and both the host and the parasite will be completely killed. Therefore, the optimal light intensity for storage is expected to be 5 μmol photons m -2 s -1 Up to 10 μmol photons m - 2 s -1 .

Claims

1. A parasitic dinoflagellate of the genus Amoeba, which has the ability to parasitize dinoflagellate of the genus Karenia.

2. A parasitic flagellate of the genus Amoeba, comprising one of the following nucleic acid molecules (1) to (12): (1) a nucleic acid molecule containing the base sequence of SEQ ID NO: 1, (2) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 1 are replaced, added or deleted, (3) a nucleic acid molecule comprising a base sequence having at least 90% identity with the base sequence of SEQ ID NO: 1, (4) a nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO: 1, (5) a nucleic acid molecule containing the base sequence of SEQ ID NO: 2, (6) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 2 are replaced, added or deleted, (7) a nucleic acid molecule comprising a base sequence that is at least 90% identical to the base sequence of SEQ ID NO: 2, (8) a nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO: 2, (9) a nucleic acid molecule containing the base sequence of SEQ ID NO: 3, (10) a nucleic acid molecule comprising a base sequence in which one or more bases in the base sequence of SEQ ID NO: 3 are replaced, added or deleted, (11) a nucleic acid molecule comprising a base sequence that is at least 90% identical to the base sequence of SEQ ID NO: 3, and (12) A nucleic acid molecule comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO:

3.

3. The parasitic dinoflagellate according to claim 1, which has the ability to kill dinoflagellates of the genus Karenia.

4. The parasitic dinoflagellate according to claim 1, which has the ability to inhibit the proliferation of dinoflagellates of the genus Karenia.

5. A composition comprising the parasitic dinoflagellate of any one of claims 1 to 4. The composition according to claim 5 , which is used for preventing red tide.

7. A microbial pesticide comprising the parasitic flagellate according to any one of claims 1 to 4.

8. A method for obtaining parasitic dinoflagellates of the genus Amoeba, comprising the following steps: inoculating dinoflagellates of the genus Karenia infected with parasitic dinoflagellates of the genus Amoeba into a culture medium in which uninfected dinoflagellates of the genus Karenia are cultured.

9. Parasitic dinoflagellates of the genus Amoeba obtained by the method according to claim 8.

10. A method for inhibiting the proliferation of algae of the genus Karenia, comprising the following steps: allowing parasitic dinoflagellates of the genus Amoeba to parasitize dinoflagellates of the genus Karenia.

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