KR101793393B1 - INHIBITOR COMPRISING DINOFLAGELLATES Alexandrium spp. AGAINST SCUTICOCILIATES - Google Patents

Abstract

The present invention relates to an inhibitor comprising dinoflagellates Alexandrium spp. against scuticociliates and, more specifically, to an inhibitor comprising Alexandrium andersonii cells and a culture medium thereof against scuticociliates. The inhibitor comprising dinoflagellates Alexandrium spp. and a culture medium thereof against scuticociliates of the present invention can control scuticociliates in a safe biological method, and is harmless to fish resources except scuticociliates.

Description

{INHIBITOR COMPRISING DINOFLAGELLATES Alexandrium spp. ≪ / RTI > AGAINST SCITICOCILIATES}

The present invention relates to a scuticacid inhibitor comprising an Alexandrium spp. Bacterium, and more particularly to a scuticacid inhibitor using Alexandrium andersonii cells and a culture thereof.

Scuticocillatosis is found in Korea, the Mediterranean, Spain, Australia, Japan, the United States, and China, and is infectious to various marine fish species and hippocampus including major flounder and rockfish do.

Cryptomeria, one of the ciliates, is the main cause of ciliates, and 10 species have been known to date. This species is invisible to the naked eye and looks like a cucumber when viewed under a microscope. The species that are infected with many fish species in Korea and Japan are Miamiensis avidus and Uronema marinum. The ciliates belonging to the scutica are predominantly fungi as food sources and appear when the concentration of dissolved oxygen is low and there are many organic matter.

This parasite infiltrates into the gill, skin, and subepithelial epithelium of the body, between the dermis and the muscle layer, and causes epithelial cell proliferation, hypertrophy, redness and even necrosis. In muscle, parasitism occurs between transverse myofiber cells, causing tissue to collapse and necrosis. In the case of domes and rocks, abrasion of the body surface may occur. In the case of perch, head bleeding may occur.

The flounder exhibits various symptoms such as inflammation of the body surface, exposure of the dorsal soft tissue and tail sack, and head injury. Head injury suggests that the mucous membrane bleeds, ocular opacity and protrusion, and congestion of the oral mucosa. The parasite also enters the brain and causes infection. It may also enter the brain directly, but it may invade the brain and enter the brain via the central nervous system. When the parasite is infected in the brain, it is known that most infected fish die.

For mild infections caused by bodily gills or gills, formalin is bathed at 100-200 ppm for 1 hour, then bathed and then cleaned and supplied with clear seawater. However, there is no effective remedy for Sukutika insect parasitic on internal organs including the brain. Therefore, in case of infected fish, they should be collected and disposed of immediately. Therefore, it is important that the treatment is carried out under the condition of infection by the confirmation of the early infection, and that it is not transferred to the brain or internal organs. Since the Sututika spp. Is present in infected seedlings including infected seedlings and serves as an infectious source, thorough disinfection and regular cleaning of the spermatozoa are essential to prevent damage to the parasites. Periodic inspection is required.

However, formalin is widely used as a disinfectant and preservative in formaldehyde, a typical carcinogen, dissolved in water to a concentration of 35-38%. In Korea, the formaldehyde is widely used in the domestic water environment preservation law (Article 29), toxic chemical substance management act ), Waste Management Act (Article 25), and it is classified as a toxic substance according to the Atmospheric Conservation Act (Article 2). Because of the harmful effects of formalin, the use of formalin as a parasitic drug for fish in the aquaculture field is now prohibited after the controversy because formalin was detected in Japan in 2003 as a form of blowfish, and in Jeju Island in July 2006, When formalin is detected in aquatic products, it is stipulated by the ordinance that it should not be exported. However, the use of formalin, which is prohibited in Japan, is made available in cities and provinces other than Jeju Island, causing conflicts with Japan and consumers. In fact, the report of the Ministry of Maritime Affairs and Fisheries in 1997 (<acute toxic effect of growing flounder (mineralized fish) following chemical treatment>) also showed that "Formalin in formalin, chlorine dioxide and hydrogen peroxide has a higher mortality rate Because it is toxic, it is effective to use hydrogen peroxide rather than formalin. "He also advised against using formalin.

Korean Patent No. 1353200 proposes a scutica infectious disease remedy composition containing chlorine dioxide and citric acid as a main component. However, since chlorine dioxide is highly volatile and explosive, the gas has an optical decomposition function that is easily decomposed by ultraviolet rays , And aquaculture workers. Korean Patent Laid-Open Publication No. 2015-0142788 proposes a vaccine composition containing streptococcus bacteria, but there is a hassle to inject the target fish.

The Sututika infectious disease caused by the outbreak pathogen, Sututika ciliates, is one of the most serious causes of diseases worldwide in the marine aquaculture industry. Therefore, removal of these ciliates is a fundamental problem for marine aquaculture managers and researchers. So far, formalin and several other toxic chemicals have been used as scuticacid inhibitors. But there are secondary side effects. As a result, it is necessary to develop safe technology for the removal of scutica.

Preceding Documents 1. Korean Registered Patent No. 1353200 (Published on January 17, 2014) Prior art document 2. Korean Patent Publication No. 2015-0142788 (Publication date: December 23, 2015)

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a biocompatible Sukitika antifungal agent which is free from secondary damage and easy to apply.

In order to solve the above problems, the present invention provides a scuticacid inhibitor comprising an Alexandrium spp.

According to a preferred embodiment of the present invention, the above-mentioned Alexandrium spp. Can be selected from the group consisting of Alexandrium andersonii, Alexandrium fraterculus, Alexandrium hiranoi, Alexandrium insuetum may be used. In addition, according to a preferred embodiment of the present invention, the scuticaccharum may be a parasite of the scuticum, and the parasitic parasite of the scuticum may be selected from the group consisting of Miamiensis avidus, Miamiensis sp ) And Uronema marinum or Anophryoides haemophilia. &Lt; / RTI &gt;

In order to solve the above problems, the present invention provides a scuticacid inhibitor comprising a culture medium of an Alexandrium spp.

According to a preferred embodiment of the present invention, the culture medium of the above Alexandrium spp. Bacterium is selected from the group consisting of Alexandrium andersonii, Alexandrium fraterculus, Alexandrium hiranoi ) Or a culture of Alexandrium insuetum may be used.

Sukutika infestation inhibitors, including the present Alexandrium spp. Mussel and its culture, can control the Sukitika infestation by a safe biological method.

In addition, the scuticacid inhibitor containing the Alexandrium spp. Of the present invention and the culture thereof has no harmful effect on useful fish resources other than the scutica.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate exemplary embodiments of the invention and, together with the description of the invention, It should not be construed as limited.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the effect of suppressing the accumulation of Aleksandrynos cell sututica according to a preferred embodiment of the present invention.
FIG. 2 is a graph showing the effect of inhibiting the accumulation of Alexandrium enterosone cellulosicus according to a preferred embodiment of the present invention.
FIG. 3 is a graph showing the effect of suppressing the accumulation of scuticacid in a culture medium of Alexandrium herder in accordance with a preferred embodiment of the present invention.
FIG. 4 is a graph showing the effect of suppressing the accumulation of scuticacid in a culture medium of Alexandrium endersony according to a preferred embodiment of the present invention.

Hereinafter, the present invention will be described in detail with reference to the drawings. The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may appropriately define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Hereinafter, the embodiments described in the present specification and the configurations shown in the drawings are merely the most preferred embodiments of the present invention, and not all of the technical ideas of the present invention are described. Therefore, various equivalents It should be understood that water and variations may be present.

The scuticacid inhibitor of the present invention includes the Alexandrium spp.

Sututika is a parasitic bacterium that is a kind of ciliate, which penetrates into the body and causes scutica. Sucutica infestation causes the necrosis and hemorrhage of cell tissue and ultimately leads to death. Sucutika sucker penetrates from the gills such as gills and fins, and these sututika suckers penetrate deeply into the body along the blood vessels and are found not only in the body but also in internal organs such as the stomach, kidney, liver and brain.

In most marine fishes, such as fish, crustaceans, and mollusks, the occurrence of scuticacidosis has been confirmed and is a worldwide disease without regional characteristics. Seasonal occurrence of parasites is relatively frequent in the hot season, such as during the summer, where the parasitoids can briskly grow, and the breeding temperature range of Sukutika is wide ranging from 5 ℃ to 30 ℃. It is a disease that frequently occurs in eutrophic coastal or aquaculture tanks where parasites can actively breed. It is not easy to get relief when infected, it has mass infectious and massive deaths, and it causes serious damage to the aquaculture industry.

The scuticaccharum which is the cause of scutichariae is a parasite of the scutica subspecies and shows the form of a ciliate of 30 ㎛ to 40 ㎛ in size. Major species causing many damages in the parasites of the above-mentioned Sucutica chinens include Miamiensis avidus, Miamiensis sp., Uronema marinum and Anoprioides And Anophryoides haemophilia.

Alexandrium spp. Is a single-celled organism with a width of 20 to 50 μm and a length of 15 to 40 μm, rounded as a whole, and having a depression at its center and surrounding the band.

The present inventors of the present invention have found that the Alexandrium spp. And the Alexandrium spp. That is, a scuticacid inhibitor including the present invention Alexandrium spp. And the culture thereof can control the scuticacid.

In addition, it has been confirmed that the scuticacid inhibitor containing the Alexandrium spp. Of the present invention and the culture thereof has no deleterious effects on useful fish resources other than the scutica.

The present invention is also directed to a method for the treatment of Alexandrium spp. Of the present invention having the effect of suppressing the scuticaceous action. Examples of the sputum include Alexandrium andersonii, Alexandrium fraterculus, Alexandrium hiranoi or Alexandrite spp. Alexandrium insuetum may be used, but the present invention is not limited thereto.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the above-described embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example

Alexandrium Enderson Sony  ( Alexandrium  andersonii)

The Alexandrium Enderson was separated purely from Jinhae Bay in May 2015, at a temperature of 15.3 ℃ and a salinity of 27.4. Alexandrite endosone was purified by single pipetting using a micropipette and transferred to 50-, 250-, and 500-mL polycarbonate bottles containing F / 2-si medium as cell density increased, The cultures were maintained in a 20 ° C incubator at a light intensity of 20 μE / m ² / s and a light period of 14:10.

Miami Meensis Of Miami's avidus  Ready

Miamisensis Evidus was isolated from the brains harvested from the infected fish (530 g) of the flounder farm in Jeju Island in February 2016. The temperature of the flounder farm was 17.3 ℃. Purely separated miamiensis evidus was cultured in seawater with 1 mg / 100 mL Proteose-peptone and yeast. The culture was incubated at 20 ° C in a culture room at 20 μE / m ² / s , And 14:10, respectively.

Miami Meensis Of the epiphyte (Miamiensis sp.)  Ready

Miami Nishi Espi was separated in May 2016 from the western part of the western part of the sea. The water temperature and salinity were 20.8 ℃ and 31.3, respectively. Miami Nishis Espie found that she was infected with the larvae of the Clithon retropictus. Mia US N-Sys SP were cultured put the photosynthesis bacteria in the sea water, the cultures were maintained in a growth chamber at 20 ℃ brightness 20 μE / m ² / s, a group of Gwangju 14:10.

Example  One. Alexandrium Enderson Sony  cell Scutica  Inhibitor ( Miami Meensis Evidus )

The concentrations of the Alexandrium endosonian cells were prepared to be 50, 100, 500, 1000, 2500 and 4500 cells / ml.

Sukutika pomiemiensis evidus was cultured with Alexandrite endosone at different cell concentrations prepared above, and the reaction was observed over time, and the survival curve was shown in Fig.

The cell concentration of Miamyensis Evidus cultivated with Alexandrium Enderson significantly decreased over time. The cells were cultured in the presence of the following concentrations of Alexandrite endosonin (500 cells / ml, after 48 hours; 1000 cells / ml, after 24 hours; 2500 cells / ml, after 12 hours; 4500 cells / Chumia Miensis Evidus died. However, when combined with the following (50-100 cells / ml, after 24 hours) of Alexandrite enderson, only 25-35% of Miamiensis evidus survived.

Example  2. Alexandrium Enderson Sony  cell Scutica  Inhibitor ( Miami Meensis Espie )

The concentrations of the Alexandrium endosonian cells were prepared to be 50, 100, 500, 1000, 2500 and 4500 cells / ml.

Sukutika pneumoniaeisispi was cultured with Alexandrite endosone at different cell concentrations prepared above, and the reaction with time was confirmed, and the survival curve was shown in Fig.

The cell concentration of cultivated Miamyensis sp. With Alexandrium Enderson decreased with time, depending on the concentration of Alexandrite enderson. On the other hand, the cell concentration of Miamiensis spp was gradually decreased in the experimental group without Alexandrium enderson. Miamyensis spp. Survived in the condition of Alexandrite enderson (<100 cells / ml). Miamiensis spp survived only 10% at the condition of Alexandrium enderson (500 cells / ml, after 48 hours). However, miamiensis spp. Was almost killed in the condition of alexandrite enderson (1000 cells / ml, after 6 hours).

Example  3. Alexandrium Enderson Sony  Culture solution Scutica  Inhibitor ( Miami Meensis  Evidus)

The concentrations of the culture medium of the Alexandrite seed culture were adjusted to 50, 100, 500, 1000, 2500 and 4500 cells / ml.

The Sukutika pomiemiensis evidus was incubated with the above-prepared Alexandrite seed culture broth at different concentrations, and the reaction was observed over time, and the survival curve was shown in Fig.

Over time as cultured with the culture medium of Alexandrite enderson, the cell concentration of Sukutika pneumoniaeensis evidus dropped sharply. At the following concentration of Alexandrite seed culture medium (2500 cells / ml, after 24 hours; 4500 cells / ml, after 24 hours) However, Sukutika pomiemiensis evidus at the concentration of the Alexandrium enderson culture medium (100 cells / ml, after 24 hours; 500 cells / ml, after 24 hours; 1000 cells / ml after 24 hours) , 40 and 20%, respectively. This survival rate was maintained after 24 hours.

Example  4. Alexandrium Enderson Sony  Cell culture medium Scutica  Inhibitor ( Miami Meensis Espie )

The concentrations of the culture medium of the Alexandrite seed culture were adjusted to 50, 100, 500, 1000, 2500 and 4500 cells / ml.

The Sukutika superabsorbate culture was incubated with the above-prepared concentrations of the Alexandrite seed culture, and the reaction was observed over time, and the survival curves were shown in Fig.

As time passed with incubation with the Alexandrite enderson culture, the cell concentration of Sukutika chlamia miensis spp. Dropped sharply. However, Miamisensis sp. Survived at low concentration of 50 cells / ml. Miamyensis spp. Showed similar survival rates under control conditions without the culture medium of Alexandrium perthersoni under the conditions of the culture medium (50-500 cells / ml, after 48 hours) of the Alexandrite seed culture. Miamyensis spp. Survived 20% at the conditions of Alexandrium enderson culture (1000 cells / ml, after 24 hours), which remained similar after 24 hours. Miamyensis spp was almost killed in the culture medium of Alexandrium herder (2500 ~ 4500 cells / ml, after 24 hours).

According to Examples 1-4, it was confirmed that the culture medium of Alexandrite endosonian cells and Alexandrite endosone adversely affects the growth of Sututika spp., And it was confirmed that complete extinction occurs at a certain concentration or higher. In other words, the suppressive effect of Suktika spp. Was confirmed by Alexandrium perthersoni, a kind of Alexandrium spp.

Reference example  One. Alexandrium Enderson Sony  cell Scutica  Toxicity test of inhibitor

For the toxicity test of the brine shrimp Artemia salina larvae of Alexandria Enderson cells, the eggs of Artemia salinina were placed in clean sea water and incubated at 20 ° C in a culture room at 20 μE / m ² / s in a culture room at 14:10 And cultured for 2 days.

Successful hatching and activity of the active aldemia salina larvae were used for toxicity testing.

 10 Alzheimer's Salinas were added to each well of a 6-well plate which had been densely cultured with Alexandrite enderson cells to a final density of 50, 100, 500, 1000, 2500, 4500 cells / mL, The final volume was made 5 mL using seawater. And 10 controls of Salmonella larvae were injected into the clean filtered seawater as a control. The number of killed S. aureus larvae was observed at 1, 2, 4, 6, and 24 hours.

After 24 hours, no dead Artemia salina larvae were found in all cases where the density of the Alexandrite endosonian cells was 50, 100, 500, 1000, 2500, 4500 cells / mL.

Reference example  2. Alexandrium Enderson Sony  Culture Scutica  Toxicity test of inhibitor

For the toxicity test of the brine shrimp Artemia salina larvae of the Alexandrite enderson culture, the eggs of Artemia salina were placed in clean sea water and incubated at 20 ° C in a culture room at 20 μE / m ² / s in a culture room at 14:10 And cultured for 2 days.

The larvae of the larvae were successfully used for experiments.

The culture medium of Alexandrium enterosyne was added to a 6-well plate at a density of 50, 100, 500, 1000, 2500 and 4500 cells / mL for each of the final concentrations of the Alexandrium enterocyte cells. 5 mL. Ten wells of Salmonella were added to each well. And 10 controls of Salmonella larvae were injected into the clean filtered seawater as a control. The number of killed S. aureus larvae was observed at 1, 2, 4, 6, and 24 hours.

After 24 hours, no dead Artemia salina larvae were found in all cases where the density of the Alexandrite endosonian cells was 50, 100, 500, 1000, 2500, 4500 cells / mL.

According to Reference Example 1-2, it was confirmed that the culture of Alexandrite endosonian cells and Alexandrite endosonian did not affect the growth of the alfalfa salina larvae, and no alfalfa salinha larvae died at any concentration. In other words, it was confirmed that the organism other than Suktika is not toxic through Alexandrium enderson which is a kind of Alexandrite genus.

Claims (8)

Alexandrium andersonii (Miamiensis spp.) Scuticacea inhibitor containing the epididymis. delete delete delete Alexandrium andersonii A subspecies inhibitor of the genus Miamiensis spp. Containing the culture of the epididymis. delete delete delete

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