Application of nannochloropsis in preparation of product for inhibiting fish parasite activity

Abstract

The invention belongs to the technical field of aquaculture, and particularly relates to application of nannochloropsis in preparation of a product for inhibiting fish parasite activity. Specifically, the invention discloses application of nannochloropsis in preparation of a product for inhibiting fish parasite activity and / or reducing fish parasite infection rate. The protochlorococcum has a remarkable killing effect on fish parasites, the infection rate of fishes to the fish parasites can be remarkably increased, and the protochlorococcum can also promote growth of the fishes. According to the invention, the protochlorococcum is used for inhibiting fish parasites, no chemical drug residue exists, and the problems of drug resistance and environmental pollution caused by the existing chemical insect repellant are effectively solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture, and particularly relates to the application of Prochlorococcus in the preparation of products with activity against fish parasites. Background Art

[0002] Fish parasitic diseases are one of the important threats faced by the aquaculture industry. Parasites represented by Uronema marinum can cause damage to the fish body surface, secondary infections, and large-scale deaths, resulting in serious economic losses. According to statistics, the annual loss of the global aquaculture industry due to parasitic diseases exceeds billions of US dollars. The reduction rate of production caused by parasite outbreaks in the main aquaculture areas of China is as high as 20%-30%, seriously restricting the sustainable development of the industry.

[0003] At present, the prevention and control of fish parasitic diseases mainly rely on chemical agents such as trichlorfon and cypermethrin. However, long-term and high-dose use has led to a significant increase in parasite resistance. For example, after continuous application of trichlorfon, the lethal concentration for Dactylogyrus spp. increased from the initial 0.5 mg / L to 5 mg / L, and the resistance increased by 10 times. The resistance of Sinibrama wui to trichlorfon increased significantly after continuous medication, resulting in a decline in the prevention and control effect. More seriously, chemical agents are likely to remain in the environment, posing a threat to non-target organisms and human health.

[0004] Biological control has become an important research direction due to its environmentally friendly characteristics. For example, the metabolites of marine fungi or soil actinomycetes can kill parasites, and have a 100% lethal effect on Sinibrama wui at a concentration of 1.2 μg / mL. The active ingredients extracted from Solanum nigrum and Periploca sepium can effectively expel and kill external parasites such as Gyrodactylus spp., and have low toxicity to the host fish and are environmentally friendly. The infection of gill parasites in grass carp will affect the intestinal microbial community through the "distant interaction dialogue" mechanism. Oral probiotics may enhance the host immunity by regulating the intestinal flora, providing a new idea for biological control. However, biological resources with high killing activity and suitable for the aquaculture environment are still scarce, and the large-scale preparation process of related preparations and the application plan in the aquaculture water body need to be urgently broken through. In addition, the biological control methods for high-value fish such as flounder and turbot are still insufficient.

[0005] In recent years, the ecological regulation role of microorganisms in the microbial loop has provided a new idea for ecological prevention and control, and the predation relationship between its prey and zooplankton contains a dynamic defense mechanism. For example, specific bacteria enhance the host defense traits through metabolites, or lyse DMSP to produce acrylic acid to directly resist predators. These mechanisms indicate that regulating the natural defense network of the microbial community can replace chemical intervention, establish an ecological barrier, and achieve sustainable prevention and control. Therefore, developing the ecological prevention and control of fish parasitic diseases and establishing an efficient and safe parasite prevention and control system are of great significance for promoting the green transformation of aquaculture. SUMMARY OF THE INVENTION

[0006] In view of the above existing technical problems, the primary object of the present invention is to provide the use of Prochlorococcus in the preparation of a product for inhibiting the activity of fish parasites and / or reducing the infection rate of fish parasites.

[0007] The second object of the present invention is to provide the use of Prochlorococcus in the preparation of a product for promoting the growth of fish.

[0008] To achieve the above objects, the present invention is realized through the following technical solutions:

[0009] The present invention claims the use of Prochlorococcus in the preparation of a product for inhibiting the activity of fish parasites and / or reducing the infection rate of fish parasites.

[0010] Through research, the present invention has found that Prochlorococcus can effectively kill fish parasites, significantly improve the survival rate of fish parasitized by fish parasites, and reduce the infection rate of fish to fish parasites. The above findings of the present invention can solve the problems of high cost, poor safety, and serious pollution existing in the prevention and treatment of fish parasite diseases with chemical drugs. Prochlorococcus can also be used as bait to promote the growth of certain fish, with significant ecological and economic benefits.

[0011] Preferably, in the present invention, the parasite is at least one of the genus Uronema, Pseudocohnilembus, Paralembus, Kudoa.

[0012] Preferably, in the present invention, the parasite is at least one of Uronema marinum, Pseudocohnilembus persalinus, Paralembus digitiformis, Kudoa rayformis.

[0013] Preferably, the fish is at least one of the genus Scophthalmus, Polyprion, Sparidae, Pleuronectiformes, Sardina.

[0014] Preferably, the fish is at least one of the genus Scophthalmus (such as Scophthalmus maximus), the genus Polyprion (such as Polyprion oxygeneios), the genus Paralichthys, the genus Cynoglossus, the genus Hippoglossus, the genus Platichthys, the genus Pagrus, the genus Sparus, the genus Acanthopagrus, the genus Rhabdosargus, the genus Parargyrrops, the genus Sardina, the genus Sardinops, and the genus Sardinella.

[0015] Preferably, the inhibition of fish parasite activity and / or the reduction of fish parasite infection rate are carried out cyclically under light conditions and dark conditions.

[0016] Preferably, the light conditions are: light intensity 35 - 100 μmole / m 2 / s.

[0017] Preferably, the Prochlorococcus is a culture of Prochlorococcus.

[0018] Preferably, in the culture, the concentration of Prochlorococcus is 2 - 5×10 8 cells / mL.

[0019] Preferably, the Prochlorococcus is preserved in the Provasoli - Guillard National Center for Marine Phytoplankton Culture Collection of the United States, with the preservation number CCMP1986 or CCMP2969.

[0020] Furthermore, the present invention also claims protection for an ecological prevention and control method for fish parasitic diseases based on the microbial food web, comprising the following steps:

[0021] S1. Inoculate Prochlorococcus into a culture medium for large - scale culture to obtain a Prochlorococcus culture;

[0022] S2. Uniformly apply the Prochlorococcus culture to a culture water body containing fish parasites, or to a culture water body in which fish containing fish parasites are cultured;

[0023] S3. The culture water body is cycled under light and dark conditions.

[0024] Preferably, in step S1, the volume ratio of Prochlorococcus to the culture medium is 1:5 - 15. More preferably, the volume ratio of Prochlorococcus to the culture medium is 1:10.

[0025] Preferably, in the step S1, the culture medium comprises: 28 - 29 g / L sodium chloride, 6 - 7 g / L magnesium sulfate heptahydrate, 5 - 6 g / L magnesium chloride hexahydrate, 0.6 - 0.7 g / L potassium chloride, 1.4 - 1.5 g / L calcium chloride dihydrate, 0.1 - 0.3 g / L sodium bicarbonate, 0.05 - 0.06 g / L ammonium sulfate, 0.03 - 0.04 mM sodium dihydrogen phosphate.

[0026] More preferably, the culture medium comprises: 28.10 g / L sodium chloride, 6.90 g / L magnesium sulfate heptahydrate, 5.49 g / L magnesium chloride hexahydrate, 0.67 g / L potassium chloride, 1.47 g / L calcium chloride dihydrate, 0.20 g / L sodium bicarbonate, 0.058 g / L ammonium sulfate, 0.036 mM sodium dihydrogen phosphate.

[0027] Preferably, in the step S1, the time for the scale-up culture is 7 - 10 days.

[0028] Preferably, in the step S1, the concentration of the Prochlorococcus culture is 2 - 5×10 8 cells / mL.

[0029] Preferably, in the step S2, the volume ratio of the Prochlorococcus culture to the aquaculture water body is 1:20 - 40. More preferably, the volume ratio of the Prochlorococcus culture to the aquaculture water body is 1:30.

[0030] Preferably, in the step S3, the aquaculture water body is circulated under 10 h light / 14 h darkness for 2 - 4 days.

[0031] Preferably, in the steps S1 to S3, the culture environment is 20 - 25 °C, and the light intensity is 35 - 100 μmole / m 2 / s.

[0032] Furthermore, the present invention also claims the application of Prochlorococcus in the preparation of products for promoting the growth of fish.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The Prochlorococcus in the present invention has an excellent inhibitory effect on the activity of fish parasites and can reduce the infection rate of fish by fish parasites. The present invention solves the problems of high cost, poor safety, and serious pollution existing in the prevention and treatment of fish parasite diseases by chemical drugs. Prochlorococcus can also be used as bait to promote the growth of certain fish, having significant ecological and economic benefits. Description of the Drawings

[0035] Figure 1 It is a schematic diagram showing the change of fish parasite concentration over time in Example 1.

[0036] Figure 2 Schematic diagram of the change of fish parasite infection rate over time in Examples 2-3 and Comparative Examples 1-2. Detailed implementation mode

[0037] The present invention will be further described below in conjunction with the specification and specific embodiments. However, the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0038] In the present invention, the culture medium used includes: 28.10 g / L sodium chloride, 6.90 g / L magnesium sulfate heptahydrate, 5.49 g / L magnesium chloride hexahydrate, 0.67 g / L potassium chloride, 1.47 g / L calcium chloride dihydrate, 0.20 g / L sodium bicarbonate, 0.058 g / L ammonium sulfate, 0.036 mM sodium dihydrogen phosphate.

[0039] Prochlorococcus A, sourced from the Provasoli-Guillard National Center for Marine Phytoplankton Culture Collection, USA, with the preservation number CCMP1986.

[0040] Prochlorococcus B, sourced from the Provasoli-Guillard National Center for Marine Phytoplankton Culture Collection, USA, with the preservation number CCMP2969.

[0041] Fish parasites (Marine trichodina, Pseudocohnilembus persalinus, Paranophrys digitata, Myxosporea) are from the Hong Kong University of Science and Technology, China, and the fish are from the Huangsha Seafood Market in Guangzhou, China.

[0042] Example 1

[0043] (1) Take 100 mL of Prochlorococcus A that has been continuously passaged 3 times under the condition of a light intensity of 70 μmole / m 2 / s, add it to 1000 mL of the culture medium, and expand the culture for 6 days to make its concentration reach 1×10 7 cells / mL to obtain a Prochlorococcus culture. Prochlorococcus B is operated in the same way according to the above method.

[0044] (2) Add Marine trichodina, Pseudocohnilembus persalinus, Paranophrys digitata, and Myxosporea to each portion of the Prochlorococcus culture in step (1) (wherein, in the Prochlorococcus culture of Prochlorococcus A, Marine trichodina and Pseudocohnilembus persalinus are added respectively; in the Prochlorococcus culture of Prochlorococcus B, Paranophrys digitata and Myxosporea are added respectively) so that the final concentration of fish parasites in the Prochlorococcus culture reaches more than 500 cells / mL.

[0045] (3) Put the original Chlorella culture in step (2) into an illumination incubator. The culture environment is 20 - 25 °C, and the light intensity is 70 μmole / m 2 / s. Set it to be turned on for 10 hours (under light conditions) and turned off for 14 hours (under dark conditions) every day, and run continuously for 2 days.

[0046] Example 2

[0047] (1) Take 200 mL of Chlorella A that has been continuously passaged 3 times under the condition of a light intensity of 70 μmole / m 2 / s, add it to 2000 mL of culture medium, and expand the culture for 9 days to make its concentration reach 1×10 7 cells / mL to obtain a Chlorella culture.

[0048] (2) Add 2 L of the Chlorella culture in step (1) to the aquaculture water body (60 L) where four turbot infected with Uronema marinum are cultured, and run an oxygen pump with a power of 3 W for 10 min to evenly distribute the Chlorella culture.

[0049] (3) Fix an LED lighting fixture with a power of 120 W on the top of the fish tank, 10 cm away from the water surface of the aquaculture water body. Set it to be turned on for 10 hours (under light conditions) and turned off for 14 hours (under dark conditions) every day, and the light intensity is 70 μmole / m 2 / s, and run continuously for 4 days.

[0050] Example 3

[0051] (1) Take 250 mL of Chlorella B that has been continuously passaged 3 times under the condition of a light intensity of 70 mole / m 2 / s, add it to 2500 mL of culture medium, and expand the culture for 8 days to make its concentration reach 1×10 7 cells / mL to obtain a Chlorella culture.

[0052] (2) Add 2.5 L of the Chlorella culture in step (1) to the aquaculture water body (75 L) where four sardines infected with Myxosporea are cultured, and run an oxygen pump with a power of 3 W for 13 min to evenly distribute the Chlorella culture.

[0053] (3) Fix an LED lighting fixture with a power of 120 W on the top of the fish tank, 10 cm away from the water surface of the aquaculture water body. Set it to be turned on for 10 hours (under light conditions) and turned off for 14 hours (under dark conditions) every day, and the light intensity is 70 μmole / m 2 / s, and run continuously for 4 days.

[0054] Comparative Example 1

[0055] The difference between this comparative example and Example 2 is as follows: In step (2), four turbot infected with Uronema marinum of the same batch as in Example 2 were selected, and the Prochlorococcus culture was not added.

[0056] Comparative Example 2

[0057] The difference between this comparative example and Example 3 is as follows: In step (2), four sardines infected with Myxosporea of the same batch as in Example 3 were selected, and the Prochlorococcus culture was not added.

[0058] Test Example

[0059] (1) After staining the culture after 2 days of continuous operation in step (3) of Example 1 with Lugol's iodine solution, a fluorescence inverted microscope was used to count the fish parasites. The results are shown in Figure 1 and Table 1.

[0060] (2) Examples 2 and 3 and Comparative Examples 1 and 2 were each repeated 3 times to determine the infection status of fish with fish parasites; among them, when at least one of the following symptoms appeared in turbot: body surface congestion, ulceration, increased mucus secretion, skin tissue necrosis, red and swollen eyes, wild swimming, rubbing against the edge of the culture pond or floating, it was determined that the fish was still infected with parasites; if none of the above symptoms appeared, it was determined that the fish was no longer infected with parasites. When at least one of the following symptoms appeared in sardines: tumor-like cysts on the body surface, swollen gill filaments, increased mucus, pustule-like protrusions, wild swimming, spinning, jumping, it was determined that the fish was still infected with parasites; if none of the above symptoms appeared, it was determined that the fish was no longer infected with parasites. Based on the above judgment, the fish parasite infection rate was calculated (retaining one decimal place). An electronic scale was used to weigh the fish. The final test results are shown in Figure 2 and Table 2.

[0061] Table 1 Changes in the concentrations of Prochlorococcus and Uronema marinum over time in Example 1

[0062]

[0063] Table 2 Changes in fish infection rate and weight over time in Examples 2 - 3 and Comparative Examples 1 - 2

[0064]

[0065] As Figure 1 and shown in Table 1, it is indicated that Prochlorococcus can effectively kill fish parasites, and the killing rate can reach over 90% in 48 hours.

[0066] As Figure 2As shown in Table 2, Prochlorococcus marinus can effectively reduce the infection rate of fish by fish parasites. Compared with the case where the Prochlorococcus marinus culture is not applied, the infection rate of turbot is reduced by 75%, and the infection rate of sardine is reduced by 66.7%. In addition, applying the Prochlorococcus marinus culture can promote the growth of sardines.

[0067] The foregoing examples are merely illustrative and are used to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as can be contemplated, and the embodiments presented herein are supported by the applicant's actual test results. Therefore, it is the applicant's intention that the appended claims not be limited by the selection of examples that illustrate the features of the present invention. Some of the numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be construed as being covered by the appended claims whenever possible.

Claims

1. Use of Prochlorococcus in preparing products for inhibiting the activity of fish parasites and / or reducing the infection rate of fish parasites.

2. The application according to claim 1, characterized in that: The parasite is at least one of the genera Caudata, Pseudoconidium, Pseudoconidium and Myxosomes.

3. The application according to claim 2, characterized in that: The parasite is at least one of marine filaria, water droplet pseudoconchocystis, digitate-shaped boatworm, and myxosporea.

4. The use according to claim 1, characterized in that: The fish is at least one of turbot, trout, sparid, flounder and sardine.

5. The application according to claim 4, characterized in that: The fish is at least one of the genera of turbot, sea bass, tooth flounder, tongue sole, tooth flounder, flatfish, red sea bream, black sea bream, yellow sea bream, flat sea bream, two-spined sea bream, sardine, pseudo-sardinella, and sardinella.

6. The use according to claim 1, characterized in that: The inhibition of fish parasite activity and / or the reduction of fish parasite infection rate is carried out under cycles of light conditions and dark conditions.

7. The use according to claim 6, characterized in that: Light conditions: light intensity 35-100 μmole / m 2 / s.

8. The use according to claim 1, characterized in that: The Prochlorococcus algae is a culture of Prochlorococcus algae.

9. The use according to claim 8, characterized in that: In the culture, the concentration of Prochlorococcus is 2-5×10 8 Pieces / mL.

10. Use of Prochlorococcus in preparing products for promoting fish growth.

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