Use of prochlorophytes in the preparation of products active against fish parasites

Abstract

The application belongs to the technical field of aquaculture, and particularly relates to application of protochloris vulgaris in preparation of products with activity of inhibiting fish parasites. Specifically, the application discloses application of protochloris vulgaris in preparation of products with activity of inhibiting fish parasites and / or reducing infection rate of fish parasites. Protochloris vulgaris has a significant killing effect on fish parasites, can significantly reduce the infection rate of fish to fish parasites, and can promote the growth of fish. The protochloris vulgaris is used to inhibit fish parasites in the application, no chemical drug residue is left, and the problems of drug resistance and environmental pollution caused by existing chemical repellents are effectively solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquaculture, and particularly relates to application of Prototheca in preparation of a product for inhibiting activity of fish parasites. BACKGROUND

[0002] Fish parasite disease is one of the important threats faced by the aquaculture industry, among which the parasites represented by Uronema marinum can cause fish body surface damage, secondary infection and large-scale death, causing serious economic losses.

[0003] Currently, the prevention and treatment of fish parasite diseases mainly relies on chemical agents such as trichlorphon and cypermethrin. However, long-term and large-dose use leads to significant increase in parasite drug resistance, for example, the lethal concentration of trichlorphon to Pseudodactylogyrus sp. increases from the initial 0.5 mg / L to 5 mg / L, with a 10-fold increase in drug resistance. The drug resistance of Chinese lizardfish to trichlorphon significantly increases after continuous use, resulting in decreased control effect. More seriously, chemical agents are prone 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 100% lethal effect on Chinese lizardfish at a concentration of 1.2 μg / mL. The active ingredients extracted from Solanum nigrum and Berchemia floribunda can effectively kill in vitro parasites such as trichodinids, and have low toxicity to host fish and are environmentally friendly. Gill parasite infection of grass carp can affect intestinal microbial community through the "distal cross-talk" mechanism, and oral administration of probiotics may enhance the host's immunity by regulating intestinal flora, providing a new idea for biological control. However, there is still a lack of biological resources with high-efficiency killing activity suitable for aquatic environments, and the large-scale preparation process of related preparations and the application scheme in aquaculture water bodies need to be broken through. In addition, the biological control methods for high-value fish such as flatfish and turbot are still insufficient.

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

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

[0007] A second object of the present application is to provide an application of Protochlorococcum in preparing a product for promoting the growth of fish.

[0008] To achieve the above object, the present application is implemented by the following technical solutions.

[0009] The present application claims an application of Protochlorococcum in preparing a product for inhibiting the activity of fish parasites and / or reducing the infection rate of fish parasites.

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

[0011] Preferably, in the present application, the parasites are at least one of Uronema, Pseudocohnilembus, Paralembus, and Kudoa.

[0012] Preferably, in the present application, the parasites are at least one of Uronema marinum, Pseudocohnilembus persalinus, Paralembus digitiformis, and Kudoa rayformis.

[0013] Preferably, the fish is at least one of Scophthalmus, Polyprion, Sparidae, Pleuronichthys, and Sardinella.

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

[0015] Preferably, the inhibiting fish parasite activity and / or reducing fish parasite infection rate is cycled under light condition and dark condition.

[0016] Preferably, the light condition is: 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 x 10 8 / mL.

[0019] Preferably, the Prochlorococcus is preserved in the U.S. Provasoli-Guillard National Center for Marine Algae and Microbiota with the preservation number of CCMP1986 or CCMP2969.

[0020] Further, the present application also claims a fish parasite disease ecological prevention and control method based on micro-food ring, comprising the following steps:

[0021] S1, inoculating Prochlorococcus into culture medium for expansion culture to obtain Prochlorococcus culture;

[0022] S2, uniformly applying the Prochlorococcus culture to the aquaculture water body containing fish parasites, or the aquaculture water body containing fish containing fish parasites;

[0023] S3, cycling the aquaculture water body under light and dark.

[0024] Preferably, in the step S1, the volume ratio of Prochlorococcus and culture medium is 1:5-15. Further preferably, the volume ratio of Prochlorococcus and 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 phosphate monobasic.

[0026] Further 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 phosphate monobasic.

[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 x 10 8 / mL.

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

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

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

[0032] Further, the present application also claims the use of Prochlorococcus in the preparation of a product for promoting the growth of fish.

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

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

[0035] Figure 1 Figure 1 is a schematic diagram of the change of the concentration of fish parasites with time in Example 1.

[0036] Figure 2 Fig. 2 is a graph showing the infection rate of fish parasites over time according to Example 2-3 and Comparative Example 1-2. DETAILED DESCRIPTION

[0037] The present application is further illustrated by the following description and examples, which do not limit the present application in any manner. Unless otherwise specified, the reagents, methods and apparatus employed in the present application are those conventional in the art.

[0038] In the present application, the culture medium 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, and 0.036 mM sodium dihydrogen phosphate.

[0039] Prochlorococcus A was obtained from the Provasoli-Guillard National Center for Marine Algae and Microbiota, USA, and has the accession number CCMP1986.

[0040] Prochlorococcus B was obtained from the Provasoli-Guillard National Center for Marine Algae and Microbiota, USA, and has the accession number CCMP2969.

[0041] The fish parasites (Marine Haemogregarina, Pseudokarlingia aquadropi, Parapontonia davisii, Myxospora) were obtained from the Hong Kong University of Science and Technology, China, and the fish were obtained from the Huangsha Seafood Market, Guangzhou, China.

[0042] Example 1

[0043] (1) 100 mL of Prochlorococcus A, which had been subcultured three times under the condition of an illumination intensity of 70 μmole / m 2 / s, was added to 1000 mL of culture medium, and expanded culture was performed for 6 days to obtain a Prochlorococcus culture having a concentration of 1 x 10 7 / mL. Prochlorococcus B was subjected to the same operation as described above.

[0044] (2) Marine Haemogregarina, Pseudokarlingia aquadropi, Parapontonia davisii, and Myxospora were added to each of the Prochlorococcus cultures of step (1) (in which Marine Haemogregarina and Pseudokarlingia aquadropi were added to the Prochlorococcus culture of Prochlorococcus A, and Parapontonia davisii and Myxospora were added to the Prochlorococcus culture of Prochlorococcus B), so that the final concentration of the fish parasites in the Prochlorococcus culture was 500 or more per mL.

[0045] (3) The Prochlorococcus culture of step (2) was placed in a light incubator, the culture environment was 20-25℃, the light intensity was 70 μmole / m 2 / s, and the daily 10-hour opening (light condition) and 14-hour closing (dark condition) were set, and the continuous operation was 2 days.

[0046] Example 2

[0047] (1) 200 mL of Prochlorococcus A which was continuously subcultured for 3 times under the light intensity of 70 μmole / m 2 / s was added into 2000 mL of culture medium, and the expansion culture was performed for 9 days to make the concentration reach 1×10 7 / mL, and the Prochlorococcus culture was obtained.

[0048] (2) 2 L of the Prochlorococcus culture of step (1) was added into the aquaculture water body (60 L) in which the four sea flounder infected with the marine filarid worm were bred, and the oxygen pump with the power of 3 W was operated for 10 min to make the Prochlorococcus culture uniformly distributed.

[0049] (3) The LED lighting fixture with the power of 120 W was fixed on the top of the fish tank, which was 10 cm away from the water surface of the aquaculture water body, the daily 10-hour opening (light condition) and 14-hour closing (dark condition) were set, the light intensity was 70 μmole / m 2 / s, and the continuous operation was 4 days.

[0050] Example 3

[0051] (1) 250 mL of Prochlorococcus B which was continuously subcultured for 3 times under the light intensity of 70 mole / m 2 / s was added into 2500 mL of culture medium, and the expansion culture was performed for 8 days to make the concentration reach 1×10 7 / mL, and the Prochlorococcus culture was obtained.

[0052] (2) 2.5 L of the Prochlorococcus culture of step (1) was added into the aquaculture water body (75 L) in which the sardines infected with the myxosporean were bred, and the oxygen pump with the power of 3 W was operated for 13 min to make the Prochlorococcus culture uniformly distributed.

[0053] (3) The LED lighting fixture with the power of 120 W was fixed on the top of the fish tank, which was 10 cm away from the water surface of the aquaculture water body, the daily 10-hour opening (light condition) and 14-hour closing (dark condition) were set, the light intensity was 70 μmole / m 2 / s, and the continuous operation was 4 days.

[0054] Comparative Example 1

[0055] The difference between the present comparative example and Example 2 is that in step (2), four infected marine threadworms of the same batch as in Example 2 are selected, and no Prochlorococcus culture is added.

[0056] Comparative Example 2

[0057] The difference between the present comparative example and Example 3 is that in step (2), four infected myxosporan of the same batch as in Example 3 are selected, and no Prochlorococcus culture is added.

[0058] Test Example

[0059] (1) After the culture after 2 days of continuous operation in step (3) of Example 1 is dyed with Lugol's iodine solution, the fish parasites are counted using a fluorescence inverted microscope, and the results are shown in Figure 1 and Table 1.

[0060] (2) Each of Example 2, Example 3, and Comparative Example 1 and Comparative Example 2 is repeated 3 times to determine the infection of fish by fish parasites; when at least one of the following symptoms appears in the sciaenops ocellatus, such as body surface congestion, ulcer, increased mucus secretion, skin tissue necrosis, eye swelling, and swimming along the edge of the culture pond or floating, it is determined that the fish is still infected with parasites; if none of the above symptoms appears, it is determined that the fish is no longer infected with parasites. When at least one of the following symptoms appears in the sardines, such as body surface tumor cysts, gill filament swelling, mucus increase, pustule-like protrusions, frantic swimming, spinning, and jumping, it is determined that the fish is still infected with parasites; if none of the above symptoms appears, it is determined that the fish is no longer infected with parasites. Based on the above determination, the fish parasite infection rate (to one decimal place) is calculated. The fish is weighed using an electronic scale. The final test results are shown in Figure 2 and Table 2.

[0061] Table 1 Concentration of Prochlorococcus and marine threadworms in Example 1 changes with time

[0062]

[0063] Table 2 Fish infection rate and weight change with time in Examples 2-3 and Comparative Examples 1-2

[0064]

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

[0066] As shown in Figure 2As shown in Table 2, the protochlorophytes can effectively reduce the infection rate of fish to fish parasites. Compared with no protochlorophyte culture, the infection rate of turbot is reduced by 75%, and the infection rate of sardines is reduced by 66.7%. In addition, the application of protochlorophyte culture can promote the growth of sardines.

[0067] The foregoing examples are illustrative only and are not meant to limit the scope of the application as described in the claims. The claims appended hereto are intended to encompass the widest array of equivalents to the features described in the examples presented herein. Accordingly, the applicant(s) reserves the right to amend the claims to affirmatively disclaim non-essential features. The use of some of the ranges set out in the claims are intended to include the whole range of values of the sub-ranges within the range. Changes in the meaning of terms used in the claims are not to be construed as overcoming the meaning of the terms used in the examples presented herein.

Claims

1. A method of reducing the prevalence of fish parasite infection, characterized in that, The method comprises the following steps: S1, inoculating Prototheca into a culture medium for expansion culture to obtain a Prototheca culture; S2, uniformly applying the Prototheca culture to a culture water body in which fish containing fish parasites are bred; S3, cyclically performing the culture water body under light and darkness; In the culture of Prochlorococcus, the concentration of Prochlorococcus is 2-5 x 10 8 cells / mL; The parasites are at least one of marine Dracunculus, Myxospora; The fish is at least one of Scophthalmus, Sardinella, Pseudosaridella, and Harengula; Light conditions: light intensity 35-100 μmole / m 2 / s; In the step S3, the culture water body is cyclically performed under 10 h light / 14 h darkness, and the cycle is performed for 2-4 days; In the step S2, the volume ratio of the Prototheca culture to the culture water body is 1:20-40.

2. The method of claim 1, wherein, In the step S1, the volume ratio of the Prototheca to the culture medium is 1:5-15.

3. The method of claim 1, wherein, 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, and 0.03-0.04 mM sodium dihydrogen phosphate.

4. The method of claim 1, wherein, In the step S1, the expansion culture is performed for 7-10 days.

5. The method of claim 1, wherein, In the steps S1-S3, the culture environment is 20-25 ℃.

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