A method for selecting sea urchin Strongylocentrotus intermedius resistant to black mouth disease

Abstract

The present invention discloses a method for selecting sea urchins of Strongylocentrotus intermedius resistant to black mouth disease, which is carried out successively according to the following steps: First, induce spawning in female and male sea urchins respectively and perform artificial insemination. After artificial insemination, use conventional methods for seedling cultivation to construct a breeding population, and then conduct seed selection. Transfer the selected candidate seedlings to a temporary culture pool for temporary culture for 24h - 72h. Prepare a dedicated infection pool, add Vibrio echinodermatis to the infection pool, transfer the candidate seedlings to the infection pool for infection, pick out the diseased individuals at regular intervals. When the proportion of diseased individuals reaches 50 - 90%, conduct the last water change, then lower the water temperature to 1 - 3°C, and add ammonium chloride to the infection pool. Under the condition of adding ammonium chloride, change the water once every three days and continuously maintain for 9 - 15 days. Then select the surviving seedlings, and cultivate these seedlings to sexual maturity by conventional methods to obtain the breeding sea urchins of Strongylocentrotus intermedius resistant to black mouth disease.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture and breeding of seafood products, and particularly to a method for selecting resistant-to-black-mouth-disease Strongylocentrotus intermedius Background Art

[0002] Strongylocentrotus intermedius is the main cultured sea urchin species in China, and its aquaculture methods are mainly raft culture and bottom seeding proliferation in natural waters. Black-mouth disease is a common disease in the aquaculture process of Strongylocentrotus intermedius. The outbreak of black-mouth disease will lead to large-scale death of Strongylocentrotus intermedius, causing huge losses to aquaculture enterprises. To prevent and control black-mouth disease, existing studies have proposed "a traditional Chinese medicine preparation for preventing and treating sea urchin black-mouth disease and red spot disease and its preparation method", and "a compound Chinese herbal medicine preparation for treating sea urchin black-mouth disease" and other methods. However, due to the open-sea aquaculture method being not suitable for administering drugs or probiotic products to control diseases, it is very necessary to breed a new variety of Strongylocentrotus intermedius with strong disease resistance.

[0003] Currently, existing studies have used the method of family selection to breed disease-resistant strains of Strongylocentrotus intermedius [Jiang Huijie, Zhang Weijie, Chen Long, et al. Screening of disease-resistant families of Strongylocentrotus intermedius and analysis of their growth performance [J]. Journal of Dalian Ocean University, 2020, 35(2): 198-204]. Through natural infection, the survival rates of each family are compared, and the families with high survival rates are selected as candidates. However, for the method of selecting seeds within a family, this method does not mention it, and due to its complex operation, large investment, long cycle, the implementation difficulty is relatively large. In the disease-resistant breeding process of many aquatic animals, the conventional method is to use pathogens to challenge the animals and then select the surviving individuals as seeds. However, in sea urchins, we found that it is difficult to accurately control the mortality rate after challenge. In most cases, the mortality rate of sea urchins after challenge is very high, almost reaching 100%. It can be said that if the mortality rate after challenge cannot be controlled, it is difficult to smoothly complete the seed selection work.

[0004] Therefore, there is a need for a method that can solve the above problems now. Summary of the Invention

[0005] The present invention is to solve the above-mentioned deficiencies existing in the prior art, and proposes a method for selecting resistant-to-black-mouth-disease Strongylocentrotus intermedius that is simple and easy to operate and can effectively reduce the mortality rate after challenge.

[0006] The technical solution of the present invention is: a method for selecting resistant-to-black-mouth-disease Strongylocentrotus intermedius, characterized in that the method is carried out in the following steps in sequence:

[0007] A. First, induce spawning in female and male sea urchins respectively and perform artificial insemination. After artificial insemination, use the conventional method for seedling cultivation to construct a breeding population.

[0008] B. Then, when the shell diameter of the seedlings in the breeding population is between 1 - 3 cm, seed selection is carried out.

[0009] C. Place the selected candidate seedlings in a temporary rearing pond for 24h - 72h. During the temporary rearing period, the water temperature is controlled at 5℃ - 9℃.

[0010] D. Prepare a dedicated infection pond. After adjusting the water temperature of the infection pond to be the same as that of the temporary rearing pond, add Vibrio echinodermatis to the infection pond until the final concentration reaches 1×10 3 ~1×10 4 cfu / mL.

[0011] E. Transfer the candidate seedlings to the infection pond for infection. Change the water once every 24h. After changing the water, add Vibrio echinodermatis to the infection pond to ensure that the final concentration of Vibrio echinodermatis in the infection pond remains unchanged. At the same time, pick out the diseased individuals.

[0012] F. When the proportion of diseased individuals reaches 50 - 90%, conduct the last water change. After changing the water, check the seedlings. If there are newly emerged diseased individuals, pick them out. Lower the water temperature to 1 - 3℃, and add ammonium chloride to the infection pond until the final concentration reaches 20.5 - 41 mg / L.

[0013] G. Under the condition of maintaining the water temperature at 1 - 3℃ and the ammonium chloride concentration at 20.5 - 41 mg / L, change the water once every three days.

[0014] H. Continuously maintain the above conditions for 9 - 15 days in step G. Then select the surviving seedlings and culture these seedlings to sexual maturity using conventional methods to obtain the broodstock of the sea urchin Strongylocentrotus intermedius resistant to black mouth disease.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] In the traditional technology, there are only reports on cultivating disease-resistant strains of the sea urchin Strongylocentrotus intermedius, but there is no seed selection method within the same family or a certain breeding population. In view of the above problems, the present invention discloses a seed selection method for the sea urchin Strongylocentrotus intermedius resistant to black mouth disease. Using this method for seed selection of the sea urchin Strongylocentrotus intermedius will not cause a large number of candidate seedlings to die in a short time. During the operation process, the infection operation can be stopped in time according to the elimination rate. After screening by this method, the disease resistance of the offspring of the seedlings is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Survival rates of the selected group and the control group after infection (repeated 3 times, mean ± standard deviation). DETAILED DESCRIPTION OF THE INVENTION

[0018] The following will describe the specific embodiments of the present invention in conjunction with the drawings as follows.Figure 1 As shown: A method for selecting strongylocentrotus intermedius resistant to black mouth disease, which is carried out successively according to the following steps:

[0019] First, induce spawning in female and male sea urchins respectively and perform artificial insemination. After artificial insemination, use conventional methods for seedling cultivation to construct a breeding population; then, when the shell diameter of the seedlings in the breeding population is between 1 - 3 cm, conduct seed selection; place the selected candidate seedlings in a temporary rearing pool for 24h - 72h, and control the water temperature during the temporary rearing period at 5°C - 9°C; prepare a dedicated infection pool. After adjusting the water temperature of the infection pool to be the same as that of the temporary rearing pool, add Vibrio echinicola to the infection pool until the final concentration reaches 1×10 3 ~1×10 4 cfu / mL; transfer the candidate seedlings to the infection pool for infection, change the water once every 24h, and add Vibrio echinicola to the infection pool after each water change to ensure that the final concentration of Vibrio echinicola in the infection pool remains unchanged, and at the same time pick out the diseased individuals; when the proportion of diseased individuals reaches 50 - 90%, conduct the last water change, and check the seedlings after the water change. If there are newly emerged diseased individuals, pick them out, lower the water temperature to 1 - 3°C, and add ammonium chloride to the infection pool until the final concentration reaches 20.5 - 41 mg / L; under the condition of maintaining the water temperature at 1 - 3°C and the ammonium chloride concentration at 20.5 - 41 mg / L, change the water once every three days; continuously maintain the above step for 9 - 15 days, and then select the surviving seedlings. Cultivate these seedlings to sexual maturity using conventional methods to obtain the broodstock of strongylocentrotus intermedius resistant to black mouth disease.

[0020] Example:

[0021] Select 20 female sea urchins and 20 male sea urchins, induce spawning in them respectively and perform artificial insemination. After artificial insemination, use conventional methods for seedling cultivation to construct a breeding population; then, when the shell diameter of the seedlings in the breeding population is 2.5 cm, conduct seed selection; select 200 candidate seedlings and place them in a temporary rearing pool for 24h, and control the water temperature during the temporary rearing period at 9°C;

[0022] Prepare a dedicated infection pool. After adjusting the water temperature of the infection pool to be the same as that of the temporary rearing pool, add Vibrio echinicola to the infection pool until the final concentration reaches 5×10 3 cfu / mL; transfer the candidate seedlings to the infection pool for infection treatment. During the infection treatment period, change the water once every 24h, and add Vibrio echinicola to the infection pool after each water change to ensure that the final concentration of Vibrio echinicola in the infection pool is always 5×10 3 cfu / mL, and at the same time pick out the diseased individuals;

[0023] On the 7th day of infection treatment, a total of 110 infected individuals were selected, and the infection rate reached 55%. At this time, the last water change was carried out, and the seedlings were inspected after the water change. If there were newly emerged diseased individuals, they were picked out. The water temperature was reduced to 2°C, and ammonium chloride was added to the infected pool to a final concentration of 20.5 mg / L;

[0024] Under the condition of maintaining a water temperature of 2°C and an ammonium chloride concentration of 20.5 mg / L, the water was changed every three days; the above step was continuously maintained for 9 days, and then a total of 85 surviving seedlings were selected, with a survival rate of 94.4%. These seedlings were cultured to sexual maturity by conventional methods, and the seed gall of the sea urchin Strongylocentrotus intermedius resistant to black mouth disease was obtained.

[0025] Using 85 sea urchins selected through the above steps as parents to breed offspring. When the offspring seedlings reached 2.5 cm, 200 offspring of the selected breeding group and 200 offspring of the control group were taken and infected with Vibrio echinodermatis again. The survival rate of the selected breeding group was 78.3%, while that of the control group was 53.7%. Through Figure 1 it can be seen that the survival rate of the selected breeding offspring increased by 24.6%.

Claims

1. A method for selecting sea urchin Strongylocentrotus intermedius resistant to black mouth disease, characterized in that, The described method is carried out successively according to the following steps: A. First, induce spawning in female and male sea urchins respectively and conduct artificial insemination. After artificial insemination, use conventional methods for seedling cultivation to construct a breeding population. B. Then, select seeds when the shell diameter of the seedlings in the breeding population is between 1 - 3 cm. C. Place the selected candidate seedlings in a temporary rearing pool for 24h - 72h, and control the water temperature during the temporary rearing period at 5°C - 9°C. D. Prepare a dedicated infection pool. After adjusting the water temperature of the infection pool to be the same as that of the temporary rearing pool, add Vibrio echinodermatis to the infection pool until the final concentration reaches 1×10 3 ~1×10 4 cfu / mL. E. Transfer the candidate seedlings to an infection pool for infection, change the water once every 24h, and add Vibrio echinodermatis to the infection pool after water change to ensure that the final concentration of Vibrio echinodermatis in the infection pool remains unchanged. At the same time, pick out the diseased individuals. F. When the proportion of diseased individuals reaches 50 - 90%, conduct the last water change, and check the seedlings after water change. Pick out any newly emerged diseased individuals, lower the water temperature to 1 - 3°C, and add ammonium chloride to the infection pool to a final concentration of 20.5 - 41 mg / L. G. Under the conditions of maintaining a water temperature of 1 - 3°C and an ammonium chloride concentration of 20.5 - 41 mg / L, change the water once every three days. H. Continuously maintain the conditions in step G for 9 - 15 days, then select the surviving seedlings and cultivate these seedlings to sexual maturity using conventional methods, thus obtaining the broodstock of Strongylocentrotus intermedius resistant to black mouth disease.

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