A method for breeding new species of razor clams resistant to Vibrio
By using Vibrio parahaemolytic stress and transgenerational immune sensitization technology, combined with the "northern breeding and south breeding" breeding method, new varieties with strong anti-Vibiral ability and rapid growth were successfully cultivated, and excellent varieties in the bristle breeding industry were solved. and the problem of insufficient disease resistance.
Patent Information
- Application Number
- CN202311840570.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-28
- Publication Date
- 2025-01-03
- Estimated Expiration
- 2043-12-28
AI Technical Summary
The lack of excellent varieties and poor disease resistance in the hairy breeding industry has led to a high breeding mortality rate. The existing immunology research has lagged, hindering the breeding process of hairy disease resistance in the hairy breeding.
The semi-lethal concentration of Vibrio parahaemolyticus stress adult hairs were used to screen anti-Vibiral species. Through cross-generation immune sensitization and 'Northern and South-nourishing' technology, the hairs were induced to produce heritable immune factors, and cultivate stable genetic and excellent traits. New varieties of Vibrio anti-Vibromi.
It significantly enhances the anti-Vibiral ability of the wool, shortens the breeding cycle, improves the breeding efficiency and the growth rate of the wool, and solves the problem of insufficient supply of excellent germplasm and disease-resistant varieties.
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Figure CN117678546B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic breeding of marine economic shellfish, in particular to a breeding method for a new Vibrio-resistant variety of razor clams. Background Art
[0002] Hairy Clam ( Scapharca subcrenata ) belongs to the phylum Mollusca, class Bivalvia, Clam ( Scapharca ) genus, commonly known as hairy clams, hemp clams, and corrugated shellfish, is an important economic shellfish in my country, mainly distributed in coastal mudflats such as Japan, North Korea and China. In China, it is distributed from the Yalu River in the north to Guangxi in the south. In the 1980s, the resources were most abundant in Laizhou Bay, Bohai Bay, Liaodong Bay and Haizhou Bay. Hairy clams are delicious, have high nutritional and medicinal value, and are deeply favored by consumers. .
[0003] In recent years, marine environmental pollution has intensified, and the quality of seawater has deteriorated year by year, leading to frequent Vibrio infections and frequent large-scale deaths of marine shellfish such as razor clams. Existing data show that the economic losses caused by the death of shellfish caused by pathogenic microorganisms such as Vibrio are as high as 2-3 billion yuan each year. There is currently a lack of targeted prevention and treatment methods. In addition, the lag in immunology-related research has hindered the selection and breeding of disease-resistant razor clams. Therefore, we urgently need to start with the razor clams themselves and use modern breeding technology to quickly cultivate high-quality, disease-resistant razor clams. New varieties (strains) to meet the needs of healthy and sustainable development of the razor clam seed industry and aquaculture industry. Summary of the invention
[0004] The invention aims to propose a breeding method for a new Vibrio-resistant variety of razor clam in view of the problems that the razor clam breeding industry lacks excellent varieties and the existing breeding varieties have poor disease resistance and high breeding mortality rate.
[0005] To achieve the above purpose, the present invention adopts the following technical solution:
[0006] A breeding method for a new Vibrio-resistant variety of razor clams comprises the following steps: using a semi-lethal concentration of Vibrio parahaemolyticus to stress adult razor clams, recovering 15% of the surviving individuals, and selecting 8% seed retention rate to obtain Vibrio-resistant razor clams, fattening the razor clams in an indoor workshop to culture them until their gonads are mature, and inducing the razor clams to produce heritable immune factors by using inactivated Vibrio parahaemolyticus before inducing spawning; then inducing the razor clams to lay eggs to obtain Vibrio-resistant strains of the F1 generation of razor clams, and then rapidly cultivating the F1 generation of razor clams to sexual maturity using the "north breeding and south raising" breeding and culture technology; thereafter, selecting seed clams by stress, enhancing the immunity of the offspring, and continuously breeding by "north breeding and south raising" every year to obtain a new Vibrio-resistant variety of razor clams with stable genetics and excellent traits.
[0007] Further,
[0008] 1) Screening Vibrio-resistant scaly clams by Vibrio stress: Use semi-lethal concentrations of Vibrio parahaemolyticus to stress adult scaly clams. When the mortality rate of scaly clams is close to 85%, stop stressing and place the scaly clams in regular aquaculture seawater to recover. Then, select large, healthy and intact scaly clams with an 8% seed retention rate as scaly clams for breeding the next generation;
[0009] 2) Enhance the immunity of the offspring of sculterella by using transgenerational immune sensitization: Cultivate the Vibrio-resistant sculterella seed shellfish until the gonads are mature, and inject inactivated Vibrio parahaemolyticus before inducing spawning to induce the sculterella to produce heritable immune factors, and then induce the sculterella to spawn by drying in the shade combined with warming seawater stimulation, that is, the Vibrio-resistant strain of the sculterella F1 generation;
[0010] 3) Adopt the breeding technology of "breeding in the north and rearing in the south" to quickly cultivate hairy clams to sexual maturity: cultivate the F1 generation of hairy clams resistant to Vibrio strain larvae obtained above in the northern hatchery indoors until metamorphosis and attachment, and then transport the hairy clams to the southern coastal mudflats for intermediate cultivation and growth, thereby realizing the "breeding in the north and rearing in the south" to cultivate hairy clams to sexual maturity;
[0011] 4) Breeding of new Vibrio-resistant varieties of razor clams: Cultivate the above-mentioned individuals to sexual maturity and continuously select them according to steps 1), 2) and 3) to obtain new Vibrio-resistant varieties of razor clams with genetic stability and excellent traits after they are genetically stable.
[0012] The adult razor clams in the above step 1 are wild adult razor clams, or disease-resistant adult razor clams; wherein the disease-resistant adult razor clams are F1 generation adults obtained by coercing the adult razor clams in step 1) and then culturing them.
[0013] In step 1), adult sculterella were placed in a solution with a final concentration of 3×10 7 cfu / ml Vibrio parahaemolyticus in a seawater culture tank. When the mortality rate of razor clams is close to 85%, the stress is stopped and the clams are placed in regular aquaculture seawater to recover. Then, large, healthy and intact clams are selected at an 8% seed retention rate as breeding shellfish for the next generation.
[0014] In the step 2), the water body for breeding the selected hairy clams is heated at a rate of 0.5-1.0°C / day, and the temperature is kept stable for 1 day after each continuous heating of 2°C. When the breeding water temperature rises to 23°C, the heating is stopped and the water temperature is kept stable at 23±0.5°C until the hairy clams gonads are fully mature, and then each hairy clams with mature gonads are injected with 3×10 8 cfu of heat-killed Vibrio parahaemolyticus were dried in the shade for 6 hours and placed in an incubation tank filled with filtered seawater at 25°C to induce spawning and sperm discharge, thus establishing a new Vibrio-resistant strain of the F1 generation of clams.
[0015] The heat-inactivated Vibrio parahaemolyticus is cultured in a liquid culture medium until the strain concentration is 3×10 8cfu, and then the suspension was treated in a water bath at 80°C for 30 minutes to obtain heat-killed Vibrio parahaemolyticus.
[0016] The Vibrio parahaemolyticus is a PL2 strain, which was donated by Qingdao Agricultural University and is described accordingly in the literature Lin, J., Ning, J., Lu, X., Chen, M., Cao, W., Wang, C., 2020. Transcriptomic analysis and expression of C-type lectins in response to Vibrio parahaemolyticus challenge in Scapharca subcrenata. Fish&Shellfish Immunology. 106, 365-373.
[0017] In the step 3), the larvae of the F1 generation of the new Vibrio-resistant strain of razor clams obtained above are cultured indoors in a northern nursery until they undergo metamorphosis and attach. After the razor clams seedlings grow to 100,000 to 150,000 grains per kilogram, they are transported to the southern coast by refrigerated trucks or by air transportation in insulated boxes with ice. The razor clams seedlings are then evenly sown on the mud flats for breeding. After the razor clams seedlings are cultured on the mud flats for 3 months, the size of the razor clams seedlings can reach 600 to 800 grains per kilogram. The large-sized razor clams seedlings are transferred to the bottom of nearby ponds for breeding. In early April of the following year, the large-sized razor clams seedlings can be cultured to sexual maturity for stress treatment and breeding of the next generation.
[0018] During the north-south transportation, the transportation temperature is controlled at 15° C. or below, and the transportation time is preferably within 20 hours.
[0019] The half-lethal concentration of Vibrio parahaemolyticus in the above-mentioned scorpion was set to a final concentration of 5×10 3 cfu / ml, 5×10 4 cfu / ml, 5×10 5 cfu / ml, 5×10 6 cfu / ml, 5×10 7 cfu / ml, 5×10 8 cfu / ml of Vibrio parahaemolyticus were used to infect adult sculter clams. Conventional marine culture was used as the control group. Three replicates were set up in each group, with 60 adult sculter clams in each replicate. The experiment lasted for 4 days. The half-lethal concentration of Vibrio parahaemolyticus in adult sculter clams was determined to be 3×10 7 cfu / ml.
[0020] The injection concentration of the inactivated Vibrio parahaemolyticus that effectively sensitizes adult sculterella is set at 3×10 5 cfu, 3×10 6 cfu, 3×107 cfu, 3×10 8 cfu, 3×10 9 cfu of inactivated Vibrio parahaemolyticus was used as the injection group, and PBS was injected as the control group. Three replicates were set up in each group, with 60 adult clams in each replicate. The experiment lasted for 4 days. The inactivated Vibrio parahaemolyticus injection concentration that effectively sensitized adult clams was determined by changes in indicators such as cell phagocytosis and apoptosis, immune enzyme activity, and gene expression. The concentration was determined to be 3×10 8 cfu.
[0021] The advantages of the present invention are:
[0022] (1) The present invention adopts a breeding technology scheme that combines stress breeding with modern molecular-assisted breeding to achieve precise breeding of target traits and significantly enhance the Vibrio resistance of the new Vibrio-resistant strain of razor clams.
[0023] (2) By using the transgenerational immunization method to significantly improve the anti-Vibrio ability of the offspring, a new variety of razor clams with broad-spectrum resistance to Vibrio can be cultivated, which significantly improves the ability of farmed razor clams to resist Vibrio infection.
[0024] (3) The present invention adopts the "north breeding and south rearing" razor clam seedling breeding and culture technology, making full use of the advantages of high water temperature, sufficient bait and few harmful organisms in the southern coastal mudflats, which can significantly increase the growth rate of razor clam. The new Vibrio-resistant razor clam strain reaches sexual maturity after 12 months of culture and can be used to breed the next generation, significantly shortening the breeding cycle. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The effect diagram of immune sensitization on the apoptosis level of blood cells of hairy clams provided in the embodiment of the present invention (PBS: control group; FRX: injection of Vibrio parahaemolyticus group, inactivated Vibrio concentration 3×10 8 cfu).
[0026] Figure 2 The effect of immune sensitization provided in the embodiment of the present invention on the phagocytic activity of blood cells of hairy clam (PBS: control group; FRX: injection of Vibrio parahaemolyticus group, inactivated Vibrio concentration 3×10 8 cfu).
[0027] Figure 3 The effect of immune sensitization on the total number of blood cells of hairy clams provided in the embodiment of the present invention (PBS: control group; FRX: injection of Vibrio parahaemolyticus group, inactivated Vibrio concentration 3×10 8 cfu).
[0028] Figure 4 The effect of immune sensitization on the immune enzyme activity of scorpion provided by the embodiment of the present invention (PBS: control group; FRX: injection of Vibrio parahaemolyticus group, inactivated Vibrio concentration 3×10 8cfu; ACP: acid phosphatase; AKP: alkaline phosphatase; NOS: nitric oxide synthase; LZM: lysozyme).
[0029] Figure 5 This is a diagram showing the effect of determining the half-lethal concentration of Vibrio in adult razor clams for 96 hours provided in an embodiment of the present invention.
[0030] Figure 6 This is a diagram showing the effect of transgenerational immune sensitization provided in an embodiment of the present invention on significantly improving the gene expression of prophenoloxidase activator and lysozyme in offspring.
[0031] Figure 7 This is a diagram showing the effect of cross-generational immune sensitization provided in an embodiment of the present invention on significantly improving the activity of progeny phenoloxidase and lysozyme.
[0032] Figure 8 This is a diagram showing the effect of cross-generational immune sensitization provided in an embodiment of the present invention on significantly improving the survival rate of D-shaped larvae of clams infected with Vibrio. DETAILED DESCRIPTION
[0033] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.
[0034] The invention uses Vibrio parahaemolyticus at a half-lethal concentration for 96 hours to stress adult sculter clams, and selects Vibrio-resistant sculter clams with a survival rate of 15% and a seed retention rate of 8%; then the selected Vibrio-resistant sculter clams are cultured until gonads mature, and inactivated Vibrio parahaemolyticus is injected before inducing spawning to induce the sculter clams to produce heritable immune factors; after the sculter clams are induced to spawn, the breeding and culture technology of "breeding in the north and raising in the south" is adopted to quickly culture the sculter clams to sexual maturity, and continuous breeding is carried out to obtain a new Vibrio-resistant sculter clam variety with stable genetics and excellent properties.
[0035] The breeding technology of "breeding in the north and raising in the south" is to cultivate the larvae indoors in the northern hatchery until they metamorphose and attach, and then transport the hairy clam juveniles to the southern coastal mudflats for intermediate cultivation and raising them to sexual maturity; the means of breeding in different regions are all achieved in accordance with conventional breeding methods.
[0036] Example 1
[0037] Determination of the half-lethal concentration of Vibrio parahaemolyticus in sculterella
[0038] The final concentration was set to 5 × 10 3 cfu / ml, 5×10 4 cfu / ml, 5×10 5 cfu / ml, 5×10 6 cfu / ml, 5×10 7 cfu / ml, 5×108 cfu / ml of different Vibrio parahaemolyticus were used to immerse and infect adult sculter clams. Conventional marine aquaculture was set as the control group. Three replicates were set in each group, with 60 adult sculter clams in each replicate. The experiment lasted for 4 days. The half-lethal concentration of Vibrio parahaemolyticus in adult sculter clams at 96 hours was determined to be about 3×10 7 cfu / ml.
[0039] The above-mentioned Vibrio parahaemolyticus was the PL2 strain provided by Qingdao Agricultural University. The Vibrio parahaemolyticus was inoculated on a solid culture medium and cultured at 28°C for 12 hours. The larger single colony was selected for liquid culture. The concentration of Vibrio was determined by dilution plate counting method and spectrophotometry, and the final concentration was 5×10 3 cfu / ml, 5×10 4 cfu / ml, 5×10 5 cfu / ml, 5×10 6 cfu / ml, 5×10 7 cfu / ml, 5×10 8 cfu / ml of different Vibrio parahaemolyticus gradient groups.
[0040] Example 2
[0041] Determination of injection concentration of inactivated Vibrio parahaemolyticus that effectively sensitizes adult sculterella
[0042] Set 3×10 5 cfu, 3×10 6 cfu, 3×10 7 cfu, 3×10 8 cfu, 3×10 9 cfu of inactivated Vibrio parahaemolyticus was used as the injection group, and PBS was injected as the control group. Three replicates were set up in each group, with 60 adult clams in each replicate. The experiment lasted for 4 days. The inactivated Vibrio parahaemolyticus injection concentration that effectively sensitized adult clams was determined by changes in indicators such as cell phagocytosis and apoptosis, immune enzyme activity, and gene expression. The concentration was determined to be 3×10 8 cfu (see Figure 1-4 ).
[0043] The inactivated Vibrio parahaemolyticus was the PL2 strain provided by Qingdao Agricultural University. The Vibrio parahaemolyticus was inoculated on a solid culture medium and cultured at 28°C for 12 hours. A larger single colony was selected for culture in a liquid culture medium. The concentration of Vibrio was determined by the dilution plate count method and the spectrophotometry method, and the final concentration was 3×10 5 cfu, 3×10 6 cfu, 3×10 7 cfu, 3×10 8 cfu, 3×10 9The Vibrio parahaemolyticus was heat-killed by placing the suspension of each group of Vibrio in a test tube and treating it in a water bath at 80℃ for 30 minutes.
[0044] Example 3
[0045] 1) Breeding by Vibrio parahaemolyticus infection: In early April 2016, the strain was infected with Vibrio parahaemolyticus at a 96-hour half-lethal concentration (3×10 7 cfu / ml) were used to treat adult clams collected from the coast of Laizhou, Shandong. When the survival rate of clams was close to 15%, the Vibrio stress was stopped and the clams were placed in regular aquaculture seawater to recover. Then, large, healthy and intact clams were selected as Vibrio-resistant clams for breeding the next generation, with a seed retention rate of about 8%.
[0046] 2) Enhance the immunity of the offspring of razor clams by using transgenerational immune sensitization: 800 razor clams selected above were cultured in seawater at a rate of 0.5°C / day. After each continuous increase of 2°C, the temperature was stabilized for 1 day. When the culture water temperature rose to 23°C, the temperature was stopped and kept stable at 23±0.5°C until the gonads of the razor clams were fully mature. Then, each razor clams with mature gonads were injected with 3×10 8 cfu of heat-killed Vibrio parahaemolyticus were dried in the shade for 6 hours and placed in an incubation tank filled with filtered seawater at 25°C to induce spawning and sperm release, thus establishing a new strain of sculta clams F1 generation resistant to Vibrio. After the above fertilization and hatching, the sculta clams larvae were cultured according to conventional methods such as marine shellfish larvae culture, placement of attachment substrates and juvenile shellfish culture. This method can significantly improve the anti-Vibrio ability of sculta clams progeny (see Figure 1-4 ), the specific data are shown in the figure below. The experimental groups were the untreated control group, the negative control group injected with PBS, and the transgenerational immune sensitization group injected with inactivated Vibrio.
[0047] 3) Use the breeding technology of "breeding in the north and raising in the south" to quickly cultivate hairy clams to sexual maturity:
[0048] 20 million larvae of the above-mentioned F1 generation of Vibrio-resistant new strain of hairy clams are cultivated indoors in the northern nursery until metamorphosis and attachment (the cultivation method can be carried out according to conventional means). After the hairy clams seedlings grow to 100,000 to 150,000 grains / jin, they are transported to the coast of Yueqing, Zhejiang Province by air transportation in an insulated box with ice, and then evenly sowed on the mudflat for intermediate cultivation, with a sowing density of 18 to 20 jin / mu. After the hairy clams seedlings are cultured on the mudflat for 3 months, the size can reach 600 to 800 grains / jin. The large-sized hairy clams seedlings are transferred to the bottom of nearby ponds for bottom sowing, and the breeding density is 100-150 grains / m 2 In early April of the following year, large-sized clams can be cultivated to sexual maturity, reaching 50-60 grains per kilogram.
[0049] During the north-south transportation, the transportation temperature is controlled at 15° C. or below, and the transportation time is preferably within 20 hours.
[0050] 4) Breeding of new Vibrio-resistant varieties of razor clams: The above-obtained individuals are cultivated to sexual maturity and continuously bred according to steps 1), 2) and 3) to obtain new Vibrio-resistant varieties of razor clams with genetic stability and excellent traits (four generations are bred in this embodiment).
[0051] Cultivate according to the above steps 1)-3) to obtain mature individuals, which can reach 50-60 grains / jin, and can be used as breeding shellfish for coercion and breeding the next generation. Then, the adults are continuously bred according to the above steps 1), 2) and 3) to obtain a new variety of razor clams resistant to Vibrio that is genetically stable and has excellent traits.
[0052] Comparative Example 1
[0053] 1) Breeding by Vibrio parahaemolyticus infection: In early April 2016, the strain was infected with Vibrio parahaemolyticus at a 96-hour half-lethal concentration (3×10 7 cfu / ml) were used to treat adult clams collected from the coast of Laizhou, Shandong. When the survival rate of clams was close to 15%, the Vibrio stress was stopped and the clams were placed in regular aquaculture seawater to recover. Then, large, healthy and intact clams were selected as Vibrio-resistant clams for breeding the next generation, with a seed retention rate of about 8%.
[0054] 2) Raise the temperature of the seawater for the cultivation of hairy clams at a rate of 0.5℃ / day, and stabilize it for 1 day after each continuous increase of 2℃. When the aquaculture water temperature rises to 23℃, stop raising the temperature and keep the water temperature stable at 23±0.5℃ until the hairy clams' gonads are fully developed and mature. Induce hairy clams to lay eggs at the Laizhou cooperative hatchery, select D-type larvae after about 24 hours of incubation, and then cultivate hairy clams larvae according to conventional methods such as marine shellfish larvae cultivation, placement of attachment substrates, and juvenile shellfish cultivation. When the hairy clams grow to 150,000-200,000 grains / jin, they are placed in incubators or nylon bags in the early morning or evening and transported to the open sea area in the north or nearby ponds for cultivation until they become adult shellfish. Using this breeding and cultivation technology, hairy clams need to grow for two years to reach sexual maturity.
[0055] It can be seen that this method has a long breeding cycle and high labor intensity, which significantly increases the breeding cost of new stress-resistant varieties of hairy clams.
[0056] Example 4
[0057] 1) Breeding by Vibrio parahaemolyticus infection stress: using the 96-hour half-lethal concentration (3×10 7 cfu / ml) was used to treat the above comparative example step 2) to obtain adult scaly clams. When the survival rate of the scaly clams was close to 15%, the Vibrio stress was stopped and the scaly clams were placed in conventional aquaculture seawater to recover their health. Then, large, healthy and intact scaly clams were selected as Vibrio-resistant scaly clams for breeding the next generation, and the seed retention rate was about 8%.
[0058] 2) Enhance the immunity of the offspring of razor clams by using transgenerational immune sensitization: 800 razor clams selected above were cultured in seawater at a rate of 0.5°C / day. After each continuous increase of 2°C, the temperature was stabilized for 1 day. When the culture water temperature rose to 23°C, the temperature was stopped and kept stable at 23±0.5°C until the gonads of the razor clams were fully mature. Then, each razor clams with mature gonads were injected with 3×10 8 cfu of heat-killed Vibrio parahaemolyticus were dried in the shade for 6 hours and placed in an incubation tank filled with filtered seawater at 25°C to induce spawning and sperm release, thus establishing a new strain of sculta clams F1 generation resistant to Vibrio. After the above fertilization and hatching, the sculta clams larvae were cultured according to conventional methods such as marine shellfish larvae culture, placement of attachment substrates and juvenile shellfish culture. This method can significantly improve the anti-Vibrio ability of sculta clams progeny (see Figure 5-8 ), the specific data are shown in the figure below. The experimental groups were the untreated control group, the negative control group injected with PBS, and the transgenerational immune sensitization group injected with inactivated Vibrio.
[0059] 3) Use the breeding technology of "breeding in the north and raising in the south" to quickly cultivate hairy clams to sexual maturity:
[0060] 20 million larvae of the above-mentioned F1 generation of Vibrio-resistant new strain of hairy clams are cultivated indoors in the northern nursery until metamorphosis and attachment (the cultivation method can be carried out according to conventional means). After the hairy clams seedlings grow to 100,000 to 150,000 grains / jin, they are transported to the coast of Yueqing, Zhejiang Province by air transportation in an insulated box with ice, and then evenly sowed on the mudflat for intermediate cultivation, with a sowing density of 18 to 20 jin / mu. After the hairy clams seedlings are cultured on the mudflat for 3 months, the size can reach 600 to 800 grains / jin. The large-sized hairy clams seedlings are transferred to the bottom of nearby ponds for bottom sowing, and the breeding density is 100-150 grains / m 2 In early April of the following year, large-sized clams can be cultivated to sexual maturity, reaching 50-60 grains per kilogram.
[0061] During the north-south transportation process, the transportation temperature is controlled at 15° C. or below, and the transportation time is preferably within 20 hours.
[0062] 4) Breeding of new Vibrio-resistant varieties of razor clams: The above-obtained individuals are cultivated to sexual maturity and continuously bred according to steps 1), 2) and 3) to obtain new Vibrio-resistant varieties of razor clams with genetic stability and excellent traits (three generations of continuous breeding in this embodiment).
[0063] According to the above steps 1)-3), resistant adults are cultivated to obtain mature individuals, which can reach 50-60 grains / jin and can be used as breeding shellfish for coercion and breeding the next generation. The adults are then continuously selected according to the above steps 1), 2) and 3) to obtain a new variety of razor clams resistant to Vibrio with genetic stability and excellent traits.
[0064] In summary, the varieties obtained by the breeding method of the present invention have the advantages of strong resistance to Vibrio, fast growth and short breeding cycle. The method can solve the current situation of insufficient supply of high-quality germplasm and high-quality seedlings in the razor clam breeding industry and aquaculture, increase the coverage rate of high-quality varieties of aquaculture razor clam, increase production and income of fishermen, and promote the quality and efficiency of the razor clam breeding industry and aquaculture industry.
[0065] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.
Claims
1. A method for breeding new varieties of scaly clams resistant to Vibrio, characterized in that: Adult razor clams were stressed with a semi-lethal concentration of Vibrio parahaemolyticus, and 15% of the surviving individuals were placed in regular aquaculture seawater to recover their health. The Vibrio-resistant razor clams were selected at an 8% seed retention rate, and the razor clams were fattened in an indoor workshop until their gonads matured. Before inducing spawning, inactivated Vibrio parahaemolyticus was used to induce the razor clams to produce heritable immune factors. The razor clams were then induced to spawn, and the Vibrio-resistant F1 strain of razor clams was obtained. The F1 generation was then rapidly cultivated to sexual maturity using the "north-breeding and south-raising" breeding and culture technology. Thereafter, new razor clams resistant to Vibrio with genetic stability and excellent traits were obtained through continuous breeding every year by screening seed clams under stress, enhancing the immunity of the offspring, and "north-breeding and south-raising".
2. The method for breeding a new Vibrio-resistant species of scaly clams according to claim 1, characterized in that: 1) Screening Vibrio-resistant scaly clams by Vibrio stress: Use semi-lethal concentrations of Vibrio parahaemolyticus to stress adult scaly clams. When the mortality rate of scaly clams is close to 85%, stop stressing and place the scaly clams in regular aquaculture seawater to recover. Then, select large, healthy and intact scaly clams with an 8% seed retention rate as scaly clams for breeding the next generation; 2) Enhance the immunity of the offspring of sculterella by using transgenerational immune sensitization: Cultivate the Vibrio-resistant sculterella seed shellfish until the gonads are mature, and inject inactivated Vibrio parahaemolyticus before inducing spawning to induce the sculterella to produce heritable immune factors, and then induce the sculterella to spawn by drying in the shade combined with warming seawater stimulation, that is, the Vibrio-resistant strain of the sculterella F1 generation; 3) Adopt the breeding technology of "breeding in the north and rearing in the south" to quickly cultivate razor clams to sexual maturity: cultivate the F1 generation of razor clams resistant to Vibrio in the northern hatchery indoors until metamorphosis and attachment, and then transport the razor clams to the southern coastal mudflats for intermediate cultivation and grow-out, thereby realizing "breeding in the north and rearing in the south" and cultivating razor clams to sexual maturity; 4) Breeding of new Vibrio-resistant varieties of razor clams: The sexually mature individuals of the F1 generation of Vibrio-resistant strains of razor clams obtained above are continuously bred according to steps 1), 2) and 3) to obtain new Vibrio-resistant varieties of razor clams with genetic stability and excellent traits after genetic stabilization.
3. The method for breeding a new Vibrio-resistant species of scaly clams according to claim 2, characterized in that: In step 1), adult sculterella were placed in a solution with a final concentration of 3×10 7 cfu / ml Vibrio parahaemolyticus in a seawater culture tank. When the mortality rate of razor clams is close to 85%, the stress is stopped and the clams are placed in regular aquaculture seawater to recover. Then, large, healthy and intact clams are selected at an 8% seed retention rate as breeding shellfish for the next generation.
4. The method for breeding a new Vibrio-resistant species of scaly clams according to claim 2, characterized in that: In the step 2), the water body for cultivating the selected hairy clams is heated at a rate of 0.5-1.0°C / day, and the temperature is kept stable for 1 day after each continuous temperature increase of 2°C. When the aquaculture water temperature rises to 23°C, the temperature is stopped and kept stable at 23±0.5°C until the hairy clams have fully developed gonads, and then 3×10 8 cfu of heat-killed Vibrio parahaemolyticus were dried in the shade for 6 hours and placed in an incubation tank filled with filtered seawater at 25°C to induce spawning and sperm release, thus establishing a new Vibrio-resistant strain of the F1 generation of clams.
5. The method for breeding a new Vibrio-resistant species of razor clams according to claim 4, characterized in that: The heat-inactivated Vibrio parahaemolyticus is cultured in a liquid culture medium until the strain concentration is 3×10 8 cfu / ml, and then the suspension was treated in a water bath at 80°C for 30 minutes to obtain heat-killed Vibrio parahaemolyticus.
6. The method for breeding a new Vibrio-resistant species of razor clams according to claim 2, characterized in that: In the step 3), the larvae of the F1 generation of the new Vibrio-resistant strain of razor clams obtained above are cultured indoors in a northern nursery until they undergo metamorphosis and attach. After the razor clams seedlings grow to 100,000 to 150,000 grains per kilogram, they are transported to the southern coast by refrigerated trucks or by air transportation in insulated boxes with ice. The razor clams seedlings are then evenly sown on the mud flats for breeding. After the razor clams seedlings are cultured on the mud flats for 3 months and the size reaches 600 to 800 grains per kilogram, the large-sized razor clams seedlings are transferred to the bottom of nearby ponds for breeding. In early April of the following year, the large-sized razor clams seedlings are cultured to sexual maturity for stress treatment and breeding of the next generation.
7. The method for breeding a new Vibrio-resistant species of razor clams according to claim 6, characterized in that: During the north-south transportation process, the transportation temperature is controlled below 15°C and the transportation time is within 20 hours.