A method for breeding fast-growing and highly resistant new varieties of hairy clams

Abstract

The present invention relates to the technical field of genetic breeding of marine economic shellfish, and in particular to a method for breeding a fast-growing and highly resistant new variety of scorpion clams. Heat-resistant and Vibrio-resistant scorpion clams are cultured until gonadal development is mature, and before inducing the mature Vibrio-resistant scorpion clams to lay eggs, inactivated Vibrio parahaemolyticus is injected to induce the scorpion clams to produce heritable immune factors, thereby inducing egg laying and hatching; the mature heat-resistant scorpion clams are induced to lay eggs, and the D-shaped larvae of the progeny are continuously acclimated at high temperature until attachment and metamorphosis; then the above-mentioned larvae are bred and cultured in a "north breeding and south rearing" manner until maturity, and then the mature scorpion clams of various properties are respectively cultured in the manner of their respective properties until a genetically stable and excellent variety is obtained, and then the two varieties are obtained for inter-strain hybrid breeding, that is, a fast-growing and highly resistant new variety of scorpion clams is obtained. The method of the present invention can significantly improve the growth rate, heat resistance and Vibrio resistance and culture survival rate of scorpion clams, and improve the preservation and supply capacity of fine varieties in the scorpion aquaculture industry.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding of marine economic shellfish, in particular to a method for breeding a fast-growing and highly resistant new variety of hairy clams. Background Art

[0002] There are many commercial shellfish on my country's tidal flats, among which Scapharca subcrenata is an important species, which is widely distributed on my country's coastal tidal flats. In the 1980s, the natural reserves of Scapharca subcrenata were most abundant in Laizhou Bay, Bohai Bay, Liaodong Bay, Haizhou Bay and other sea areas. Scapharca subcrenata is rich in flavor amino acids and vitamin B. 12 , delicious meat, high nutritional value and medicinal value, deeply loved by consumers. In recent years, with the improvement of people's living standards, the demand for marine delicacies such as hairy clams has increased year by year. However, the current fishing and breeding output of hairy clams in my country has been declining year by year, and it can no longer meet market demand, resulting in the market price of hairy clams hitting new highs. In addition, in the process of promoting the full artificial breeding of hairy clams, the awareness of breeding good varieties is weak, and the non-standard breeding technology has led to the degradation of the characteristics of hairy clam breeding varieties, and the sustainability of breeding has been affected. In summary, the lack of stress-resistant varieties and high-quality seedlings of hairy clams has become a bottleneck problem restricting the healthy development of the hairy clam seed industry and breeding industry. Therefore, we urgently need to cultivate new varieties of hairy clams that are resistant to high temperatures and diseases through modern breeding technology to meet the needs of rapid industrial development such as hairy clam seedling and breeding. Summary of the invention

[0003] Aiming at the problems that industries such as seedling and breeding of hairy clams lack excellent varieties and existing breeding varieties have poor stress resistance and high breeding mortality, the present invention proposes a method for breeding fast-growing and highly resistant new varieties of hairy clams.

[0004] To achieve the above purpose, the present invention adopts the following technical solution:

[0005] A method for breeding a fast-growing and highly resistant new variety of razor clams, comprising the steps of: culturing heat-resistant and Vibrio-resistant razor clams respectively until gonadal development is mature; injecting inactivated Vibrio parahaemolyticus into the mature Vibrio-resistant razor clams before inducing them to lay eggs, so as to induce the razor clams to produce heritable immune factors, thereby inducing them to lay eggs and hatch; inducing the mature heat-resistant razor clams to lay eggs, and then continuing to acclimate the D-shaped larvae of the progeny to high temperature until they undergo attachment and metamorphosis; then breeding and culturing the larvae in the north and raising them in the south until they mature; and then repeating the breeding methods of the mature razor clams of each performance until a genetically stable variety with excellent performance is obtained; and then performing inter-strain hybrid breeding on the two varieties, so as to obtain a fast-growing and highly resistant new variety of razor clams.

[0006] The heat-resistant razor clam molluscs are obtained by stressing razor clam adults with semi-lethal thermophilic bacteria; the Vibrio-resistant razor clam molluscs are obtained by stressing razor clam adults with semi-lethal concentrations of Vibrio parahaemolyticus.

[0007] Further,

[0008] 1) Screening of heat-resistant and Vibrio-resistant scallops: Adult scallops were selected and divided into two groups. One group was subjected to a 96-hour high-temperature semi-lethal temperature stress, and the other group was subjected to a 96-hour semi-lethal concentration stress of Vibrio parahaemolyticus infection. When the mortality rate of scallops in each group was close to 85%, the stress was stopped and the scallops were placed in conventional aquaculture seawater to recover. Then, scallops with large individuals and intact health were selected as scallops with a seed retention rate of 8% to breed the next generation;

[0009] 2) Enhancement of immune factors: The Vibrio-resistant scaly clams obtained above are cultured until the gonads are mature, and inactivated Vibrio parahaemolyticus is injected before inducing spawning to induce the scaly clams to produce heritable immune factors, and then the scaly clams are induced to spawn by drying in the shade combined with warming seawater stimulation, that is, the Vibrio-resistant strain of the scaly clams F1 generation;

[0010] 3) High temperature acclimation of planktonic larvae of hairy clams: The above-selected high temperature resistant hairy clams are cultured until their gonads mature, and spawning is induced by drying in the shade combined with warming seawater stimulation to establish a new high temperature resistant strain of hairy clams of the F1 generation; after hatching, the selected D-shaped larvae are acclimated at a culture water temperature of 28±0.5℃, and the high temperature acclimation is stopped before the hairy clams are transported to the open sea for cultivation;

[0011] 4) using the breeding and culture technology of "breeding in the north and rearing in the south" to quickly cultivate hairy clams to sexual maturity: the hairy clams larvae obtained in the above steps 2) and 3) are respectively cultivated in the northern hatchery to 100,000 to 150,000 juveniles per kilogram, and then the hairy clams are transported to the southern coastal mudflats for intermediate cultivation and rearing to sexual maturity;

[0012] 5) Breeding of new varieties: Repeat the above steps for each trait of the above-mentioned cultured varieties until the varieties with genetic stability and good traits are obtained, that is, the fast-growing and high-temperature-resistant varieties of razor clams and the fast-growing and Vibrio-resistant varieties of razor clams;

[0013] 6) Breeding of new fast-growing and highly resistant varieties of razor clams: The fast-growing and high-temperature resistant varieties of razor clams obtained above are crossbred with the fast-growing and Vibrio resistant varieties of razor clams to obtain fast-growing and highly resistant varieties of razor clams.

[0014] In step 1), adult sculterella are 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 razor clams are placed in regular aquaculture seawater to recover. Then, large, healthy and intact razor clams are selected with an 8% seed retention rate as breeding shellfish for the next generation.

[0015] In the step 1), the adult razor clams are placed in a 31.6°C high temperature seawater culture tank for stress. When the mortality rate of the razor clams is close to 85%, the stress is stopped and the razor clams are placed in regular culture seawater to recover. Then, large and healthy razor clams are selected as seed shellfish with an 8% seed retention rate to breed the next generation.

[0016] 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 placed in a hatching pond filled with filtered seawater at 25°C for 6 hours to induce spawning and spermatogenesis, thus establishing a new strain of Vibrio resistance in the F1 generation of sculta clams. After hatching, sculta clams were cultured according to conventional methods such as marine shellfish larvae culture, placement of attachment substrates and juvenile shellfish culture.

[0017] In the step 3), the selected heat-resistant hairy clam larvae are cultured until gonads mature, dried in the shade for 6 hours, and then placed in an incubation pond filled with filtered seawater at 27°C to induce spawning and ejaculation, thereby establishing a new heat-resistant F1 strain of hairy clam; after hatching, the selected D-shaped larvae are acclimated at a culture water temperature of 28±0.5°C, and the hairy clam larvae are cultured according to conventional methods such as seawater shellfish larvae culture, placement of attachment substrates, and juvenile shellfish culture, and high-temperature acclimation is stopped before the hairy clam seedlings are transported to open sea areas for culture.

[0018] In the step 4), the larvae obtained in the above steps 2) and 3) are respectively cultured indoors in a northern nursery until metamorphosis and attachment. After the larvae of the hairy clam grow to 100,000 to 150,000 grains / catties, they are transported to the southern coast by refrigerated trucks or by air transportation in insulated boxes with ice. The hairy clam seedlings are then evenly sown on the mud flat for breeding. After the hairy clam seedlings are cultured on the mud flat for 3 months, the size can reach 600 to 800 grains / catties. The large-sized hairy clam seedlings are transferred to the bottom of a nearby pond for breeding until they are sexually mature, and are used for stress treatment and breeding the next generation.

[0019] During the north-south transportation, the transportation temperature is controlled at 15° C. or below, and the transportation time is preferably within 20 hours.

[0020] In the step 6), female individuals (or male individuals) of the fast-growing, high-temperature-resistant new variety of razor clams and male individuals (or female individuals) of the fast-growing, Vibrio-resistant new variety of razor clams are selected, and after being dried in the shade for 6 hours, they are placed in an incubation pool filled with filtered seawater at 25° C. to induce spawning and spermatogenesis, and inter-strain hybridization is performed, and the hybrid offspring is the fast-growing, highly resistant new variety of razor clams.

[0021] The above-mentioned, the half-lethal concentration of Vibrio parahaemolyticus infection at 96 hours was confirmed: the concentration gradient of Vibrio parahaemolyticus was 5×10 3cfu / 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, conventional marine aquaculture was used as the control group, and 3 replicates were set up in each group, with 60 adult clams in each replicate. The stress experiment lasted for 4 days, and the half-lethal concentration of Vibrio parahaemolyticus in adult clams was determined to be 3×10 7 cfu / ml.

[0022] Among them, Vibrio parahaemolyticus is the PL2 strain, which was donated by Qingdao Agricultural University and was 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.

[0023] The temperature during the 96-hour high temperature semi-lethal temperature stress is determined as follows: different temperature gradients are set to 22°C (control group), 24°C, 26°C, 28°C, 30°C, 32°C and 34°C, conventional seawater aquaculture is used as the control group, 3 replicates are set for each group, 60 adult scultera in each replicate, the stress experiment lasts for 4 days, and the 96-hour high temperature semi-lethal temperature of adult scultera is determined to be 31.6°C.

[0024] Determination of the injection concentration of the inactivated Vibrio parahaemolyticus: set 3×10 5 cfu, 3×10 6 cfu, 3×10 7 cfu, 3×10 8 cfu, 3×10 9 cfu of heat-killed Vibrio parahaemolyticus injection, and PBS injection as the control group. Each group was set up with 3 replicates, and each replicate had 60 adult clams. The experiment lasted for 4 days. Through the changes in indicators such as cell phagocytosis and apoptosis, immune enzyme activity and gene expression, it was determined that the injection concentration of inactivated Vibrio parahaemolyticus that effectively sensitized adult clams was 3×10 8 cfu.

[0025] The advantages of the present invention are:

[0026] (1) The present invention adopts a breeding technology scheme that combines traditional stress breeding with modern molecular-assisted breeding to achieve precise breeding of target traits.

[0027] (2) The cross-generational immunization method was used to significantly improve the offspring's ability to resist Vibrio. The high-temperature acclimation method of planktonic larvae can significantly enhance the high-temperature resistance of the offspring of razor clams. Then, the inter-strain hybridization breeding technology was used to significantly enhance the high-temperature resistance and Vibrio resistance of the new high-resistant strain of razor clams.

[0028] (3) The present invention adopts the "north breeding and south rearing" razor clam seedling breeding and culture technology, which fully utilizes the advantages of high water temperature, sufficient bait and few harmful organisms in the mudflats and ponds along the southern coast, and can significantly increase the growth rate of razor clam. The razor clam seedlings reach 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

[0029] Figure 1 The half-lethal concentration of Vibrio parahaemolyticus in adult scaly clams for 96 hours provided by the embodiments of the present invention.

[0030] Figure 2 This is a diagram showing the effect of 96-hour semi-lethal high temperature on razor clams provided in an embodiment of the present invention.

[0031] Figure 3 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.

[0032] Figure 4 This is a diagram showing the effect of transgenerational immune sensitization provided in an embodiment of the present invention on significantly improving the growth rate of D-shaped larvae of clams infected with Vibrio.

[0033] Figure 5 The invention provides a test on the high temperature stress resistance of razor clams.

[0034] Figure 6 The invention provides a Vibrio parahaemolyticus infection stress test. DETAILED DESCRIPTION

[0035] 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.

[0036] The invention firstly uses 96-hour half-lethal high temperature and 96-hour half-lethal concentration of Vibrio parahaemolyticus to stress sculter clams, and selects heat-resistant and Vibrio-resistant sculter clams at a survival rate of 15% and a seed retention rate of 8%; then the selected heat-resistant and Vibrio-resistant sculter clams are cultured until gonads mature, and inactivated Vibrio parahaemolyticus is injected into the Vibrio-resistant sculter clams before inducing spawning to induce the sculter clams to produce heritable immune factors, and after the induced spawning and hatching, the D-shaped larvae of the heat-resistant sculter clams continue to be acclimated at high temperature until attachment and metamorphosis; the breeding and culture technology of "breeding in the north and raising in the south" is adopted to quickly cultivate the sculter clams to sexual maturity, and continuous breeding is carried out to obtain a fast-growing heat-resistant and fast-growing Vibrio-resistant new sculter clams with stable genetics and excellent properties; finally, the inter-strain hybrid breeding technology is adopted to select and breed a fast-growing and highly resistant new sculter clams.

[0037] Embodiment 1:

[0038] Confirmation of the half-lethal concentration of Vibrio parahaemolyticus infection: The concentration gradient of Vibrio parahaemolyticus 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, conventional marine aquaculture was used as the control group, and 3 replicates were set up in each group, with 60 adult clams in each replicate. The Vibrio stress experiment lasted for 4 days, and the half-lethal concentration of Vibrio parahaemolyticus in adult clams was determined to be 3×10 7 cfu / ml (see Figure 1 ).

[0039] Among them, Vibrio parahaemolyticus was the PL2 strain provided by Qingdao Agricultural University. Vibrio parahaemolyticus was inoculated on solid culture medium and cultured at 28°C for 12 hours. The larger single colony was selected for liquid culture medium. The concentration of Vibrio was determined by dilution plate counting method and spectrophotometry, and the final concentration was 3×10 5 cfu, 3×10 6 cfu, 3×10 7 cfu, 3×10 8 CFU of different Vibrio parahaemolyticus gradient groups.

[0040] Determination of the temperature during the 96-hour high temperature semi-lethal temperature stress: different temperature gradients were set to 22°C (control group), 24°C, 26°C, 28°C, 30°C, 32°C and 34°C, 3 replicates were set for each group, and 60 adult sculterella were set for each replicate. The temperature stress experiment lasted for 4 days, and the 96-hour high temperature semi-lethal temperature of adult sculterella was determined to be 31.6°C (see Figure 2 ).

[0041] Determination of the injection concentration of the inactivated Vibrio parahaemolyticus: set 3×10 5 cfu, 3×10 6 cfu, 3×10 7 cfu, 3×10 8 cfu, 3×10 9 cfu of heat-killed Vibrio parahaemolyticus injection, and PBS injection as the control group. Each group was set up with 3 replicates, and each replicate had 60 adult clams. The experiment lasted for 4 days. Through the changes in indicators such as cell phagocytosis and apoptosis, immune enzyme activity and gene expression, it was determined that the injection concentration of inactivated Vibrio parahaemolyticus that effectively sensitized adult clams was 3×10 8 cfu.

[0042] Example 2

[0043] 1) Breeding of new strains of scaly clams resistant to high temperature and Vibrio: In early April 2016, the 96-hour semi-lethal high temperature (31.6°C) and the 96-hour semi-lethal concentration of Vibrio parahaemolyticus (3×10 7 cfu / ml) were used to treat adult clams collected from the coast of Laizhou, Shandong. Each treatment group had 10,000 clams. When the survival rate of the clams was close to 15%, the stress was stopped and the clams were placed in regular aquaculture seawater to recover. Then, large, healthy and intact clams were selected as breeding shellfish for the next generation, and the seed retention rate was about 8%.

[0044] 2) Enhancement of immune factors: The water body for cultivating the selected hairy clams was heated at a rate of 0.5-1.0°C / day, and the temperature was kept stable for 1 day after each continuous temperature increase of 2°C. When the water temperature rose to 23°C, the temperature was stopped and kept stable at 23±0.5°C until the gonads of the hairy clams were fully mature. Then, each hairy clams with mature gonads were injected with 3×10 8 cfu of heat-killed Vibrio parahaemolyticus were placed in a hatching pond filled with filtered seawater at 25°C for 6 hours to induce spawning and spermatogenesis, thus establishing a new strain of Vibrio resistance in the F1 generation of sculta clams. After hatching, sculta clams were cultured according to conventional methods such as marine shellfish larvae culture, placement of attachment substrates and juvenile shellfish culture.

[0045] 3) High temperature acclimation of planktonic larvae of hairy clams: The above-screened high temperature resistant hairy clams are cultured until the gonads are mature (the seawater for the cultivation of hairy clams is heated at a rate of 0.5°C / day, and the temperature is stabilized for 1 day after each continuous temperature increase of 2°C. When the culture water temperature rises to 25°C, the temperature is stopped and the water temperature is kept stable at 25±0.5°C until the gonads of the hairy clams are fully mature), and after being dried in the shade for 6 hours, they are placed in an incubation pool filled with filtered seawater at 27°C to induce spawning and spermatogenesis, and a new high temperature resistant strain of hairy clams F1 generation is established; after hatching, the selected D-shaped larvae are acclimated at a culture water temperature of 28±0.5°C, and the hairy clams larvae are cultured according to conventional methods such as seawater shellfish larvae culture, placement of attachment substrates and juvenile shell culture, and the high temperature acclimation is stopped before the hairy clams are transported to the open sea for cultivation.

[0046] 4) Adopting the breeding and culture technology of "breeding in the north and rearing in the south" to quickly cultivate hairy clams to sexual maturity: the larvae obtained in the above steps 2) and 3) are respectively cultured in the northern nursery indoors until metamorphosis and attachment, and when the hairy clams seedlings grow to 100,000 to 150,000 grains / jin, they are transported to the southern coast by refrigerated trucks or insulated boxes with ice and air transportation, and then the hairy clams seedlings are evenly sown on the mud flats for culture. After the hairy clams seedlings are cultured on the mud flats for 3 months, the size can reach 600 to 800 grains / jin, and the large-sized hairy clams seedlings are transferred to the bottom of nearby ponds for culture until sexual maturity, and are used for stress treatment and breeding of the next generation.

[0047] During the north-south transportation, the transportation temperature is controlled at 15° C. or below, and the transportation time is preferably within 20 hours.

[0048] 5) New variety breeding: The F1 generation Vibrio-resistant new strain of razor clams obtained above is continuously bred according to steps 1), 2) and 4) until a fast-growing, Vibrio-resistant new razor clams variety with genetic stability and excellent traits is obtained (three generations are bred in this embodiment).

[0049] The above-obtained new heat-resistant strain of the F1 generation of razor clams is continuously bred according to steps 1), 3) and 4) until a fast-growing, heat-resistant new variety of razor clams with genetic stability and excellent traits is obtained (three generations are bred in this embodiment).

[0050] 6) Breeding of fast-growing, highly resistant new varieties of razor clams: Select female individuals (or male individuals) from the above-mentioned fast-growing, high-temperature resistant new varieties of razor clams and male individuals (or female individuals) from the fast-growing, Vibrio resistant new varieties of razor clams, dry them in the shade for 6 hours, place them in an incubation pond filled with filtered seawater at 25°C to induce spawning and spermatogenesis, and perform inter-strain hybridization. The hybrid offspring is the fast-growing, highly resistant new variety of razor clams.

[0051] Cultivate according to the above steps 1)-4) 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), 3) and 4) to obtain a new variety of razor clams resistant to Vibrio with genetic stability and excellent traits. Then, the new variety is hybridized between strains, and the hybrid offspring is a new variety of razor clams with fast growth and high resistance.

[0052] Comparative Example

[0053] 1) Breeding of new strains of scaly clams resistant to high temperature and Vibrio: In early April 2016, the 96-hour semi-lethal high temperature and 96-hour semi-lethal concentration of Vibrio parahaemolyticus (3×10 7 cfu / ml) were used to treat adult clams collected from the coast of Laizhou, Shandong. Each treatment group had 10,000 clams. When the survival rate of the clams was close to 15%, the stress was stopped and the clams were placed in regular aquaculture seawater to recover. Then, large, healthy and intact clams were selected as breeding shellfish for the next generation, and the seed retention rate was about 8%.

[0054] 2) Cultivation: 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 cultivation water temperature rises to 25℃, stop heating and keep the water temperature stable at 25±0.5℃ until the gonads of hairy clams 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 according to conventional methods such as marine shellfish larvae cultivation, placement of attachment substrates and juvenile shellfish cultivation. The D-shaped larvae of the offspring of high-temperature resistant hairy clams are acclimated at a cultivation water temperature of 28±0.5℃ until the high-temperature acclimation is stopped before the hairy clams are shipped out of the warehouse. When the hairy clams grow to 150,000-200,000 grains / jin, they are loaded into incubators in the early morning and transported to the open sea areas in the north and nearby ponds for cultivation until they become shellfish. Using this breeding and farming technology, hairy clam seedlings 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 3

[0057] 1) Breeding of new strains of scaly clams resistant to high temperature and Vibrio: In early April 2016, the 96-hour semi-lethal high temperature and 96-hour semi-lethal concentration of Vibrio parahaemolyticus (5×10 8cfu / ml) were used to treat adult clams collected from the coast of Laizhou, Shandong Province. Each treatment group had 10,000 clams. When the survival rate of clams was close to 15%, the stress was stopped and the clams were placed in conventional aquaculture seawater to recover. Then, large, healthy and intact clams were selected as seed shells to breed the next generation, and the seed retention rate was about 8%. The seawater for clams breeding was heated at a rate of 0.5℃ / day, and the temperature was stabilized for 1 day after each continuous 2℃ increase. When the aquaculture water temperature rose to 25℃, the temperature was stopped and kept stable at 25±0.5℃ until the gonads of clams were fully developed and mature. Induced clams to lay eggs in Laizhou cooperative hatchery, D-type larvae were selected after about 24 hours of incubation, and then the clams larvae were cultivated according to conventional methods such as seawater shellfish larvae cultivation, placement of attachment substrates and juvenile shellfish cultivation. The D-shaped larvae of the heat-resistant hairy clam larvae are acclimated at a culture water temperature of 28±0.5℃ until the hairy clam seedlings are shipped out of the warehouse. When the hairy clam seedlings grow to 150,000-200,000 grains / jin, they are loaded into incubators in the early morning and transported to the open sea areas in the north and nearby ponds for culture until they become adult shellfish.

[0058] 2) In early April 2020, 10,000 scallops of heat-resistant and Vibrio-resistant strains with well-developed gonads and large individuals were selected and kept for seed using the above-mentioned high temperature and Vibrio stress methods. In mid-to-late May, the scallops were cultured in the Laizhou cooperative hatchery until their gonads matured using the above-mentioned temperature-raising method, and the scallops were induced to lay eggs using shade drying combined with temperature-raising stimulation. Then, the scallop larvae were cultured using conventional methods such as marine shellfish larvae culture, placement of attachment substrates, and juvenile culture.

[0059] 3) Enhancement of immune factors: In order to further improve the anti-Vibrio properties and broad-spectrum disease resistance of the antibacterial strain of sculta clams, the effective concentration of sensitized adult sculta clams for inactivating Vibrio parahaemolyticus was first determined to be 3×10 8 cfu, and then culture the Vibrio-resistant razor clams until their gonads mature, inject inactivated Vibrio parahaemolyticus before inducing spawning to induce the razor clams to produce heritable immune factors, dry in the shade for 6 hours, and place in a hatching pond filled with 26°C filtered seawater to induce spawning and sperm release, and after fertilization and hatching, culture the razor clams larvae according to conventional methods such as marine shellfish larvae culture, placement of attachment substrates and juvenile shellfish culture.

[0060] Meanwhile, 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; is the untreated control group, PBS treated refers to the negative control group injected with PBS, and TGIP treated refers to the transgenerational immune sensitization group injected with inactivated Vibrio

[0061] Depend on Figure 3-4 It can be seen that the anti-Vibrio ability of the offspring of hairy clams can be significantly improved.

[0062] 4) High temperature acclimation of planktonic larvae of hairy clams: the high temperature resistant hairy clams selected in step 2) are cultivated until the gonads are mature (the seawater for cultivating hairy clams is heated at a rate of 0.5°C / day, and the temperature is stabilized for 1 day after each continuous temperature increase of 2°C. When the culture water temperature rises to 25°C, the temperature is stopped and kept stable at 25±0.5°C until the gonads of hairy clams are fully mature), and spawning is induced by a combination of shade drying and heated seawater stimulation to establish a new high temperature resistant strain of hairy clams of the F1 generation; after hatching, the selected D-shaped larvae are acclimated at a culture water temperature of 28±0.5°C, and the high temperature acclimation is stopped before the hairy clams are transported to the open sea for cultivation;

[0063] 5) Use the breeding and farming technology of "breeding in the north and raising in the south" to quickly cultivate hairy clams to sexual maturity: The hairy clam larvae obtained in the above steps 2) and 3) are separately cultivated indoors in the northern nursery. In mid-to-late August, the hairy clam seedlings grow to 150,000-200,000 grains / jin of juveniles. The hairy clam seedlings are packed in nylon bags and transported to the coastal mudflats of Yueqing, Zhejiang for artificial seeding and medium-term cultivation. The sowing density is 20-25 jin / mu. Hairy clam seedlings are transported to the southern coast by air transportation in insulated boxes with ice. The transportation temperature is controlled at 15°C or below, and the transportation time is within 20 hours. During the medium-term cultivation of hairy clam seedlings, technical personnel will carry out daily management and pest control. The size of hairy clam seedlings can reach 600-800 grains / jin after being cultured on the mudflats for 3 months. At the end of November, most of the large-sized hairy clam seedlings are transferred to the bottom of nearby ponds for bottom sowing and culture, with a culture density of 100-150 grains / m 2 By adopting the breeding and farming technology of "breeding in the north and raising in the south", large-sized razor clams can be cultivated to sexual maturity in early April of the following year for stress treatment and breeding of the next generation.

[0064] 6) Breeding of fast-growing and Vibrio-resistant new varieties of razor clams: The F1 generation Vibrio-resistant new strains of razor clams obtained above are continuously bred according to steps 1), 2), 3) and 5) until a fast-growing and Vibrio-resistant new variety of razor clams with genetic stability and excellent traits is obtained (three generations are bred in this embodiment).

[0065] Breeding of fast-growing, high-temperature-resistant new varieties of razor clams: The above-obtained new high-temperature-resistant strain of the F1 generation of razor clams is continuously bred according to steps 1), 2), 4) and 5) until a fast-growing, high-temperature-resistant new variety of razor clams with genetic stability and excellent traits is obtained (three generations are bred in this embodiment).

[0066] 7) Analysis of stress resistance of the new strain of hairy clams with high resistance: On June 15, 2021, the above-mentioned new strains of hairy clams with high temperature resistance and new strains with resistance to Vibrio were used as seed shellfish. The shade drying combined with the stimulation of warmed seawater was used to induce the spawning of hairy clams for inter-group hybridization, and the new strain of hairy clams with high temperature resistance and resistance to Vibrio (high resistance strain) was constructed. The local wild population in Laizhou that had not been selected was used as a control. When the hairy clams grew to 100 grains / jin, the hairy clams with high resistance and control groups were stressed by 96 hours of semi-lethal high temperature and 96 hours of semi-lethal concentration of Vibrio parahaemolyticus, respectively. Three parallels were set for each group, and each parallel had 60 hairy clams. After four consecutive days of high temperature stress at 32.3℃, the average mortality rate of the new high-resistant strain of razor clams was 60.3±1.4%, while the average mortality rate of the unselected razor clams cultured during the same period was 81.0±2.6%. The difference in the mortality rates of the two groups of razor clams was significant, indicating that the high-resistant strain of razor clams has significantly improved its ability to withstand high temperatures.

[0067] The half-lethal concentration of 3×10 7 cfu / mL of Vibrio parahaemolyticus for 9 consecutive days. The results showed that the mortality rate of the highly resistant new strain of razor clams was 47.7±2.5%, while the mortality rate of the unselected razor clams was 60±3.4%. There was a significant difference in mortality between the selected group and the control group, indicating that the highly resistant new strain of razor clams has significantly improved its anti-Vibrio ability.

[0068] Cultivate according to the above steps 1)-5) 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), 3), 4) and 5) to obtain a new variety of razor clams resistant to Vibrio with genetic stability and excellent traits. Then, the new variety is hybridized among strains, and the hybrid offspring is a new variety of razor clams with fast growth and high resistance.

[0069] In summary, the fast-growing and highly resistant varieties of razor clams obtained by the breeding method of the present invention have the advantages of high temperature resistance and 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 improved varieties of aquaculture razor clams, increase production and income of fishermen, and promote the quality and efficiency of the razor clam breeding industry and aquaculture industry.

[0070] 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 a fast-growing and highly resistant new variety of razor clams, characterized in that: The heat-resistant and Vibrio-resistant razor clams are cultured until their gonads mature, and the mature Vibrio-resistant razor clams are injected with inactivated Vibrio parahaemolyticus before inducing them to lay eggs, so as to induce the razor clams to produce heritable immune factors, thereby inducing them to lay eggs and hatch razor clams larvae; the mature heat-resistant razor clams are induced to lay eggs, and the D-shaped larvae are further acclimated at high temperature until they undergo attachment and metamorphosis; the above-mentioned larvae are then bred and cultured in a "north-rearing and south-rearing" manner until they mature, and the mature razor clams of each performance are cultured in the same manner until a genetically stable variety with excellent performance is obtained, and the two varieties are then crossbred to obtain a fast-growing, highly resistant new variety of razor clams.

2. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 1, characterized in that: The heat-resistant scaly clam seed shellfish is obtained by stressing the adult scaly clam with semi-lethal thermophilic bacteria; The Vibrio-resistant sculta clams are obtained by stressing adult sculta clams with Vibrio parahaemolyticus at a semi-lethal concentration.

3. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 1 or 2, characterized in that: The following steps are involved: 1) Screening of heat-resistant and Vibrio-resistant scallops: Adult scallops were selected and divided into two groups. One group was subjected to a 96-hour high-temperature semi-lethal temperature stress, and the other group was subjected to a 96-hour semi-lethal concentration stress of Vibrio parahaemolyticus infection. When the mortality rate of scallops in each group was close to 85%, the stress was stopped and the scallops were placed in conventional aquaculture seawater to recover. Then, scallops with large individuals and intact health were selected as scallops with a seed retention rate of 8% to breed the next generation; 2) Enhancement of immune factors: The Vibrio-resistant scaly clams selected in step 1) are cultured until gonadal maturity is achieved, and inactivated Vibrio parahaemolyticus is injected before induction of spawning to induce the scaly clams to produce heritable immune factors, and then the scaly clams are induced to spawn by drying in the shade combined with warmed seawater stimulation, thus obtaining the Vibrio-resistant strain of the scaly clams F1 generation; 3) High-temperature acclimation of planktonic larvae of hairy clams: The above-selected high-temperature resistant hairy clams are cultured until their gonads mature, and spawning is induced by drying in the shade combined with warm seawater stimulation to establish a new high-temperature resistant strain of hairy clams of the F1 generation; after hatching, the selected D-shaped larvae are acclimated at a culture water temperature of 28±0.5℃, and the high-temperature acclimation is stopped before the hairy clams are transported to the open sea for culture; 4) Adopt the breeding and culture technology of "breeding in the north and rearing in the south" to quickly cultivate hairy clams to sexual maturity: the hairy clams larvae obtained in the above steps 2) and 3) are respectively cultivated in the northern hatchery to 100,000-150,000 juveniles per kilogram, and then the hairy clams are transported to the southern coastal mudflats for intermediate cultivation and rearing to sexual maturity; 5) Breeding of new varieties: Repeat the corresponding steps for breeding the varieties with different traits after cultivation in step 4) until obtaining varieties with different traits that are genetically stable and have good traits, i.e., fast-growing and high-temperature-resistant varieties of razor clams and fast-growing and Vibrio-resistant varieties of razor clams; 6) Breeding of new fast-growing and highly resistant varieties of razor clams: The fast-growing and high-temperature resistant varieties of razor clams obtained in step 5) are crossbred with the fast-growing and Vibrio resistant varieties of razor clams to obtain fast-growing and highly resistant varieties of razor clams.

4. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 3, 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.

5. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 3, characterized in that: In the step 1), the adult razor clams are placed in a 31.6°C high temperature seawater culture tank for stress. When the mortality rate of the razor clams is close to 85%, the stress is stopped and the razor clams are placed in regular culture seawater to recover. Then, large and healthy razor clams are selected as seed shellfish with an 8% seed retention rate to breed the next generation.

6. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 3, 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 culture 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.

7. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 3, characterized in that: In the step 3), the selected heat-resistant hairy clam larvae are cultured until gonads mature, and after being dried in the shade for 6 hours, they are placed in an incubation pond filled with filtered seawater at 27°C to induce spawning and ejaculation, so as to establish a new heat-resistant F1 strain of hairy clam; after hatching, the selected D-shaped larvae are acclimated at a culture water temperature of 28±0.5°C, and the hairy clam larvae are cultured according to conventional methods for culturing marine shellfish larvae, placing attachment substrates and juvenile shellfish, and the high-temperature acclimation is stopped before the hairy clam seedlings are transported to open sea areas for cultivation.

8. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 3, characterized in that: In the step 4), the larvae obtained in the steps 2) and 3) are respectively cultured indoors in a northern nursery until metamorphosis and attachment. After the larvae of the hairy clam grow to 100,000 to 150,000 grains per kilogram, they are transported to the southern coast by refrigerated trucks or by air transport in insulated boxes with ice. The hairy clam seedlings are then evenly sown on the mud flat for breeding. After the hairy clam seedlings are cultured on the mud flat for 3 months, the size can reach 600 to 800 grains per kilogram. The large-sized hairy clam seedlings are transferred to the bottom of a nearby pond for breeding until they are sexually mature, and are used for stress treatment and breeding the next generation.

9. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 8, characterized in that: During north-south transportation, the transportation temperature should be controlled at 15°C or below, and the transportation time should be within 20 hours.

10. The method for breeding a fast-growing and highly resistant new variety of razor clams according to claim 3, characterized in that: In the step 6), female individuals of the new fast-growing and heat-resistant varieties of razor clams and male individuals of the new fast-growing and Vibrio-resistant varieties of razor clams are selected, or male individuals of the new fast-growing and heat-resistant varieties of razor clams and female individuals of the new fast-growing and Vibrio-resistant varieties of razor clams are selected, and after being dried in the shade for 6 hours, they are placed in an incubation pool filled with filtered seawater at 25° C. to induce spawning and spermatogenesis, and inter-strain hybridization is performed, and the hybrid offspring is the new fast-growing and highly resistant variety of razor clams.

Images