Method for controlling the culture of nereid polychaetes without specific pathogen shrimp

Abstract

The present application relates to a kind of cultivation process control method without specific pathogen of shrimp Nereis, belong to aquaculture technical field, the method includes cultivation site management, cultivation water treatment and management, sediment treatment, insemination, hatching and placement, Nereis seed disinfection, bait treatment and feeding;Invention method carries out all-round effect management from the air in cultivation site, cultivation appliance, sediment laid in cultivation pond, cultivation water etc., and further strictly controls spawning parent, fertilized egg etc., so as to be able to produce the Nereis without main pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a method for controlling the aquaculture process of sandworms free from specific shrimp pathogens. Background Technology

[0002] Sandworms are benthic marine organisms with high economic and nutritional value, and are commonly used in aquaculture as live feed for raising and maturing broodstock shrimp. However, studies have shown that during the breeding of broodstock shrimp, sandworms, as high-quality feed, often carry pathogenic bacteria and viruses, leading to large-scale disease outbreaks and hindering the healthy development of the shrimp farming industry. Pathogens not only adhere to the surface of sandworms but also hide within their bodies. Simply disinfecting the surface of the sandworms is insufficient to completely eliminate pathogens within them. Therefore, farming sandworms free of pathogens is a crucial industry requirement. These pathogens generally originate from the sandworms' habitat. By implementing biosafety measures to rationally control the sandworm farming process and environment, the risk of pathogen transmission can be reduced, increasing the commercial and application value of sandworms and providing strong support for improving the quality of shrimp and other seedling cultivation. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a method for controlling the breeding process of sandworms free from specific shrimp pathogens. This method addresses the problem that sandworms often carry aquatic pathogens in the prior art, which can directly or indirectly cause diseases in marine farmed animals. This method for breeding sandworms free from major pathogens can maintain an excellent breeding environment and obtain sandworms free from pathogens (i.e., SPF sandworms) by eliminating pathogens in the sediment and water inhabited by sandworms and controlling the breeding process. This has important practical significance for the shrimp and fish farming industries.

[0004] This invention is achieved through the following technical solution:

[0005] A method for controlling the breeding process of sandworms free from specific shrimp pathogens, the method includes breeding site management, breeding water treatment and management, sediment treatment, fertilization, hatching and release, sandworm seedling disinfection, and feed treatment and feeding.

[0006] The method includes farm management, enclosing the farm, and equipping it with ultraviolet light with an intensity greater than 100 μW / cm². 2 The ultraviolet lamps are used to disinfect the breeding environment. The ultraviolet lamps are placed at a height of about 100cm above the breeding pond. The ultraviolet lamps are turned on for 1 hour every 6 hours to kill pathogenic microorganisms in the environment. All breeding operation tools and personnel entering the breeding workshop must be disinfected.

[0007] Furthermore, aquaculture equipment should be placed in a location within the aquaculture area where it can be exposed to ultraviolet light, or immersed in a chlorine dioxide disinfection tank.

[0008] Furthermore, the breeding site is equipped with a dedicated pre-disinfection room for the disinfection of operators.

[0009] Furthermore, the inner walls of the aquaculture ponds are treated with a smooth finish to facilitate thorough disinfection.

[0010] Furthermore, the aforementioned aquaculture water treatment and management: seawater used in the aquaculture process is sterilized by high-temperature heating (100℃, 30min) or by using ClO2 with a concentration of 20mg / L, and then allowed to return to normal temperature or be aerated for 3-4 days before use; during non-use periods, the containers holding the sterilized seawater are covered, and the sterilized seawater and storage containers are kept in the aquaculture site throughout the entire process.

[0011] The sediment treatment involves sterilizing the sediments using a combination of high temperature and chlorine dioxide.

[0012] Further, dried marine sediments exposed to the sun are sieved and thoroughly mixed with seawater that has been sterilized at high temperatures and then cooled to room temperature. The mixture is then heated to above 90°C and maintained for at least 30 minutes, followed by natural cooling to room temperature. After 24 hours, 300 mg / L ClO2 is added to the overlying water and surface sediments for secondary sterilization. During the treatment, ultraviolet lamps are used to kill pathogens in the environment. Pathogen status needs to be tested the day after sterilization and before stocking. If the pathogens are not completely eliminated, the above operation needs to be repeated. Sediments that have passed the pathogen test are then laid in the aquaculture pond.

[0013] The fertilization, hatching, and release process is as follows: Prepare male and female polychaete bodies at a ratio of approximately 3:1. Sterilize their bodies by soaking them in 6 mg / L ClO2 seawater for 10 seconds to obtain sperm and eggs, and fertilize them. Collect the fertilized eggs and rinse them 2-3 times with sterilized seawater. Place the fertilized eggs in a small hatching tank and keep them at 20-25℃ for about 8-9 days until the larvae reach the 5-segment larvae stage. During this period, slightly aerate the seawater and change the water once a day. When the ultraviolet lamp is on during incubation, the hatching tank should be covered. When the larvae reach the 3-segment larvae stage, a small amount of spirulina powder can be fed. After reaching the 5-segment larvae stage, release the larvae into the breeding pond.

[0014] Furthermore, disinfection of sandworm seedlings: Sandworm seedlings are first soaked and rinsed three times with sterile seawater, then soaked in seawater with a concentration of 4 mg / L ClO2 for 10 seconds for surface disinfection, and then rinsed with sterile seawater three to four times before being placed in the breeding pond for normal breeding.

[0015] Furthermore, the processing and feeding of the feed: feed the prepared feed once every 1-2 days (the actual amount of feed can be adjusted according to the feeding situation of the sandworms). Before feeding, dry the feed in a 100℃ oven for 10 minutes to ensure that the feed is free of pathogens.

[0016] The beneficial effects of this invention compared with the prior art are as follows: The method of this invention manages the air, breeding equipment, sediments in the breeding pond, and breeding water in an all-round way, and further strictly controls the egg-laying parent and fertilized eggs, thereby enabling the production of sandworms free of major pathogens.

[0017] This invention uses ultraviolet lamps to kill pathogens in the aquaculture environment, promptly eliminating pathogenic microorganisms that settle on the surface of the aquaculture water and aquaculture equipment from the air, thus strictly preventing the risk of pathogens brought in from the air.

[0018] This invention disinfects seawater used in aquaculture. In existing technologies, disinfected seawater is placed in a pool without considering the risk of airborne pathogens accumulating in the water. Therefore, existing technologies do not cover the water storage containers. This invention creatively covers the sterilized seawater to reduce the risk of airborne pathogens contaminating the seawater.

[0019] Since sandworm farming requires the laying of seabed sediments, i.e., sea mud, the existing technology generally treats sea mud by sieving and sun-drying it, followed by high-temperature or chlorine dioxide treatment, which is considered to be thorough disinfection and sterilization. However, this invention has found through testing that a single sterilization method cannot completely eliminate pathogens in the sediments. Even if the pathogen test is negative after sterilization, pathogens can still grow in the sediments themselves over time, even if external pathogens are completely eliminated. This invention uses two disinfection methods in synergy to completely eliminate pathogens in the sediments. Detailed Implementation

[0020] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0021] The main pathogens in aquaculture are Covert mortality noda virus (CMNV), Vibrio parahaemolyticus (VpAHPND), Shrimp hemocyte iridescent virus (SHIV), White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Yellow head virus (YHV), Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, and Streptococcus agalactiae. The shrimp-specific pathogen-free method mentioned in this invention refers to the above-mentioned pathogens, and the virus detection method is qPCR.

[0022] The breeding farm is constructed using large tents measuring 430×290×300cm, with the top and four sides covered by waterproof tarpaulins. The interior height is 200cm. The breeding farm is under closed management and equipped with ventilation openings.

[0023] Example 1: Experiment on the duration of ultraviolet lamp irradiation in the environment

[0024] This embodiment uses an experimental method to measure the duration of ultraviolet lamp irradiation in a closed breeding farm environment. The specific implementation steps are as follows:

[0025] Equipped with a UV intensity of 30W (UV intensity greater than 100μW / cm). 2 The aquaculture environment was disinfected by irradiating it with ultraviolet lamps. Before the formal experiment, the aquaculture ponds and all aquaculture equipment were washed and disinfected. The ultraviolet lamps were then turned on and the aquaculture area was sealed for 24 hours to kill pathogenic microorganisms in the air and on the surfaces of objects. During normal aquaculture, the ultraviolet lamps were turned on for 1 hour every 6 hours to kill pathogenic microorganisms in the environment. During sediment treatment, the ultraviolet lamps were turned on throughout the process to kill pathogens in the environment.

[0026] Existing technologies typically sterilize airborne and surface organisms during the breeding period every 12 hours. However, the inventors monitored pathogens on the surface of the environment during the operation and found that when the interval was longer than 6 hours, pathogens accumulated on the surface of the breeding water and operating equipment. If sterilization is not carried out in time, the pathogens will sink into the water and infect the breeding sandworms. Therefore, ultraviolet lamp disinfection every 6 hours is the optimal technical solution.

[0027] Example 2: Method for disinfecting sediments

[0028] This embodiment employs the method for disinfecting sediments described above, and its specific implementation steps are as follows:

[0029] For the sterilization of sediments, existing technologies generally employ high temperature or chlorine dioxide. This embodiment explores the use of high temperature alone (100℃ for 30 min), chlorine dioxide alone, and a combination of high temperature and chlorine dioxide for sterilization. Sun-exposed dried marine sediments were collected and passed through a 12-mesh (1.4 mm) sieve to remove impurities. Seawater, sterilized at high temperature and then cooled to room temperature, was thoroughly mixed with the dried marine sediments and soaked for 1 hour. The mixture was then heated to 100℃ in a water bath for 30 min, allowed to cool naturally to room temperature, and samples were collected for pathogen detection. Simultaneously, the sterilized sediments were placed in a sealed container and tested again for pathogens after 15 days.

[0030] Alternatively, after soaking for 1 hour, add 300 mg / L ClO2 to the sediment for sterilization, take samples for pathogen detection, and place the sterilized sediment in a sealed container for pathogen detection again after 15 days.

[0031] Alternatively, after high-temperature sterilization, the mixture is cooled for 24 hours, and then ClO2 at a concentration of 300 mg / L is added to perform secondary sterilization on the overlying water and surface sediment. Samples are taken for pathogen detection, and the sterilized sediment is placed in a sealed container and pathogen detection is performed again after 15 days.

[0032] The test results were as follows: when sterilization was performed immediately, all pathogen tests were negative. However, after 15 days of sealed treatment, pathogens could be detected in both the high-temperature and ClO2 sterilization groups alone, while no pathogens were found in the sediment when both sterilization methods were used in combination.

[0033] Example 3

[0034] A method for controlling the breeding process of sandworms, a specific pathogen of shrimp, includes the following: management of breeding sites, treatment and management of breeding water, sediment treatment, fertilization, hatching and release, disinfection of sandworm seedlings, and treatment and feeding of feed.

[0035] The aforementioned breeding site management: The breeding site is constructed using large tents measuring 430×290×300cm, with the top and four sides covered by waterproof tarpaulins. The interior height is 200cm. Every 18m... 3 Set an ultraviolet intensity greater than 100 μW / cm 2 Ultraviolet (UV) lamps are used to disinfect the shed. Two UV lamps are installed in each shed, with the lamps positioned 100cm above the breeding ponds. The UV lamps are turned on for 1 hour every 6 hours to kill pathogenic microorganisms in the air and on object surfaces. All breeding equipment must be disinfected. Ideally, breeding equipment should be placed in the breeding shed where it can be exposed to UV lamps or immersed in a chlorine dioxide disinfection tank.

[0036] All aquaculture equipment and personnel entering the aquaculture workshop must be disinfected; as a preferred aquaculture site, a dedicated pre-disinfection room is provided for the disinfection of operators. Before entering, personnel are provided with a dedicated pre-disinfection room (preparation room), equipped with ultraviolet lamps, disinfectant, and special disinfected clothing. After changing into the clothing, they enter the aquaculture workshop.

[0037] As a preferred method, to ensure thorough disinfection during aquaculture, the inner walls of the aquaculture pond were smoothed, while other aspects remained the same as conventional aquaculture ponds. Before the formal experiment, the aquaculture pond was thoroughly cleaned with a brush, then repeatedly washed and rinsed with ClO2-sterilized seawater. After 24 hours of irradiation with ultraviolet lamps turned on and the tent closed, sterilized sediment was introduced.

[0038] The aforementioned aquaculture water treatment and management: Sterilized seawater used in the aquaculture process is preferably sterilized by high-temperature heating (100℃, 30 min) or by using ClO2 at a concentration of 20 mg / L, and then allowed to return to room temperature or aerated for 3-4 days before use. Sterilized seawater is stored in dedicated containers, which are kept sealed when not in use. The sterilized seawater and storage containers are kept within the aquaculture area at all times.

[0039] The sediment treatment involves a combined high-temperature and chlorine dioxide sterilization process. Preferably, sun-dried marine sediments are passed through a 12-mesh (1.4mm) sieve to remove impurities. Seawater, cooled to room temperature after high-temperature sterilization, is thoroughly mixed with the dried sediments and soaked for 1 hour. The mixture is then heated to 100℃ in a water bath and maintained for 30 minutes, followed by natural cooling to room temperature. After 24 hours, 300mg / L ClO2 is added to the overlying water and surface sediments for secondary sterilization. Ultraviolet lamps are used during this process to kill pathogens in the environment. Pathogen detection is performed the day after sterilization and before stocking. Results show no pathogens in the sediments and only a very small amount in the overlying water. The pathogen-tested sediments are then spread in a 15-20cm layer in the aquaculture pond and allowed to settle naturally for one week before use.

[0040] The fertilization, hatching, and release process is as follows: Prepare male and female polychaete bodies at a ratio of 3:1. Sterilize the body surface by soaking in 6 mg / L ClO2 seawater for 10 seconds. Then, cut open the body wall of the parent polychaete and place them in a net bag in a hatching tank filled with sterilized seawater. After about 10 minutes, remove the body wall of the parent polychaete. After another 20 minutes, collect the fertilized eggs using a 200-300 mesh sieve and rinse the fertilized eggs 2-3 times with sterilized seawater. Place the fertilized eggs in a small hatching tank and keep them at 20-25℃ for about 8-9 days until the larvae reach the 5-segment larvae stage. During this period, slightly aerate the seawater and change the water once a day. When using the UV lamp during hatching, cover the hatching tank. When the larvae reach the 3-segment larvae stage, feed them a small amount of spirulina powder. After reaching the 5-segment larvae stage, release the larvae into the rearing pond.

[0041] Disinfection of the sandworm seedlings: Preferably, sandworm seedlings with a size of about 0.5g-1.0g are selected. They are first soaked and rinsed three times with sterile seawater, then soaked in seawater with a concentration of 4mg / L ClO2 for 10 seconds for surface disinfection. After that, their surface is rinsed with sterile seawater three to four times. Then, sandworm seedlings with better vitality are selected and put into the breeding pond for normal breeding.

[0042] The processing and feeding of the feed: Preferably, feed the feed once every 1-2 days (the actual amount of feed can be adjusted according to the feeding situation of the sandworms). Before feeding, dry the feed in a 100℃ oven for 10 minutes to ensure that the feed is free of pathogens.

[0043] According to the method described above, in this embodiment, seawater, sediment, and overlying water, as well as sandworm (homogeneous material), were periodically sampled during the aquaculture period for detection of major pathogens. Specifically, the pathogens were Noda-mura virus, Vibrio parahaemolyticus causing acute hepatopancreatic necrosis, shrimp hemocytic iridovirus, white spot syndrome virus, infectious hypodermal and hematopoietic tissue necrosis virus, as well as yellow head virus, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, and Streptococcus agalactiae. The test results showed that no pathogens were found in the seawater, sediment, and sandworm (homogeneous material), and only a very small amount of pathogens were found in the overlying water.

Claims

1. A method for controlling the culture process of *Nematodesmus spp.* free from specific shrimp pathogens, characterized in that... The methods described include aquaculture site management, aquaculture water treatment and management, sediment treatment, insemination, hatching and release, disinfection of sandworm seedlings, and feed treatment and feeding; the specific pathogens of shrimp mentioned are Nodamura virus, Vibrio parahaemolyticus causing acute hepatopancreatic necrosis, shrimp hemocytic iridovirus, white spot syndrome virus, infectious hypodermal and hematopoietic tissue necrosis virus, as well as yellow head virus, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus and Streptococcus agalactiae; The method includes farm management, enclosing the farm, and equipping it with ultraviolet light with an intensity greater than 100 μW / cm². 2 The ultraviolet lamps are used to disinfect the breeding environment. The ultraviolet lamps are placed at a height of about 100 cm above the breeding pond. The ultraviolet lamps are turned on for 1 hour every 6 hours to kill pathogenic microorganisms in the environment. All breeding operation tools and personnel entering the breeding workshop must be disinfected. The aforementioned aquaculture water treatment and management: Seawater used in the aquaculture process is sterilized at 100℃ for 30 minutes or with ClO2 at a concentration of 20mg / L, and then allowed to return to normal temperature or be aerated for 3-4 days before use; During non-use periods, the containers holding the sterilized seawater are covered, and the sterilized seawater and storage containers are kept in the aquaculture site throughout the entire process; The sediment treatment involves a combination of high-temperature and chlorine dioxide sterilization. Sun-dried marine sediments are sieved and thoroughly mixed with seawater (after high-temperature sterilization and subsequent return to room temperature) for soaking. The mixture is then heated to above 90°C and maintained for at least 30 minutes, followed by natural cooling to room temperature. After 24 hours, 300 mg / L ClO2 is added to the overlying water and surface sediments for secondary sterilization. Ultraviolet lamps are used during the treatment to kill pathogens in the environment. Pathogen detection is performed the day after sterilization and before stocking. If pathogens are not completely eliminated, the above steps are repeated. Sediments that pass pathogen detection are then laid in the aquaculture pond. The fertilization, hatching, and release process is as follows: Prepare male and female polychaete bodies at a ratio of 3:

1. Sterilize the body surface by soaking in 6 mg / L ClO2 seawater for 10 seconds to obtain sperm and eggs, and fertilize them. Collect the fertilized eggs and rinse them 2-3 times with sterilized seawater. Place the fertilized eggs in a small hatching tank and keep them at 20-25℃ for 8-9 days until the larvae reach the 5-segment larvae stage. During this period, slightly aerate the seawater and change the water once a day. When the ultraviolet lamp is on during incubation, the hatching tank should be covered. When the larvae reach the 3-segment larvae stage, a small amount of spirulina powder can be fed. After reaching the 5-segment larvae stage, release the larvae into the breeding pond. Disinfection of the sandworm seedlings: The sandworm seedlings are first soaked and rinsed three times with sterile seawater, then soaked in seawater with a concentration of 4 mg / L ClO2 for 10 seconds for surface disinfection, then rinsed with sterile seawater three to four times, and then placed in the breeding pond for normal breeding. The processing and feeding of the feed: Feed the feed once every 1-2 days. Before feeding, dry the feed in a 100℃ oven for 10 minutes to ensure that the feed is free of pathogens.

2. The method for controlling the culture process of *Nematostella asiatica* free from specific shrimp pathogens according to claim 1, characterized in that, The breeding equipment should be placed in a location within the breeding area where it can be exposed to ultraviolet light, or placed in a chlorine dioxide immersion disinfection tank.

3. The method for controlling the culture process of *Nematodes edulis* free from specific shrimp pathogens according to claim 1, characterized in that, The breeding site has a dedicated pre-disinfection room for the disinfection of operators.

4. The method for controlling the culture process of *Nematostella asiatica* free from specific shrimp pathogens according to claim 1, characterized in that, The inner walls of the aquaculture pond are smooth to facilitate thorough disinfection.