A method for applying microalgae to prevent and treat Vibrio diseases in shrimp farming

Abstract

The invention discloses a microalgae application method for preventing and treating Vibrio diseases in shrimp farming, which relates to the technical field of shrimp farming, and comprises the following steps: S1, culturing Porphyridium cruentum and Phaeochromis triangularis; S2, mixing Porphyridium cruentum and Phaeochromis triangularis to form a composite algae liquid, and spreading it in the shrimp farming water body. The microalgae application method for preventing and treating Vibrio diseases in shrimp farming can effectively improve the survival rate and growth rate of shrimps after suffering from Vibrio diseases, realize green and efficient shrimp farming, and reduce farming risks.

Description

Technical Field

[0001] The invention relates to the technical field of shrimp farming, and in particular to a method for applying microalgae to prevent and treat Vibrio diseases in shrimp farming. Background Art

[0002] Microalgae are a type of algae with tiny individuals, most of which are aquatic eukaryotic organisms, single-celled or composed of several cells. Microalgae are rich in nutrients, rich in substances such as protein, vitamins and polyunsaturated fatty acids, and have fast growth rates and high yields per unit area. Based on these characteristics, microalgae are widely used in industrial fields such as bait, health food, pharmaceuticals and biofuels. At present, microalgae are often used as water regulators in aquaculture to reduce water quality indicators such as ammonia nitrogen and nitrite nitrogen in water bodies and increase dissolved oxygen. The main algae used are Chlorella and Oocystis. Usually, the main purpose is to improve water quality and inhibit cyanobacteria, while there are almost no reports on the application of microalgae to increase the growth rate of shrimp and prevent and control diseases.

[0003] In the process of shrimp farming, with the increase of breeding density, there are problems such as degradation of germplasm resources, deterioration of water quality, difficulty in cultivating shrimp seedlings and uneven feed quality, which seriously restrict the healthy and sustainable development of the shrimp farming industry. The growth and disease resistance of shrimp larvae of different strains vary under different environmental conditions. Shrimp larvae are more active from the zoanthid stage to the mysid stage, grow fast and are easily affected by the external environment and invaded by pathogens (such as Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio alginolyticus and other bacteria), resulting in low survival rate of shrimp larvae. The problem of seedling production being affected by not feeding the appropriate starter feed on time and in the right amount is common. At the same time, most of the feeds on the market have a single nutrient composition, which affects the growth of shrimp.

[0004] It can be seen that developing a microalgae application method that can improve the survival rate and growth rate of shrimp after suffering from Vibrio disease has important practical significance for healthy aquaculture and reducing aquaculture risks. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for applying microalgae to prevent and treat Vibrio diseases in shrimp farming. The method can effectively improve the survival rate and growth rate of shrimp after suffering from Vibrio diseases, realize green and efficient shrimp farming, and reduce farming risks.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for applying microalgae to prevent and treat Vibrio diseases in shrimp farming, comprising the following steps:

[0007] S1, culturing Porphyridium cruentum and Phaeochromis triangularis;

[0008] S2. Mix Porphyridium crusgalli and Phaeochromis triangularis to form a compound algae liquid, and spread it in the shrimp farming water.

[0009] Preferably, the culture medium used for culturing Porphyridium and Phaeochromis tricornutum in step S1 comprises components with the following concentrations: 0.1-1 g / L glycerol, 15-30 g / L sodium chloride, 3-7 g / L magnesium sulfate heptahydrate, 3-8 g / L magnesium chloride hexahydrate, 0.04-1 g / L dipotassium hydrogen phosphate, 0.3-0.8 g / L potassium chloride, 0.5-1 g / L sodium nitrate, and 0.5-2 ml / L trace element mother solution.

[0010] Preferably, the trace element mother solution comprises the following components in concentration: EDTA 0.45-0.55 g / L, copper sulfate pentahydrate 0.007-0.009 g / L, zinc sulfate heptahydrate 0.220-0.224 g / L, cobalt nitrate hexahydrate 0.004-0.006 g / L, manganese chloride tetrahydrate 1.80-1.82 g / L, cobalt nitrate dihydrate 0.38-0.40 g / L, and boric acid 2.85-2.87 g / L.

[0011] Preferably, the culture medium used in culturing Porphyridium cruentum and Phaeochromis tricornutum in step S1 is sterilized at high temperature.

[0012] Preferably, the conditions for high temperature sterilization are: temperature 110-120° C., and sterilization time 18-22 minutes.

[0013] Preferably, the concentration ratio of Porphyridium cruentum and Phaeochromis tricornutum in the composite algae liquid in step S2 is (1-3):1.

[0014] Preferably, the feeding amount of the composite algae solution in step S2 is 5×10 4 -20×10 4 cells / mL.

[0015] Due to the application of the above technical solution, the present invention has the following beneficial effects: the present invention adopts the compound of Porphyridium and Phaeochromis triangularis to form a multifunctional mixed algae liquid, which is rich in Porphyridium exopolysaccharides and Phaeochromis triangularis. Porphyridium exopolysaccharide is a β-1,3 glucan with antiviral and antibacterial functions. Phaeochromis triangularis is a diatom, rich in kelp polysaccharides and unsaturated fatty acids, and can be used as bait for aquatic animals to promote the growth of aquatic animals. The mixture of the two microalgae simultaneously promotes the growth of shrimp and improves the survival rate of shrimp after Vibrio infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Comparison of the body lengths of prawns in different treatment groups in Example 1 of the present invention;

[0017] Figure 2 A graph showing the survival rate of shrimp infected with Vibrio parahaemolyticus in Example 1 of the present invention;

[0018] Figure 3Figure 2 shows the body length of prawns after 20 days of culture and the survival rate after infection with Vibrio parahaemolyticus in Example 2 of the present invention. DETAILED DESCRIPTION

[0019] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0020] Example 1

[0021] Both Porphyridium and P. triangularis were cultured in a 1L column photobioreactor (liquid volume 800mL), the culture temperature was controlled at 25°C, sterile air (filtered with a 0.22μm membrane filter head) was introduced, the ventilation volume was 0.2vvm, and LED lamps were used to provide light to the column photobioreactor, with a light intensity of 4000lux. The culture medium of Porphyridium and P. triangularis was glycerol 0.1g·L -1 , sodium chloride 20g·L -1 , magnesium sulfate heptahydrate 6g·L -1 , magnesium chloride hexahydrate 5g·L -1 , Dipotassium hydrogen phosphate 0.05 g·L -1 , Potassium chloride 0.4 g·L -1 , Sodium nitrate 0.65 g·L -1 、Trace element mother solution 1ml·L -1 The prepared culture medium was placed in a 1L glass column photoreactor (800mL of liquid) and sterilized in an autoclave. The sterilization temperature was set to 115°C for 20 minutes. After sterilization and cooling, 20% of the inoculation amount of Porphyridium and Phaeochromis triangularis was inoculated in the clean bench workbench in the sterile room, and then placed in a temperature-controlled small room for the cultivation of Porphyridium and Phaeochromis triangularis.

[0022] At the same time, four groups of sorting boxes (40cm×30cm×20cm) were set up to culture shrimps, including the control group (no microalgae added), the purple algae group, the purple algae and triangular brown finger algae (1:1) group, and the triangular brown finger algae group. Artificial seawater was used with a salinity of 10‰. A small air pump was connected to a hose and an air stone to aerate the culture water to provide sufficient oxygen to the shrimp. Fifty white shrimp seedlings (average body length of about 0.4cm) were cultured in each experimental group. A certain amount of seedling feed was fed every day, and the residual bait, feces and dead shrimp at the bottom of the culture water were cleaned with a sewage suction device. Except for the control group, purple algae and / or triangular brown finger algae were added to the culture water, so that after the addition of microalgae, the algal cell concentration in the water was, the purple algae group (15×10 4 cells / ml), Porphyridium and Phaeochromis triangularis groups (7.5×10 4 cells / mL and 7.5×10 4cells / mL), Phaeochromis triangularis (15×10 4 cells / ml). Microalgae were added twice at the beginning of culture and 15 days after culture. After 20 days of culture, the Vibrio parahaemolyticus challenge (infection period) was carried out. The shrimps in each group were challenged with Vibrio parahaemolyticus by immersion bath. The shrimps were first taken out of the culture water with a net bag and placed in 2.13×10 7 CFU / ml Vibrio parahaemolyticus liquid was soaked for 15 min, then put back, and then Vibrio parahaemolyticus liquid was added to the aquaculture water to make the concentration of Vibrio in the water reach 2.13×10 5 CFU / ml, and the culture time of the challenge phase was 9 days. The length of shrimp in each treatment group was measured after 20 days of shrimp culture. After the challenge, the number of surviving shrimp was determined and the survival rate of shrimp was calculated. Adding Porphyridium cruciferum and Phaeochromis triangularis to the culture water can promote the growth of shrimp. Phaeochromis triangularis has the most obvious growth-promoting effect, followed by Porphyridium cruciferum and Phaeochromis triangularis mixed algae, and Porphyridium cruciferum has the lowest growth effect (see Figure 1 ). As for the effect on the survival rate of shrimp infected with Vibrio parahaemolyticus, Porphyridium cruzi was the best (see Figure 2 ). Porphyridium can significantly improve the survival rate of shrimp after infection with Vibrio parahaemolyticus, which is related to the ability of Porphyridium to produce extracellular glucan. Studies have shown that Porphyridium glucan has antiviral and antibacterial functions, thus improving the resistance of shrimp to the toxicity of Vibrio parahaemolyticus and improving the survival rate. In the mixed algae group of Porphyridium and Phaeochromis triangularis, the survival rate of shrimp after the attack was also high, and the survival rate value decreased slowly, while the survival rate of shrimp in the Phaeochromis triangularis group was not significantly different from that in the control group. It can be seen that the addition of Porphyridium can significantly improve the survival rate of shrimp, while Phaeochromis triangularis has basically no effect on the survival rate of shrimp after infection with Vibrio parahaemolyticus, and does not have an anti-Vibrio effect. Therefore, the composite mixed algae of Porphyridium and Phaeochromis triangularis can not only improve the growth rate of shrimp, but also fight against Vibrio at the same time, significantly improving the survival rate of shrimp after infection with Vibrio parahaemolyticus.

[0023] Example 2

[0024] Both Porphyridium and P. triangularis were cultured in 5L triangular shake flasks (liquid volume 2L), the culture temperature was controlled at 25°C, sterile air (filtered with a 0.22μm membrane filter head) was introduced, the ventilation volume was 0.2vvm, and LED lamps were used to provide illumination for the column photobioreactor, with an incident light intensity of 6000lux. The culture medium for Porphyridium and P. triangularis was 0.1g·L glycerol -1 , sodium chloride 20g·L -1 , magnesium sulfate heptahydrate 6g·L -1 , magnesium chloride hexahydrate 5g·L -1 , Dipotassium hydrogen phosphate 0.05 g·L -1 , Potassium chloride 0.4 g·L -1, Sodium nitrate 0.65 g·L -1 、Trace element mother solution 1ml·L -1 The prepared culture medium was placed in a 5L triangular shake flask (liquid volume 2L) and placed in an autoclave for sterilization. The sterilization temperature was set to 115°C for 20 minutes. After sterilization and cooling, 15% of the inoculation amount of Porphyridium and Phaeochromis triangularis was inoculated on the clean bench workbench in the sterile room, and then placed in a temperature-controlled small room for the cultivation of Porphyridium and Phaeochromis triangularis.

[0025] At the same time, 4 groups of sorting boxes (40cm×30cm×20cm) were set up to culture shrimps, using artificial seawater with a salinity of 10‰. A small air pump was connected to a hose and an air stone to aerate the culture water to provide sufficient oxygen to the shrimps. Mixed algae group 1 (purpurogenous algae: triangular brown finger algae = 0.5:1), mixed algae group 2 (purpurogenous algae: triangular brown finger algae = 1:1), mixed algae group 3 (purpurogenous algae: triangular brown finger algae = 3:1), mixed algae group 4 (purpurogenous algae: triangular brown finger algae = 4:1), 30 white shrimps (average body length 1.2cm) were cultured in each experimental group, a certain amount of seedling feed was fed every day, and the residual bait, feces and dead shrimps at the bottom of the culture water were cleaned with a sewage suction device. Porphyridium and triangular brown finger algae were added to the culture water, so that after the addition of microalgae, the algae cell concentration in the water was 10×10 4 cells / ml). Microalgae were added at the beginning of culture. After 20 days of culture, the Vibrio parahaemolyticus challenge (infection period) was carried out. The shrimps in each group were challenged with Vibrio parahaemolyticus. The shrimps were first taken out of the culture water with a net bag and placed in a 2.13×10 7 CFU / ml Vibrio parahaemolyticus liquid was soaked for 15 min, then put back, and then Vibrio parahaemolyticus liquid was added to the aquaculture water to make the concentration of Vibrio in the water reach 2.13×10 5 CFU / ml, and the cultivation time of the attack phase is 5 days. The length of the shrimp in each treatment group was measured after 25 days of shrimp cultivation. After the attack, the number of surviving shrimps was determined and the survival rate of the shrimps was calculated. The experimental results show that within the range of (1-3):1 of the concentration ratio of Porphyridium and Phaeochromis triangularis, not only can the shrimps grow rapidly, but also the survival rate of the shrimps after infection with Vibrio parahaemolyticus can be maintained. When the concentration ratio of Porphyridium and Phaeochromis triangularis is 0.5:1 (less Porphyridium and more Phaeochromis triangularis) or 4:1 (more Porphyridium and less Phaeochromis triangularis), a lower survival rate or insignificant growth-promoting effect occurs. Therefore, the condition for simultaneously ensuring the improvement of the growth rate of shrimps and the improvement of the survival rate of shrimps is that the ratio of the number of Porphyridium and Phaeochromis triangularis is (1-3):1.

[0026] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for applying microalgae to prevent and treat Vibrio diseases in shrimp farming, characterized in that: The steps include: S1, culturing Porphyridium cruentum and Phaeochromis triangularis; S2, mixing Porphyridium crusgalli and Phaeochromis triangularis to form a composite algae liquid, and applying the composite algae liquid to the shrimp aquaculture water body; the concentration ratio of Porphyridium crusgalli and Phaeochromis triangularis in the composite algae liquid is (1-3):1; The culture medium used for culturing Porphyridium and Phaeochromis triangularis in step S1 includes the following components in concentration: 0.1-1 g / L glycerol, 15-30 g / L sodium chloride, 3-7 g / L magnesium sulfate heptahydrate, 3-8 g / L magnesium chloride hexahydrate, 0.04-1 g / L dipotassium hydrogen phosphate, 0.3-0.8 g / L potassium chloride, 0.5-1 g / L sodium nitrate, and 0.5-2 ml / L trace element mother solution.

2. The microalgae application method for preventing and treating Vibrio diseases in shrimp culture according to claim 1, characterized in that: The trace element mother solution comprises components with the following concentrations: 0.45-0.55 g / L EDTA, 0.007-0.009 g / L copper sulfate pentahydrate, 0.220-0.224 g / L zinc sulfate heptahydrate, 0.004-0.006 g / L cobalt nitrate hexahydrate, 1.80-1.82 g / L manganese chloride tetrahydrate, 0.38-0.40 g / L cobalt nitrate dihydrate, and 2.85-2.87 g / L boric acid.

3. The microalgae application method for preventing and treating Vibrio diseases in shrimp culture according to claim 1, characterized in that: The culture medium used in culturing Porphyridium cruentum and Phaeochromis tricornutum in step S1 is sterilized at high temperature.

4. The microalgae application method for preventing and treating Vibrio diseases in shrimp culture according to claim 3, characterized in that: The conditions for high temperature sterilization are: temperature 110-120° C., and sterilization time 18-22 minutes.

5. The microalgae application method for preventing and treating Vibrio diseases in shrimp culture according to claim 1, characterized in that: The feeding amount of the composite algae solution in step S2 is 5×10 4 -20×10 4 cells / mL.

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