Application of nonanoic acid in breeding of artemia and a method for breeding artemia

Abstract

The application provides application of nonanoic acid in breeding of artemia. In the application, the nonanoic acid is directly added into a breeding water body in a form of a carrier of silica or chitosan. When the nonanoic acid is in the form of the carrier of silica, the use concentration of the nonanoic acid in the water body is 150-300 μM; when the nonanoic acid is in the form of the carrier of chitosan, the use concentration of the nonanoic acid in the water body is 55-90 μM. The inventors have unexpectedly found that, after the nonanoic acid is mixed with the silica or the chitosan and then added into the breeding water body, the pH of the water body does not suddenly decrease, so that an acid-base regulator does not need to be added and the hatching and growth of the artemia are not affected; meanwhile, the use of the carrier realizes uniform dispersion and local high concentration of the nonanoic acid in the water body, which reduces the use amount of the acid and significantly improves the bacteriostatic effect. The application further provides a breeding method of the artemia using the nonanoic acid as a substitute product. The artemia obtained by using the breeding method has the following advantages: the hatching rate and survival rate of the artemia are significantly improved, the number of vibrio carried by the artemia is effectively reduced, the expression of growth and immune related genes of the artemia is up-regulated, the development and growth of the artemia are promoted, the ecological safety of the artemia is improved, and unexpected technical effects are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of aquaculture, and relates to a breeding method of Artemia, in particular to a method for reducing the hidden danger of Artemia carrying conditional pathogenic bacteria by using nonanoic acid, and realizing rapid and healthy, stable and sustainable breeding of Artemia. BACKGROUND

[0002] Artemia, commonly known as brine shrimp, saltwater shrimp, is a small invertebrate crustacean. Artemia has the characteristics of small individual, high nutritional value, strong environmental adaptability, fast reproduction, and long-term storage, and is an important biological bait for economic animals in aquaculture. In terms of market demand, Artemia has always been in a state of shortage; high-density and healthy breeding of Artemia has important application prospects and great economic benefits. The non-selective feeding characteristics of Artemia are often used to enrich different nutrients and improve the nutritional value of Artemia. However, it is also easy to enrich conditional pathogenic bacteria, which reduces the hatching rate and survival rate of Artemia, and causes sudden collapse of high-density Artemia breeding system, resulting in instability of Artemia breeding. Once Artemia carries conditional pathogenic bacteria, it can also infect the larvae of economic animals such as fish, shrimp and crab through the food chain, causing the failure of seed production. Therefore, effectively controlling the conditional pathogenic bacteria carried by Artemia (especially pathogenic Vibrio, such as Vibrio harveyi, Vibrio parahaemolyticus, Vibrio campbellii, etc.), is crucial for ensuring the high-density and sustainable breeding of Artemia, as well as the seed production of economic animals in aquaculture and the healthy breeding of aquatic animals.

[0003] Antibiotics are commonly used in aquaculture to control the conditionally pathogenic bacteria carried by brine shrimp. However, the use of antibiotics not only has limited effect, but also promotes the generation of drug-resistant pathogenic microorganisms and is transmitted through the food chain, posing a great threat to human health and the ecological environment. At present, acidifiers (such as formic acid, acetic acid), microecological preparations (such as marine bacillus), and plant extracts (such as essential oils) have been used as alternatives to antibiotics in the culture of brine shrimp. Among them, acidifiers refer to the general term of organic acids that can be used as additives, which belong to non-nutritional feed additives. However, the bacteriostatic effect of acid weakens with the increase of pH, which is contrary to the natural environment of brine shrimp, which is neutral to alkaline. Previous studies have shown that the optimal hatching pH of brine shrimp is 7.5-8.5, and if the pH <7, it will cause eggshell softening, embryo malformation, and significantly reduce the hatching rate. Although the adaptation range of adult brine shrimp to pH is wide, they can survive at pH 7.0-9.5, but the optimal growth pH is 7.5-8.5. In addition, sudden changes in pH can trigger stress responses in brine shrimp (such as abnormal molting, decreased immunity, and susceptibility to infection). Immanuel et al. studied the effects of caprylic acid, acetic acid, propionic acid, and butyric acid on the survival rate of brine shrimp artificially infected with Vibrio harveyi and Vibrio parahaemolyticus under experimental conditions. The mortality rate of brine shrimp infected with V. harveyi significantly decreased by 16.30% after adding caprylic acid (10 mM), and the mortality rate of brine shrimp infected with V. parahaemolyticus significantly decreased by 20.61% (P<0.05). The mortality rates of brine shrimp nauplii infected with V. parahaemolyticus cultured in seawater added with acetic acid (20 mM), propionic acid (20 mM), and butyric acid (20 mM) were 16.7%, 11.7%, and 10.0% lower than the control group, respectively. However, although the mortality rate of brine shrimp decreased, the use of the aforementioned acids at high concentrations resulted in a low pH in the water, which needed to be adjusted to 7 by adding acid-base regulators to meet the environmental needs of brine shrimp. In actual production, this significantly increases the cost and operation steps, which is not conducive to the growth of brine shrimp and industrial application. As mentioned earlier, this study targets artificially infected sterile brine shrimp, and the bacterial flora carried is simpler compared to the actual farming system. In addition, Gao et al. evaluated the toxic effects of nonanoic acid on the growth and reproduction of planktonic animals and found that the mortality rate of Daphnia magna increased with the increase of nonanoic acid concentration and the extension of exposure time. The safe concentration of nonanoic acid for D. magna was 3.05 mg / L (19.3 μM). In actual production, obviously increasing the concentration of nonanoic acid increases the insecurity of the farming ecosystem.

[0004] Nonanoic acid is a natural nine-carbon fatty acid containing carboxyl group, with a molecular weight of 158.238 (g / mol), usually in the form of colorless oily liquid, weakly acidic, with certain irritation; insoluble in water, easily soluble in ethanol, diethyl ether and chloroform, and detected in varying degrees in a variety of plants, fruits and vegetables. De Smet et al. studied the antibacterial activity of nonanoic acid in the stomach and intestine of piglets, and the addition of nonanoic acid in the daily diet of piglets reduced the number of streptococci in the intestine. Nurettin et al. studied the antibacterial activity of methylated nonanoic acid derivatives on gram-positive bacteria (Bacillus subtilis, Salmonella, Escherichia coli, etc.), among which the methylated nonanoic acid derivatives at positions 4, 7 and 8 had antibacterial activity against streptomyces. Jin-Hyung et al. found that nonanoic acid could effectively inhibit the formation of Candida albicans biofilm, and significantly improve the survival rate of Caenorhabditis elegans infected by Candida albicans (50-80%, P<0.05). At present, there are few reports on the application of nonanoic acid in aquaculture, and Yao and Yu et al. found that nonanoic acid participated in the regulation of immune response by activating the MAPK / ERK signal pathway of carp tissue cells and the ERK1 / 2 signal pathway of Chinese giant salamander tissue cells. If nonanoic acid is directly applied to the culture water, there are a series of problems such as strong hydrophobicity, difficulty in dispersion, irritation, sensitivity to alkaline environment, etc. Moreover, high hardness water, alkaline and organic matter can also neutralize organic acids, thereby weakening the antibacterial effect. However, increasing the concentration of nonanoic acid will increase the ecological safety risk of Artemia, inhibit its hatching and growth, and even cause death, which can also cause the pH of the water body to decrease, further increasing the production cost and safety risk.

[0005] Based on the foregoing technical problems, there is no related report on the use of nonanoic acid in Artemia cultivation at present. SUMMARY

[0006] In view of the problems of complicated steps and high cost in the prior art of using organic acid as an alternative product in Artemia cultivation, the application of nonanoic acid in Artemia cultivation is provided. The application realizes uniform dispersion and antibacterial effect of nonanoic acid under the premise of strong hydrophobicity, difficulty in dispersion and ecological safety risk, and improves the ecological safety, overcoming the technical prejudice that increasing the concentration of nonanoic acid leads to difficulty in dispersion, decrease of water body pH and ecological safety risk in the prior art. On this basis, the application also provides a cultivation method of Artemia using nonanoic acid as an alternative product. The Artemia obtained by the cultivation method not only significantly improves the hatching rate and survival rate of Artemia, effectively reduces the number of Vibrio carried by Artemia, but also up-regulates the expression of growth and immune related genes, promotes the development and growth of Artemia and improves the ecological safety, achieving unexpected technical effects.

[0007] Technical scheme of the application:

[0008] The application of nonanoic acid in brine shrimp farming involves directly adding nonanoic acid to the farming water using silica or chitosan as a carrier. When silica is used as a carrier, the concentration of nonanoic acid in the water is 150-300 μM; when chitosan is used as a carrier, the concentration is 55-90 μM. The inventors unexpectedly discovered that mixing nonanoic acid with the aforementioned silica or chitosan before adding it to the farming water does not cause a sudden drop in pH, thus eliminating the need for acid-base regulators and not affecting the hatching and growth of brine shrimp, thereby improving ecological safety. Simultaneously, the use of a carrier achieves uniform dispersion in the water and maintains a locally high concentration of acid, reducing the amount of acid used while significantly enhancing the antibacterial effect. Therefore, the application described in this application achieves a balance between the antibacterial effect of nonanoic acid, stable water pH, and ecological safety, overcoming the technical bias of prior art that increases the concentration of nonanoic acid to achieve antibacterial effects, which leads to difficulties in water dispersion, pH drops, and ecological safety risks.

[0009] Preferably, when the nonanoic acid is supported by silica, the weight-to-volume ratio of SiO2 to nonanoic acid is (0.5~3):1 (g:ml), and the particle size of the silica is 10-40 μm; when the nonanoic acid is supported by chitosan, the weight-to-volume ratio of chitosan to nonanoic acid is (0.01~0.1):1 (g:ml), and the degree of deacetylation of the chitosan is 75-88%, with a molecular weight of 100-180 kDa. The inventors have discovered that the silanol groups (Si-OH) on the surface of SiO2 can interact with the carboxylic acid groups (-COOH or -COO-) of organic acids through hydrogen bonds, and chitosan in organic acid solutions can react by protonating amino groups (-NH3). + ) and the carboxyl ion (-COO) of organic acids - This forms ionic complexes, thereby controlling the release rate of organic acids, prolonging their action time, and achieving pH stability and ecological safety in the water. However, excessively high SiO2 or chitosan concentrations can affect the hatching rate of Artemia eggs and the survival rate of nauplii, while excessively low SiO2 or chitosan concentrations may prevent uniform dispersion and pH stability in the water.

[0010] Preferably, the density of Artemia eggs in the culture water does not exceed 1000 eggs / mL. This wide range of Artemia egg densities makes it suitable not only for experimental conditions but also for large-scale practical culture, thus possessing significant practical application value.

[0011] A simple and healthy method for cultivating artichokes includes the following steps:

[0012] (1) According to the weight-volume ratio, the carrier SiO2 or chitosan is mixed evenly with nonanoic acid to obtain a nonanoic acid product in the form of emulsion or powder, wherein the weight-volume ratio of SiO2 to nonanoic acid is (0.5~3):1 (g:ml); the weight-volume ratio of chitosan to nonanoic acid is (0.01~0.1):1 (g:ml).

[0013] (2) Add the nonanoic acid product described in step (1) to the water used for hatching and raising brine shrimp, so that the concentration of nonanoic acid in the water reaches the required level, thereby obtaining nonanoic acid aquaculture water. Wherein, when the nonanoic acid is carried by silica, the concentration of nonanoic acid in the water is 150-300 μM, and the particle size of the silica is 10-40 μm; when the nonanoic acid is carried by chitosan, the concentration of nonanoic acid in the water is 55-90 μM, and the degree of deacetylation of the chitosan is 75-88%, and the molecular weight is 100-180 kDa.

[0014] This step solves the problem of difficult dispersion in water by the interaction between the carrier and nonanoic acid, slows down the release rate of organic acids, maintains a high local concentration of acid (effective antibacterial effect), prolongs the action time and improves stability, avoids a sharp drop in the pH value of the water, and keeps the pH value of the water stable within the range of 7.5-8.5 suitable for the survival of Artemia, thus ensuring ecological safety.

[0015] (3) Artemia eggs are introduced into the nonanoic acid culture water body described in step (2), and incubated at 28-30℃ for 18-24 h. Basic feed is provided under continuous light conditions to obtain Artemia. The density of Artemia eggs in the water body is 6-1000 eggs / mL; the continuous light condition is 1000-2500 Lux; the basic feed is Chlorella, yeast powder, Spirulina powder, rice bran, soybean meal, or artificial microparticle feed.

[0016] The brine shrimp eggs used in this step are commercially farmed brine shrimp eggs, unlike the sterile brine shrimp eggs used in the previous studies. Therefore, they themselves carry opportunistic pathogens, represented by Vibrio spp. Through the aforementioned farming process, not only was the number of opportunistic pathogens carried by the brine shrimp significantly reduced, but the diversity of the carried microbiota and the relative abundance of beneficial bacteria were also increased. This not only improved the health of the brine shrimp themselves and reduced mortality due to Vibrio infection, but also optimized the entire farming microecological environment, reduced the risk of disease in aquatic animals, and reduced the use of antibiotics and other drugs, aligning with the development trend of green and healthy aquaculture. The inventors believe that this is because nonanoic acid itself has antibacterial activity, and the dispersion and sustained-release effect of the carrier allows it to safely and continuously act on the brine shrimp's living environment and on its surface and inside its body for a relatively long period.

[0017] Furthermore, the brine shrimp cultured in this step exhibited a significantly faster growth rate compared to conventional methods, producing unexpected technological benefits. The inventors conducted transcriptomics sequencing analysis and qRT-PCR verification, concluding that nonanoic acid, through the dispersion and sustained-release effects of the vector system, regulated the expression of growth- and immune-related genes in brine shrimp, comprehensively promoting their growth, development, and survival. This is a first-time discovery by the inventors, providing a theoretical basis for further optimizing brine shrimp culture technology; it not only shortens the culture cycle but also ensures high-density, sustainable, and healthy brine shrimp culture, possessing significant practical application value.

[0018] An application of brine shrimp in aquaculture is disclosed, wherein the brine shrimp are obtained using the aforementioned cultivation method. Compared with existing technologies, the brine shrimp obtained using the aforementioned cultivation method not only carry fewer pathogenic bacteria but also increase the diversity of the carried microbial community and the stability of the micro-ecological environment in aquaculture, exhibiting good ecological safety. Therefore, applying the brine shrimp to aquaculture reduces the risk of disease in farmed animals while minimizing antibiotic use, aligning with the development trend of green and healthy aquaculture. Furthermore, the brine shrimp obtained using the aforementioned cultivation method not only significantly improves the hatching and survival rates but also, due to its healthier growth and the presence of healthier carried microbial communities, becomes a high-quality biological feed for aquaculture.

[0019] The beneficial effects of this invention are:

[0020] (1) This application firstly provides the application of nonanoic acid in brine shrimp farming. The application achieves both the antibacterial effect of nonanoic acid and the stability of water pH without the addition of acid-base regulators, overcoming the technical problems in the prior art, such as the difficulty in increasing the concentration and dispersion of nonanoic acid, the resulting decrease in water pH, the insufficient concentration to produce antibacterial effect, and the ecological safety risks.

[0021] (2) This application provides a method for culturing Artemia using nonanoic acid as an antibiotic alternative. The culturing method significantly improves the hatching rate and survival rate of Artemia and effectively reduces the number of Vibrio bacteria carried by Artemia. This provides more healthy Artemia larvae for aquaculture, ensures the seedling production of aquatic economic animals, and improves the efficiency of aquaculture from the source.

[0022] (3) The breeding method described in this application promotes the growth and development of brine shrimp by regulating the expression of genes related to growth and immunity, enhances the immunity of brine shrimp, and ensures the healthy and sustainable breeding of brine shrimp. Compared with the existing technology that only utilizes the antibacterial properties of organic acids, the breeding method described achieves unpredictable technical effects and provides a theoretical basis for further optimizing brine shrimp breeding technology. Attached Figure Description

[0023] Appendix Figure 1The effect of nonanoic acid concentration in aquaculture water (without carrier) on the hatching rate and survival rate of Artemia salina;

[0024] Appendix Figure 2 The emulsions prepared from nonanoic acid using SiO2 as a carrier in Examples 1-3 of this application;

[0025] Appendix Figure 3 The nonanoic acid powder prepared using chitosan as a carrier in Examples 4-6 of this application;

[0026] Appendix Figure 4 The relative abundance of bacterial species carried by Artemia spp. in Example 7 is shown in the SiO2-based nonanoic acid group (NS), the chitosan-based nonanoic acid group (NC), and the blank control group.

[0027] Appendix Figure 5 The effect of nonanoic acid group (NS) with SiO2 as carrier and nonanoic acid group (NC) with chitosan as carrier on the number of Vibrio carried by Artemia esculenta (day 7).

[0028] Appendix Figure 6 This is a KEGG enrichment analysis diagram of differentially expressed genes in the nonanoic acid group (NS) and the control group of Artemia using SiO2 as a carrier in Example 10.

[0029] Appendix Figure 7 This is a KEGG enrichment analysis diagram of the differentially expressed genes of Artemia var. nonanoic acid (NC) and the control group using chitosan as a carrier in Example 10.

[0030] Appendix Figure 8 The results of qRT-PCR for differentially expressed genes in Artemia salina in Example 10 are as follows: A (left figure): Nonanoic acid group (NS) with SiO2 as vector and blank control group; B (right figure): Nonanoic acid group (NS) with SiO2 as vector and blank control group. Detailed Implementation

[0031] The invention will be further described below with reference to the embodiments. The Artemia eggs (80% hatching rate), food-grade SiO2, chitosan, and nonanoic acid used in the embodiments were all commercially available. The pH of the water used for Artemia hatching and culture was 8.23. 1. The effect of nonanoic acid on Artemia hatching and survival without a carrier.

[0032] To determine the safe concentration of nonanoic acid for the hatching and survival rates of Artemia, the following experiment was conducted (experimental group and control group).

[0033] The experimental groups were treated with different concentrations of nonanoic acid (0, 0.6, 6, 8, 10, 12, 18, 24, 30, 60, 100 μM) in sterile seawater; the control group received no treatment and underwent Artemia hatching and cultivation. Specifically, Artemia eggs were hatched at a density of 30 eggs / mL in 5 mL of sterile seawater for 24 h, with the culture water temperature maintained at 28-30 °C and continuous illumination (2000 Lux). Chlorella was fed twice daily at 7:00 AM and 7:00 PM. The total number of Artemia eggs (H), the total number of hatched Artemia (h), and the total number of nauplius surviving at 48 h (s) were recorded. Each group was divided into three replicates. See below for detailed results. Figure 3 Since nonanoic acid is insoluble in water, it should be diluted with DMSO before use to prepare a nonanoic acid dilution solution.

[0034] like Figure 1 As shown, nonanoic acid concentrations below 8 μM had no significant effect on the hatching rate of Artemia eggs (P > 0.05), and concentrations below 18 μM had no significant effect on the nauplius survival rate of Artemia eggs (P > 0.05). With increasing nonanoic acid concentration, the hatching rate of Artemia eggs and the survival rate of nauplius larvae gradually decreased. The LC50 of the effect of nonanoic acid on the hatching rate of Artemia eggs was calculated based on the number of dead Artemia eggs and nauplius larvae. 50 Values ​​and LC for nauplius survival 50 The values ​​were 58.22 μM and 47.89 μM, respectively.

[0035] Based on the Turubell formula for calculating safe concentrations, the safe concentration of nonanoic acid for Artemia is 9.72 μM.

[0036]

[0037] Example 1: Hatching and rearing of Artemia (nonanoic acid-SiO2 as an antibiotic alternative)

[0038] A simple and healthy method for cultivating artichokes includes the following steps:

[0039] (1) Preparation of nonanoic acid products: SiO2 and nonanoic acid are mixed evenly according to the weight-volume ratio to obtain nonanoic acid products in the form of emulsions (e.g. Figure 2 (As shown); the weight-to-volume ratio of SiO2 to nonanoic acid is 0.6:1 (g:ml).

[0040] (2) Preparation of nonanoic acid aquaculture water: The nonanoic acid product described in step (1) is added to the water used for brine shrimp hatching and aquaculture to achieve the required concentration of nonanoic acid in the aquaculture water, thus obtaining nonanoic acid aquaculture water. The concentration of nonanoic acid in the water is 150 μM, and the particle size of the silica is 10 μm. The initial pH value of the system is measured.

[0041] (3) Artemia hatching and rearing: Artemia eggs are placed in the nonanoic acid rearing water body described in step (2) and hatched at 28-30℃ for a period of time. Basic feed is provided under continuous light conditions to obtain Artemia. The hatching time is 18 hours, the density of Artemia eggs in the water body is 100 eggs / mL, the continuous light condition is 1000 Lux, and the basic feed is Chlorella, which is fed twice a day at 7:00 and 19:00.

[0042] (4) Detection of brine shrimp hatching rate, survival rate and body length:

[0043] Artemia hatching rate and survival rate: Hatching was completed according to step (3), and the total number of artemia eggs (H), the total number of hatched artemia (h), and the total number of nauplii surviving at 48 h (s) were recorded. Three parallels were set up for each group. The hatching rate was expressed as the percentage of the total number of hatched artemia (h) to the total number of artemia eggs (H), and the survival rate was expressed as the percentage of the total number of nauplii surviving at 48 h to the total number of artemia eggs (H), as shown in the following formula:

[0044]

[0045]

[0046] Note: h is the total number of hatched artichokes; H is the total number of artichoke eggs; s is the total number of nauplii surviving after 48 hours.

[0047] Body length measurement: Starting from the hatching of the artichokes, 10 artichokes were randomly selected from each experimental group and control group every day. The body length of the artichokes was measured using a stereomicroscope once a day. The body length was the distance from the front end of the head to the bottom of the tail fork. The body length of the artichokes was recorded and the average value was calculated.

[0048] Example 2: Hatching and rearing of Artemia (nonanoic acid-SiO2 as an antibiotic alternative)

[0049] Unlike Example 1, a simple and healthy method for cultivating brine shrimp includes the following steps:

[0050] (1) Preparation of nonanoic acid products: The weight-volume ratio of SiO2 to nonanoic acid is 1.2:1 (g:ml).

[0051] (2) Preparation of nonanoic acid aquaculture water: The concentration of nonanoic acid used in the water is 200 μM, and the particle size of the silica is 20 μm.

[0052] (3) Artemia hatching and culture: The hatching time is 24 hours, the density of Artemia eggs in the water is 1000 eggs / mL; the continuous light condition is 2000 Lux; the basic feed is spirulina powder, which is fed twice a day at 7:00 and 19:00.

[0053] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0054] Example 3: Hatching and rearing of Artemia (nonanoic acid-SiO2 as an antibiotic alternative)

[0055] Unlike Example 1, a simple and healthy method for cultivating brine shrimp includes the following steps:

[0056] (1) Preparation of nonanoic acid products: The weight-volume ratio of SiO2 to nonanoic acid is 2.2:1 (g:ml).

[0057] (2) Preparation of nonanoic acid aquaculture water: The concentration of nonanoic acid used in the water is 300 μM, and the particle size of the silica is 40 μm.

[0058] (3) Artemia hatching and culture: The hatching time is 24 hours, the density of Artemia eggs in the water is 500 eggs / mL; the continuous light condition is 2500 Lux; the basic feed is yeast powder, which is fed twice a day at 7:00 and 19:00.

[0059] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0060] Comparative Example 1: Hatching and rearing of Artemia (nonanoic acid is an alternative to antibiotics)

[0061] Unlike Example 2, no SiO2 was added.

[0062] Example 4: Hatching and rearing of Artemia (nonanoic acid-chitosan is an antibiotic alternative)

[0063] A simple and healthy method for cultivating artichokes includes the following steps:

[0064] (1) Preparation of nonanoic acid products: Chitosan and nonanoic acid are mixed evenly according to the weight-volume ratio to obtain nonanoic acid products in powder form (e.g. Figure 3 (As shown); the weight-to-volume ratio of chitosan to nonanoic acid is 0.01:1 (g:ml).

[0065] (2) Preparation of nonanoic acid aquaculture water: The nonanoic acid product described in step (1) is added to the water used for the hatching and aquaculture of brine shrimp to achieve the required concentration of nonanoic acid in the aquaculture water, thus obtaining nonanoic acid aquaculture water. The concentration of nonanoic acid used in the water is 55 μM, the degree of deacetylation of the chitosan is 75%, and the molecular weight is 180 kDa.

[0066] (3) Artemia hatching and rearing: Artemia eggs are placed in the nonanoic acid rearing water body described in step (2) and hatched at 28-30℃ for a period of time. Basic feed is provided under continuous light conditions to obtain Artemia. The hatching time is 18 hours, the density of Artemia eggs in the water body is 100 eggs / mL, the continuous light condition is 1000 Lux, and the basic feed is Chlorella, which is fed twice a day at 7:00 and 19:00.

[0067] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0068] Example 5: Hatching and rearing of Artemia (nonanoic acid-chitosan as an antibiotic alternative)

[0069] Unlike Example 4, a simple and healthy method for cultivating brine shrimp includes the following steps:

[0070] (1) Preparation of nonanoic acid products: The weight-volume ratio of chitosan to nonanoic acid is 0.05:1 (g:ml).

[0071] (2) Preparation of nonanoic acid aquaculture water: The concentration of nonanoic acid used in the water is 70 μM, the degree of deacetylation of chitosan is 80%, and the molecular weight is 100 kDa.

[0072] (3) Artemia hatching and culture: The hatching time is 24 hours, the density of Artemia eggs in the water is 1000 eggs / mL; the continuous light condition is 2000 Lux; the basic feed is spirulina powder, which is fed twice a day at 7:00 and 19:00.

[0073] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0074] Example 6: Hatching and rearing of Artemia (nonanoic acid-chitosan as an antibiotic alternative)

[0075] Unlike Example 4, a simple and healthy method for cultivating brine shrimp includes the following steps:

[0076] (1) Preparation of nonanoic acid products: The weight-volume ratio of chitosan to nonanoic acid is 0.1:1 (g:ml).

[0077] (2) Preparation of nonanoic acid aquaculture water: The concentration of nonanoic acid used in the water is 85 μM, the degree of deacetylation of chitosan is 88%, and the molecular weight is 120 kDa.

[0078] (3) Artemia hatching and culture: The hatching time is 24 hours, the density of Artemia eggs in the water is 500 eggs / mL; the continuous light condition is 2500 Lux; the basic feed is yeast powder, which is fed twice a day at 7:00 and 19:00.

[0079] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0080] Comparative Example 2: Hatching and rearing of Artemia (nonanoic acid as an alternative to antibiotics)

[0081] Unlike Example 5, chitosan was not added.

[0082] Comparative Example 3: Hatching and rearing of brine shrimp (blank control group)

[0083] Unlike Example 5, nonanoic acid, SiO2, and chitosan were not added.

[0084] Table 1. Hatching rate, survival rate, and body length of cultured artichokes in Examples 1-6 and Comparative Examples 1-2

[0085]

[0086] As shown in Table 1, compared with Example 3, the blank control (without nonanoic acid and carrier addition) had a culture water pH of 8.23, an Artemia hatching rate of 81% and a 48-hour survival rate of 78%, and an average body length of 3.46 mm on the 7th day.

[0087] Based on the Turubell safe concentration calculation formula, the safe concentration (9.72 μM) of nonanoic acid for the hatching and survival rate of Artemia larvae was obtained. In Examples 1-3, which used nonanoic acid-SiO2 as an antibiotic alternative, the initial pH of the culture water was 8.23. Adding nonanoic acid-SiO2 at concentrations far exceeding the safe concentration (150-300 μM) had minimal impact on the pH of the culture water, remaining between 7.80 and 8.03. The hatching rate of Artemia larvae eggs was 77-80%, the 48-hour survival rate was 75-77%, and the average body length on day 7 was 3.63-3.75 mm. In contrast, in Comparative Example 1, which used only nonanoic acid as an antibiotic alternative, 200 μM nonanoic acid reduced the final pH of the culture water to 7.65. Although the pH of the culture water remained within the suitable range for the hatching and growth of Artemia salina, the presence of nonanoic acid, far exceeding the tolerance range of Artemia salina (safe concentration 9.72 μM), significantly negatively impacted the hatching of Artemia salina eggs and the survival rate of larvae, causing the hatching rate and 48-hour survival rate to drop below 10%. The hatching and survival rates of Artemia salina in Examples 1-3 were significantly higher than those in Comparative Example 1, but showed no significant difference compared to Comparative Example 3. The Artemia salina body length in Examples 1-3 showed a significant promoting effect compared to Comparative Example 3.

[0088] In Examples 4-6, which used nonanoic acid-chitosan as an antibiotic alternative, the initial pH of the culture water was 8.23. Adding nonanoic acid-chitosan at concentrations exceeding the safe usage level (55-90 μM) had minimal impact on the pH of the culture water, resulting in a final pH between 8.05 and 8.10. The hatching rate of Artemia eggs was 77-80%, the survival rate was 73-76%, and the average body length on day 7 was 3.77-3.99 mm. In contrast, in Comparative Example 2, which used only nonanoic acid as an antibiotic alternative, 70 μM nonanoic acid only lowered the final pH of the culture water to 7.92, which was within the suitable pH range for Artemia hatching and growth. However, 70 μM nonanoic acid exceeded the tolerance range of Artemia to nonanoic acid (safe usage concentration 9.72 μM), significantly negatively impacting the hatching of Artemia eggs and the survival rate of larvae, with the hatching rate and 48-hour survival rate decreasing to 48 ± 5% and 41 ± 5%, respectively. The hatching and survival rates of the brine shrimp in Examples 4-6 were significantly higher than those in Comparative Example 2, but slightly lower than those in Comparative Example 3. The increase in body length of the brine shrimp in Examples 4-6 was more significant than that in Comparative Example 3.

[0089] Therefore, compared with the carrier-free system, (1) after adding 55-300 μM nonanoic acid-carrier to the culture water in Examples 1-6 of this application, the final pH was 7.80-8.10, and the pH of the culture water remained stable, which was suitable for the growth and reproduction of Artemia; (2) the Artemia obtained by the culture method in Examples 1-6 of this application had significantly improved hatching rate and survival rate (P<0.05), maintaining good ecological safety; (3) compared with the blank control system (without nonanoic acid and carrier addition), without significantly affecting the hatching rate and survival rate of Artemia, it not only promoted the growth of Artemia (significantly increased body length), but also increased the safe concentration of nonanoic acid (i.e., improved the antibacterial effect), achieving unexpected technical effects. This shows that the nonanoic acid-SiO2 and nonanoic acid-chitosan are antibiotic alternatives, which, without adding acid-base regulators, overcome the technical problem in the prior art that increasing the concentration of nonanoic acid has a negative impact on the hatching and survival of Artemia, and achieve the unexpected technical effect of promoting the growth and development of Artemia.

[0090] Example 7: Microbial Community Analysis of Cultured Artemia

[0091] This embodiment performs high-throughput sequencing analysis based on 16S rRNA on the bacterial communities carried by cultured Artemia fowl in Examples 1-6, and the sequencing results are consistent with the actual results. The following detailed explanation uses Examples 2 (NS group) and Example 5 (NC group) as examples, combined with the blank control group (Control, without nonanoic acid and vector):

[0092] (1) Sample preparation

[0093] Starting from the hatching of the artichokes, samples were taken on day 1 (nauplius stage), day 3 (pseudo-adult stage), and day 7 (adult stage). From each group's three replicates, 200 artichokes were randomly selected, mixed, repeatedly rinsed with ultrapure water, placed in 1.5 mL EP tubes, flash-frozen in liquid nitrogen for 30 minutes, and then stored at -80 °C for later use. After all samples were collected, Shanghai Meiji Biotechnology Co., Ltd. performed 16S rRNA-based microbial community analysis of the artichokes.

[0094] (2) Data Analysis

[0095] Pair-end reads obtained from Illumina sequencing were spliced ​​together based on overlap, and sequence quality was controlled and filtered. After sample differentiation, OTU (Operational Taxonomic Unit) clustering and taxonomic analysis were performed. Bioinformatics statistical analysis was typically conducted on OTUs with a similarity level of 97%. Based on taxonomic information, statistical analysis of community structure was performed at each taxonomic level.

[0096] Alpha diversity analysis was performed based on the high-throughput sequencing results of each group of Artemia samples, resulting in a diversity index statistical table (Table 2). The Chao, Ace, and Sob indices reflect the number of species in the samples, but do not consider the abundance of each species; the Shannon and Simpson indices reflect the influence of species abundance and evenness on the diversity of the samples. That is, under the same species abundance, the greater the evenness of each species in the sample, the greater its diversity is considered.

[0097] (3) The dilution plating method was used to verify the effect of nonanoic acid-carrier on the number of Vibrio carried by Artemia esculenta.

[0098] On day 7 (adult stage), 30 Artemia worms were collected from each of Example 2 (LS group), Example 5 (LC group), and the blank control group (Control, without nonanoic acid and carrier). In a clean bench, the Artemia worms were repeatedly rinsed with ultrapure water, and then 1 mL of physiological saline was added to a glass homogenizer for tissue homogenization until the Artemia tissue was no longer visible. The homogenized tissue solution was diluted 10 times and spread on TCBS plates. The plates were incubated at 32 °C for 2 days, and the morphological characteristics of the bacteria were observed and counted.

[0099] Nonanoic acid (NS group) with SiO2 as a carrier and nonanoic acid (NC group) with chitosan as a carrier were added to the brine shrimp culture water for brine shrimp culture. The relative abundance of bacterial community composition at the genus level of each group during the nauplius larval stage (day 1), pseudo-adult stage (day 3), and adult stage (day 7) of brine shrimp was analyzed. See details. Figure 4 .likeFigure 4 As shown, the dominant bacterial groups with the highest relative abundance were analyzed. It was found that during the nauplius stage, the dominant bacterial groups carried by Artemia in the control group were *Exiguobacterium* and *Acinetobacter*, with relative abundances of 52.2% and 12.5%, respectively; the dominant bacterial groups carried by Artemia in the NS group were *Acinetobacter* and *Rhodobacteraceae*, with relative abundances of 15.5% and 11.2%, respectively; and the dominant bacterial groups carried by Artemia in the NC group were *Psychrobacter* and *Exiguobacterium*, with relative abundances of 54.8% and 43.4%, respectively. Vibrio was not found as a dominant bacterial group in any of the three groups during the nauplius stage.

[0100] During the pseudo-adult stage, the dominant bacterial species carried by Artemia in the control group were *Psychrobacter* and *Microbacterium*, with relative abundances of 79.43% and 6.4%, respectively; the dominant bacterial species carried by Artemia in the NS group were *Marinobacter* and *Rhodobacter*, with relative abundances of 17.4% and 15.1%, respectively; and the dominant bacterial species carried by Artemia in the NC group were *Psychrobacter* and *Pseudoalteromonas*, with relative abundances of 14.4% and 8.0%, respectively. Vibrio was not observed as a dominant bacterial species in any of the three groups during the pseudo-adult stage.

[0101] However, during the adult stage, both the blank control group and the NC group showed a large number of Vibrio bacteria, which became the dominant flora. The dominant flora in the blank control group were Vibrio and Psychrophilic Bacillus, with relative abundances of 56.5% and 21.7%, respectively; the dominant flora in the NC group were Psychrophilic Bacillus and Vibrio, with relative abundances of 79.5% and 10.4%, respectively; while the dominant flora in the NS group were Acinetobacter and SM1A02, with relative abundances of 11.1% and 9.2%, respectively, and Vibrio relative abundance was only 4.1%. Previous studies have reported that Microbacteria, Psychrophilic Bacillus, Marine Bacillus, and Rhodobulb are potential probiotics, and their increase can help enhance the intestinal health of farmed animals, improve their immunity and disease resistance.

[0102] Table 2 shows that the estimated values ​​of Sobs (155, 94, 116), Chao (156.2, 105.77, 116), and Ace (156.1, 111.33, 126.70) indices of the Artemia larvae in the NS group at each growth stage were significantly higher than those in the NC group and the blank control group, indicating that nonanoic acid, with SiO2 as a carrier, enriched the number of bacterial species carried by the Artemia larvae. The Shannon index (3.17, 2.05, 2.83) of Artemia larvae in the NS group at each growth stage was higher than that in the NC group and the blank control group, indicating better species evenness in the NS group; and its Simpson index (0.07, 0.19, 0.1) was lower than that in the NC group and the blank control group, indicating fewer dominant species and higher bacterial diversity. Compared with the NC group and the blank control group, the culture micro-ecosystem of the NS group was more stable and healthier.

[0103] Table 2. Alpha diversity analysis of the microbial communities carried by Artemia foetida at different growth stages in NS, NC, and blank control groups.

[0104]

[0105] Furthermore, the effect of nonanoic acid on the number of Vibrio carried by Artemia esculents on day 7 was verified using the dilution plating method. See details below. Figure 5 .like Figure 5 The results showed that the number of Vibrio bacteria carried by Artemia spp. in the NS group was 1.05 × 10⁻⁶. 3 The number of Vibrio bacteria carried by the NC group was 3.22 × 10⁻⁶ CFU / tail. 3 The CFU / tail count was significantly lower than that of the control group (4.03 × 10⁻⁶). 5 CFU / tail (P < 0.05). The plating results were consistent with the high-throughput sequencing results. On day 7, the survival rate of Artemia in the NS group (77 ± 3%) and the NC group (74 ± 6%) was significantly higher than that in the control group (68 ± 8%).

[0106] In summary, in this application, nonanoic acid with SiO2 as the carrier can significantly improve the hatching rate and survival rate of artichokes and effectively control the Vibrio carried by artichokes, and is superior to nonanoic acid with chitosan as the carrier.

[0107] Example 8: Anti-Vibrio Infection Experiment

[0108] Data from Examples 1-6 and Comparative Examples 1-2 show that using nonanoic acid-SiO2 and nonanoic acid-chitosan as antibiotic alternatives in Artemia culturing significantly improved the hatching rate, survival rate, and body length of the Artemia worms. Analysis of the microbial community carried by the cultured Artemia worms in Example 7 shows that both nonanoic acid-SiO2 and nonanoic acid-chitosan significantly reduced the number of conditionally pathogenic bacteria carried by the Artemia worms. Nonanoic acid-SiO2 carried a richer variety of microorganisms with higher evenness and diversity, resulting in a more stable and healthy culture microecology.

[0109] Furthermore, an artesian challenge experiment was conducted using Vibrio campbelli BB120 as the pathogen to verify the antibacterial effects of nonanoic acid-SiO2 and nonanoic acid-chitosan on the pathogenic Vibrio. The following details the use of Examples 2 (NS group) and 5 (NC group) as examples, where Vibrio campbelli BB120 was added during artesian hatching, combined with the Vibrio group (only Vibrio campbelli BB120 added) and the blank control group (no antibiotic alternative and Vibrio campbelli BB120 added).

[0110] Three experimental groups were established: Example 2 (nonanoic acid-SiO2 + Vibrio campbellii BB120), Example 5 (nonanoic acid-chitosan + Vibrio campbellii BB120), and a Vibrio group (only Vibrio campbellii BB120 was added). During the hatching of Artemia elegans eggs, Vibrio campbellii BB120 was added to the culture water of each experimental group at a concentration of 1×10⁻⁶. 7 Challenge with a final concentration of cell / mL, while the blank control group received no treatment. The total number of Artemia eggs (H), the total number of hatched Artemia (h), and the total number of nauplius surviving at 48 h (s) were recorded. Each group had three replicates. Hatching rate was expressed as the percentage of hatched Artemia (h) to the total number of Artemia eggs (H), and survival rate was expressed as the percentage of surviving nauplius (s) to the total number of Artemia eggs (H), calculated using the same formula as in Example 1.

[0111] As shown in Table 3, at 1×10 7 Under challenge with Vibrio campeosa BB120 at cell / mL, the hatching rate and nauplius survival rate of Artemia eggs with added nonanoic acid-SiO2 or nonanoic acid-chitosan were significantly higher than those in the Vibrio group (P < 0.05), indicating that the nonanoic acid-carrier can significantly reduce the adverse effects of Vibrio on Artemia hatching and growth, and improve the safety of nonanoic acid for Artemia. Furthermore, the hatching rate and nauplius survival rate of Artemia in the NS and NC groups with added nonanoic acid-carrier were not significantly different from the blank control group (P > 0.05), consistent with the experimental results in Example 7. This further demonstrates that the nonanoic acid-carrier can effectively inhibit the pathogenic Vibrio carried by Artemia without affecting the normal growth and development of Artemia, exhibiting good ecological safety.

[0112] Table 3. Effects of nonanoic acid-carrier on hatching rate and survival rate of Artemia under Vibrio campestris challenge conditions.

[0113]

[0114] Note: Different letters indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05).

[0115] Example 9: Determination of Artemia growth rate

[0116] Based on the data from Examples 1-6 and Comparative Examples 1-2, it is evident that using nonanoic acid-SiO2 and nonanoic acid-chitosan as antibiotic alternatives significantly increased the body length of Artemia larvae during cultivation. Using Examples 2 (NS group) and 5 (NC group) as examples, the inventors further investigated the impact on Artemia growth rate in conjunction with a blank control group. The specific procedures are as follows:

[0117] Ten artichokes were randomly selected from each experimental group and control group. The body length of the artichokes was measured using a stereomicroscope once daily. The body length was measured as the distance from the front of the head to the base of the tail fork. The body length was recorded, and the average, average daily growth rate (ADG, mm / d), and specific growth rate (SGR, %) were calculated. The formulas for calculating the average daily growth rate and specific growth rate are as follows:

[0118]

[0119]

[0120] Note: ADG is the average daily growth rate, mm / d; SGR is the specific growth rate, %; L t L0 represents the average body length of artichokes in each group on day 7 (mm); L0 represents the average body length of artichokes in each group on day 1 (mm); and t represents time (d).

[0121] As shown in Tables 1 and 4, nonanoic acid can improve the average daily growth rate and specific growth rate of Artemia. Among them, nonanoic acid with chitosan as a carrier has the best promoting effect on Artemia growth, with the average daily growth rate and specific growth rate of Artemia reaching 0.47 mm / d and 25.90%, respectively.

[0122] Table 4. Effects of nonanoic acid-carrier on Artemia growth parameters

[0123]

[0124] In summary, nonanoic acid can improve the hatching and development of artichokes, their average daily growth rate, and specific growth rate.

[0125] Example 10: Transcriptome sequencing analysis and qRT-PCR verification of cultured Artemia worms from Examples 1-6 and Comparative Examples 1-2

[0126] According to Examples 1-6 and 9, nonanoic acid-chitosan or nonanoic acid-SiO2 was used as an antibiotic alternative for Artemia hymen culture, resulting in a significant increase in both body length and growth rate, achieving unexpected technical effects. To investigate the underlying mechanisms, the inventors conducted transcriptome sequencing analysis on the Artemia hymens cultured in Examples 1-6. The transcriptome sequencing results, for the first time, elucidated the molecular mechanism by which nonanoic acid regulates Artemia growth and immune protection at the gene level, revealing the physiological response characteristics of Artemia to nonanoic acid.

[0127] The following detailed explanation uses Example 2 (NS group) and Example 5 (NC group) as examples, combined with the blank control group (without the addition of anti-alternative products) as an example:

[0128] (1) Transcriptome sequencing analysis of cultured Artemia worms in Examples 1-6 and Comparative Examples 1-2

[0129] Starting from the hatching of the artichokes, sampling was conducted on day 7 for each experimental group. 300 artichokes were randomly selected from three replicates in each group, mixed, repeatedly rinsed with ultrapure water, placed in 1.5 mL EP tubes, flash-frozen in liquid nitrogen for 30 minutes, and then stored at -80 ℃ for later use. After all sampling was completed, Shanghai Meiji Biotechnology Co., Ltd. performed artichoke transcriptome analysis. 1) Functional annotation statistics: The obtained transcripts were compared in six databases, including NR, Swiss-Prot, Pfam, EggNOG, GO, and KEGG; 2) Expression analysis: Based on the quantitative expression results, differential gene analysis was performed between groups to identify differentially expressed genes. Differential analysis was conducted using DEGseq software; further KEGG enrichment analysis was performed on the differentially expressed genes. (See details...) Figures 6-8 .

[0130] Appendix Figure 6 This is a KEGG enrichment analysis diagram of differentially expressed genes in Artemia salina between the nonanoic acid-SiO2 group (NS) and the control group. Figure 6As shown, in the comparison between the NS group and the blank control group, differentially expressed genes were annotated into 137 pathways. Among the top 30 pathways with high enrichment, 27 were related to growth and metabolism, with the top three enriched pathways being ascorbic acid metabolism, steroid biosynthesis, and cytochrome P450 metabolism; three were related to immunity: cGMP-PKG signaling pathway, cAMP signaling pathway, and C-type lectin receptor signaling pathway. Specifically, two genes (MRC and GNAO) in the NS group were significantly upregulated in the phagosome, melanin production, and estrogen signaling pathways (P < 0.05). MRC genes are involved in regulating innate immunity in animals, playing an important role in antigen processing and presentation, inflammatory responses, and intracellular signal transduction. Guanine nucleotide-binding protein (GNAO) plays a key role in signal transduction in invertebrates, regulating various physiological processes, including metabolism, development, and response to environmental stress, by transmitting extracellular signals and activating downstream effector molecules. Upregulation of MRC and GNAO gene expression enhances the pathogen recognition and immune response capabilities of Artemia salina, promotes signal transduction, and helps it clear foreign pathogens more quickly, maintaining a healthy state. This indicates that nonanoic acid, with SiO2 as a carrier, can enhance the immune defense mechanism of Artemia salina, enabling it to better cope with external pressures and pathogen infections, thereby improving its survival rate.

[0131] Appendix Figure 7 This is a KEGG enrichment analysis diagram of differentially expressed genes in Artemia salina between the nonanoic acid-chitosan group (NC) and the control group. Figure 7As shown, in the comparison between the NC group and the control group, differentially expressed genes were annotated into 337 pathways. Among the top 30 pathways with high enrichment, 21 were signaling pathways related to growth and metabolism. The top three pathways in terms of enrichment were the insulin signaling pathway, carbohydrate digestion and absorption, and starch and sucrose metabolism. Nine were signaling pathways related to immunity. The top three pathways in terms of enrichment were EMC receptor interaction, AMPK signaling pathway, and PI3K-Akt signaling pathway. Specifically, in the NC group, the gene PI3KCA was significantly upregulated in both the insulin signaling pathway and the carbohydrate digestion and absorption pathway (P < 0.05). The gene MGAM was significantly upregulated in both carbohydrate digestion and absorption and starch and sucrose metabolism (P < 0.05). Research reports that in invertebrates, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3KCA) primarily regulates various physiological processes, including cell growth, metabolism, and proliferation, through its involvement in the PI3K-Akt signaling pathway. PI3KCA upregulation activates protein kinase C (PRKCI), nodule protein complex (TSC2), and target of rapamycin (mTOR) genes, and further promotes the expression of sterol regulatory element-binding protein 1 (SREBP1), glycogen synthase (GYS), glycogen phosphorylase (PYG), phosphoenolpyruvate carboxylkinase (PCKA), hexokinase (HK), and acetyl-CoA carboxylase α (ACACA) genes, activating downstream pathways such as lipid synthesis, glycolysis for glucose production, and starch and sucrose metabolism. Mechanistic target of rapamycin (mTOR) is a key regulatory protein in cell signaling pathways. It regulates cell growth in response to changes in nutrient levels and is a central regulator of cell growth and metabolism. Upstream regulation of the PI3KCA gene upregulates mTOR. mTOR binds to the LST8 target site (MLST8) of the rapamycin complex subunit, phosphorylating and activating serine / threonine protein kinase 1 (ULK1) and eukaryotic initiation factor (eIF4B). This further activates downstream autophagy and protein synthesis pathways, regulating fundamental biological processes such as cell proliferation, differentiation, metabolism, and survival, thereby promoting animal growth and development. Maltase-glucoamylase (MGAM) is an important carbohydrate digestive enzyme, primarily involved in the digestion and absorption of carbohydrates. Upregulation of MGAM promotes carbohydrate digestion and utilization, increases glucose production and absorption, and plays a crucial role in cell proliferation and tissue development.Upregulation of PI3KCA, mTOR, and MGAM increases the efficiency of carbohydrate digestion and absorption, enhances insulin signaling, further promotes glucose uptake and utilization, protein synthesis, and cell proliferation, and enhances the energy metabolism and cell growth of Artemia esculents. This enables Artemia esculents to accelerate growth and development and maintain a high survival rate under conditions of sufficient nutrition and high-density farming. The upregulation of these genes allows Artemia esculents to utilize energy more efficiently, supporting their growth and development and enhancing their immune response.

[0132] Compared to the NS group, the NC group showed a significantly higher enrichment of the KEGG pathway, a result consistent with the superior body length-promoting effect of the NC group on the experimental animals. This indicates that nonanoic acid, carried by chitosan, elicits a broader biological response, activating more signaling and metabolic pathways (such as the insulin signaling pathway, carbohydrate digestion and absorption), further promoting cell proliferation, protein synthesis, and energy metabolism, thereby supporting the growth of Artemia elegans body length. In contrast, the NS group showed a lesser effect in promoting growth and development.

[0133] (2) qRT-PCR verification

[0134] To verify the reliability of the transcriptome sequencing results, one differentially expressed gene closely related to metabolic or immune pathways was selected from each experimental group for qRT-PCR verification. The specific method was as follows: Total RNA was extracted from Artemia elegans tissue using the Trizol method, and the RNA was reverse transcribed into cDNA using the PrimeScript™ FAST RT reagent Kit with gDNA Eraser (TAKARA). qRT-PCR was performed on a LightCyeler 96 (Roche, Germany) using the TB Green® Premix Ex Taq™ II FAST qPCR (TAKARA) kit. PCR primers were designed and synthesized by Qingdao Platinum Biotechnology Co., Ltd., with β-actin as the internal reference gene. Primer sequences are shown in Table 5. Relative quantification methods (2...) were used... -ΔΔCT The fold change of the target gene was determined. A t-test was then performed using SPSS 26.0 software, with P < 0.05 considered statistically significant. See details for the results. Figure 8 .

[0135] Table 5. Primer sequences used for qRT-PCR

[0136]

[0137] Compared with the blank control group, the MRC gene was significantly upregulated in the NS group (P < 0.05). Figure 8(Left figure); ACACA gene was significantly upregulated in the NC group (P < 0.05) Figure 8 (See right figure). This demonstrates that the PCR validation results are consistent with the differentially expressed gene upregulation results from transcriptome sequencing analysis, confirming the reliability of the transcriptome sequencing analysis results in this application.

[0138] In summary, this application first provides the application of nonanoic acid in brine shrimp farming. This application achieves the antibacterial effect of nonanoic acid without adding acid-base regulators, simultaneously ensuring the ecological safety of brine shrimp and stabilizing the pH of the water. It overcomes the technical problems in existing technologies where increasing the concentration of nonanoic acid leads to ecological safety risks for brine shrimp, while decreasing the pH of the water and reducing the concentration of nonanoic acid is insufficient to produce an antibacterial effect. Based on this, this application provides a method for farming brine shrimp using nonanoic acid as an alternative antibiotic. This farming method, by regulating the expression of growth, immunity, and other related genes, not only significantly improves the hatching rate and survival rate of brine shrimp and effectively reduces the number of Vibrio bacteria carried by brine shrimp, but also promotes the growth and development of brine shrimp, enhances their immunity, and ensures the healthy and sustainable farming of brine shrimp. Compared with existing technologies that only utilize the antibacterial properties of organic acids, this farming method achieves unpredictable technical effects, providing a theoretical basis for further optimizing brine shrimp farming technology.

Claims

1. The application of nonanoic acid in brine shrimp farming, characterized by: The nonanoic acid is added directly to the aquaculture water using silica or chitosan as a carrier. When silica is used as a carrier, the concentration of nonanoic acid in the water is 130-300 μM. The weight-to-volume ratio of SiO2 to nonanoic acid is (0.5-3):1, and the particle size of the silica is 10-40 μm. When chitosan is used as a carrier, the concentration of nonanoic acid in the water is 55-90 μM. The weight-to-volume ratio of chitosan to nonanoic acid is (0.01-0.1):1, and the degree of deacetylation of the chitosan is 75-88%, with a molecular weight of 100-180 kDa.

2. The application according to claim 1, characterized in that: The density of Artemia eggs in the culture water shall not exceed 1000 eggs / mL.

3. A method for cultivating brine shrimp, characterized in that: Includes the following steps: (1) Mix SiO2 or chitosan with nonanoic acid in a certain weight-to-volume ratio to obtain a nonanoic acid product in the form of an emulsion or powder; when the nonanoic acid is carried by silica, the concentration of nonanoic acid in the water is 130-300 μM; the weight-to-volume ratio of SiO2 to nonanoic acid is (0.5~3):1, and the particle size of the silica is 10-40 μm; when the nonanoic acid is carried by chitosan, the concentration of nonanoic acid in the water is 55-90 μM, the weight-to-volume ratio of chitosan to nonanoic acid is (0.01~0.1):1, and the degree of deacetylation of the chitosan is 75-88%, and the molecular weight is 100-180 kDa; (2) Add the nonanoic acid product obtained in step (1) to the water used for the hatching and breeding of brine shrimp, so that the concentration of nonanoic acid in the breeding water is increased. As described in claim 2, a nonanoic acid culture water body is obtained; (3) Artemia eggs are put into the nonanoic acid culture water body described in step (2), and hatched for 18-24 hours under the conditions of temperature of 28-30℃, continuous light and feeding of basic feed, so as to obtain Artemia.

4. The method for cultivating brine shrimp according to claim 3, characterized in that: The density of Artemia eggs in the water body in step (3) is no more than 1000 eggs / mL.

5. The method for cultivating brine shrimp according to claim 3, characterized in that: The continuous light conditions in step (3) are 500-2500 Lux; the basic feed is Chlorella, yeast powder, Spirulina powder, rice bran, soybean meal or artificial microparticle feed.

6. An application of brine shrimp in fish and shrimp farming, characterized in that: The brine shrimp are obtained using the culture method described in any one of claims 3-5.

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