Application of nonanoic acid in artemia breeding and simple, convenient and healthy artemia breeding method

Abstract

The invention firstly provides application of nonanoic acid in artemia culture. In the application, the nonanoic acid takes silicon dioxide or chitosan as a carrier and is directly added into a culture water body. Wherein when the nonanoic acid takes silicon dioxide as a carrier, the use concentration of the nonanoic acid in a water body is 150-300 micrometers; when chitosan is used as a carrier of the nonanoic acid, the use concentration of the nonanoic acid in the water body is 55-90 micrometers. The inventor accidentally finds that after the nonanoic acid is mixed with the silicon dioxide or the chitosan and then added into the culture water body, the sudden drop of the pH value of the water body cannot be caused, so that an acid-base regulator does not need to be added, and the incubation and growth of artemia cannot be influenced; meanwhile, uniform dispersion and local high concentration of the nonanoic acid in the water body are realized by using the carrier, so that the use amount of the acid is reduced, and the antibacterial effect is remarkably improved. The invention further provides an artemia breeding method adopting the nonanoic acid as the antibody replacement product. According to the artemia obtained through the breeding method, the hatching rate and the survival rate of the artemia are remarkably increased, the number of vibrios carried by the artemia is effectively reduced, expression of related genes such as growth and immunity of the artemia is up-regulated, development and growth of the artemia are promoted, ecological safety is improved, and unexpected technical effects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, relates to a method for cultivating Artemia, and specifically relates to a method for using nonanoic acid to reduce the hidden dangers of Artemia carrying opportunistic pathogens, thereby realizing rapid, healthy, stable and sustainable cultivation of Artemia. Background Art

[0002] Artemia, commonly known as brine shrimp, brine shrimp, and brine shrimp, is a small, lower crustacean. Its small size, high nutritional value, strong environmental adaptability, rapid reproduction, and long-term storage make it an important biological feed for aquaculture's commercial animals. Market demand for Artemia has always been in short supply, and high-density, healthy Artemia farming offers significant application prospects and enormous economic benefits. Artemia's non-selective filter-feeding properties allow it to be used to enrich various nutrients and enhance its nutritional value. However, this can also easily lead to the accumulation of opportunistic pathogens, reducing the hatching and survival rates of Artemia, causing sudden collapse of high-density Artemia farming systems, and resulting in instability. Once Artemia carries opportunistic pathogens, they can infect the larvae of commercial aquaculture animals such as fish, shrimp, and crabs through the food chain, leading to the failure of seedling production. Therefore, effective control of opportunistic pathogens carried by Artemia (especially pathogenic Vibrio, such as Vibrio harveyi, Vibrio parahaemolyticus, Vibrio campbellii, etc.) is crucial to ensuring the high-density sustainable farming of Artemia, as well as the breeding of economic aquaculture animals and the healthy farming of aquatic animals.

[0003] Antibiotics are commonly used in aquaculture to control opportunistic pathogens carried by Artemia. However, antibiotic use is not only limited in effectiveness but also promotes the development of drug-resistant pathogens, which are then transmitted through the food chain, posing a significant threat to human health and the ecological environment. Currently, acidifiers (such as formic acid and acetic acid), probiotics (such as Bacillus marineus), and plant extracts (such as essential oils) are being used in Artemia aquaculture as alternatives to antibiotics. Acidifiers are a general term for organic acids that can be used as additives and are considered non-nutritional feed additives. However, the antibacterial effect of acids decreases with increasing pH, which is inconsistent with the neutral to slightly alkaline environment in which Artemia naturally lives. Studies have shown that the optimal pH for Artemia hatching is 7.5-8.5. A pH below 7 results in softened eggshells, embryonic deformities, and significantly reduced hatchability. Although adult Artemia have a wide pH range and can survive in pH ranges of 7.0-9.5, their optimal growth is between 7.5-8.5. Furthermore, sudden pH changes can trigger stress responses in Artemia (e.g., abnormal molting, decreased immunity, and susceptibility to infection). Immanuel et al. studied the effects of octanoic acid, acetic acid, propionic acid, and butyric acid on Artemia survival after artificial infection of sterile Artemia with Vibrio harveyi and Vibrio parahaemolyticus under experimental conditions. Adding octanoic acid (10 mM) to the culture water significantly reduced mortality by 16.30% for Artemia infected with Vibrio harveyi and 20.61% for Artemia infected with Vibrio parahaemolyticus (P < 0.05). Furthermore, incubation of Artemia nauplii infected with Vibrio parahaemolyticus in seawater supplemented with acetic acid (20 mM), propionic acid (20 mM), and butyric acid (20 mM) reduced mortality by 16.7%, 11.7%, and 10.0%, respectively, compared to the control group. However, despite the reduction in Artemia mortality, the high concentrations of these acids resulted in a low pH in the water, necessitating the addition of acid-base regulators to adjust the pH to 7 to meet Artemia's environmental needs. In actual aquaculture production, this significantly increases costs and operational steps, hindering the growth and industrial application of Artemia. As mentioned earlier, this study focused on artificially infected sterile Artemia, which carries a simpler bacterial flora than actual aquaculture systems. Furthermore, Gao Yunni et al. evaluated the toxic effects of nonanoic acid on zooplankton growth and reproduction and found that the mortality rate of Daphnia magna increased with increasing nonanoic acid concentration and prolonged exposure. The safe concentration of nonanoic acid for Daphnia magna is 3.05 mg / L (19.3 μM). In actual aquaculture production, increasing the nonanoic acid content clearly increases the ecological insecurity of the aquaculture.

[0004] Nonanoic acid is a naturally occurring nine-carbon fatty acid containing a carboxyl group with a molecular weight of 158.238 (g / mol). It typically exists as a colorless, oily liquid, is weakly acidic, and has a certain irritant property. It is insoluble in water but readily soluble in ethanol, ether, and chloroform. It has been detected to varying degrees in a variety of plants, fruits, and vegetables. Nonanoic acid and its derivatives can be added to foods as spices and are permitted for use in food flavorings under the National Food Safety Standard (GB 2760-2014). Nonanoic acid exhibits certain antibacterial properties and can be safely added to animal feed as an antibacterial agent. De Smet et al. studied the antibacterial activity of nonanoic acid in the stomach and intestines of piglets and found that the addition of nonanoic acid to piglet diets reduced the number of Streptococci in the intestine. Nurettin et al. studied the antibacterial activity of methylated nonanoic acid derivatives against Gram-positive bacteria (Bacillus subtilis, Salmonella, and Escherichia coli). Methylated nonanoic acid derivatives at positions 4, 7, and 8 exhibited activity against Streptomyces. Jin-Hyung et al. found that nonanoic acid effectively inhibited Candida albicans biofilm formation and significantly increased the survival rate of Caenorhabditis elegans infected with C. albicans (50-80%, P < 0.05). Currently, the application of nonanoic acid in aquaculture has been rarely reported. Yao and Yu et al. found that nonanoic acid regulates immune responses by activating the MAPK / ERK signaling pathway in carp tissue cells and the ERK1 / 2 signaling pathway in Chinese giant salamander tissue cells. Direct application of nonanoic acid to aquaculture water presents a number of challenges, including its strong hydrophobicity, poor dispersion, irritation, and sensitivity to alkaline environments. Furthermore, high water hardness, alkalinity, and organic matter can neutralize organic acids, weakening their antimicrobial activity. However, increasing nonanoic acid concentrations can increase the ecological safety risks of Artemia, inhibiting their hatching and growth, and even causing mortality. It can also cause a decrease in water pH, further increasing production costs and safety risks.

[0005] Due to the aforementioned technical problems, there are currently no reports on the use of nonanoic acid in Artemia aquaculture. Summary of the Invention

[0006] In view of the problems of cumbersome steps and high costs in Artemia cultivation using organic acids as alternative antibiotics in the prior art, the present invention first provides the application of nonanoic acid in Artemia cultivation. The application achieves the dual purpose of uniform dispersion and antibacterial effect of nonanoic acid and improved ecological safety under the premise of strong hydrophobicity, difficulty in dispersion and ecological safety risk, overcoming the technical prejudice of increasing the concentration of nonanoic acid in the prior art, resulting in dispersion difficulties, decreased pH of water bodies and ecological safety risks. On this basis, the present application also provides a method for cultivating Artemia using nonanoic acid as an alternative antibiotic. 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 upregulates the expression of its growth and immunity related genes, promotes the development and growth of Artemia and improves ecological safety, and achieves unexpected technical effects.

[0007] The technical solution of the present invention:

[0008] The application of nonanoic acid in Artemia aquaculture, wherein the nonanoic acid is directly added to the aquaculture water body with silica or chitosan as a carrier. Wherein, when the nonanoic acid is carried by silica, the concentration of nonanoic acid in the water body is 150-300 μM; when the nonanoic acid is carried by chitosan, the concentration of nonanoic acid in the water body is 55-90 μM. The inventor unexpectedly discovered that after mixing nonanoic acid with the aforementioned silica or chitosan and then adding it to the aquaculture water body, it will not cause the pH of the water body to drop sharply, so there is no need to add an acid-base regulator, and it will not affect the hatching and growth of Artemia, thereby improving ecological safety; at the same time, the use of a carrier achieves uniform dispersion in the water body and maintains a high local concentration of the acid in the water body, which not only reduces the amount of acid used, but also significantly improves the antibacterial effect. Therefore, in the application described in the present application, the antibacterial effect of nonanoic acid is achieved, and the pH value of the water body is stable and ecological safety is achieved, overcoming the technical bias of increasing the concentration of nonanoic acid in the prior art to achieve antibacterial effect, thereby causing difficulty in water dispersion, pH drop and ecological safety risks.

[0009] Preferably, when the nonanoic acid is carried 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 carried by chitosan, the weight-to-volume ratio of chitosan to nonanoic acid is (0.01-0.1):1 (g:ml), and the deacetylation degree of the chitosan is 75-88% and the molecular weight is 100-180 kDa. The inventors have found that the silanol groups (Si-OH) on the surface of SiO2 can interact with the carboxylic acid groups (-COOH or -COO-) of the organic acid through hydrogen bonds, and chitosan can react with the carboxylic acid groups (-COOH or -COO-) of the organic acid in the organic acid solution through protonated amino groups (-NH3 + ) and the carboxylate ion of the organic acid (-COO -) to form ion complexes; thereby controlling the release rate of organic acids, prolonging the duration of their action, 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 in the water and prevent pH stability.

[0010] Preferably, the density of Artemia ova in the aquaculture water body does not exceed 1000 / mL. The density range of Artemia ova in the aquaculture water body is large, which is not only suitable for experimental conditions, but also for large-scale actual aquaculture, and has important practical application value.

[0011] A simple and healthy method for cultivating Artemia comprises the following steps:

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

[0013] (2) Adding the nonanoic acid product described in step (1) to water used for hatching and cultivating Artemia salina so that the concentration of nonanoic acid in the cultivation water reaches the required level, thereby obtaining nonanoic acid cultivation water. 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 chitosan has a degree of deacetylation of 75-88% and a molecular weight of 100-180 kDa.

[0014] This step solves the problem of difficult dispersion in water bodies through the interaction between the carrier and nonanoic acid and slows down the release rate of organic acids, maintaining a local high concentration of the acid (effective antibacterial effect), extending the action time and improving stability, avoiding a sharp drop in the pH value of the water body, and stabilizing the pH value of the water body within the range of 7.5-8.5 suitable for the survival of Artemia, thereby ensuring ecological safety.

[0015] (3) Adding Artemia salina eggs to the nonanoic acid aquaculture water described in step (2), incubating at 28-30°C for 18-24 hours, and feeding a basic feed under continuous light conditions to obtain Artemia salina. The Artemia salina eggs have a density of 6-1000 eggs / mL in the water; the continuous light conditions are 1000-2500 Lux; and the basic feed is chlorella, yeast powder, spirulina powder, rice bran, soybean meal, or artificial microparticle feed.

[0016] The Artemia ova adopted in this step is the commodity Artemia ova of actual cultivation, is different from the sterile Artemia ova in aforementioned research, and therefore itself carries the conditional pathogens represented by pathogenic Vibrio.Through aforementioned cultivation process, not only significantly reduced the quantity of the conditional pathogens carried by Artemia, but also improved the diversity of carrying flora and the relative abundance of beneficial bacteria simultaneously.This not only improves the health of Artemia itself, reduces the death caused by Vibrio infection, but also optimizes whole cultivation microecological environment, reduces the risk of aquaculture animal disease, reduces the use of medicines such as antibiotics, meets the development trend of green, healthy cultivation.The inventor believes, through analysis, that this is because nonanoic acid itself has antimicrobial activity, and the dispersion and sustained-release effect of carrier enable them to act on Artemia living environment and Artemia body surface and body safely and continuously over a long period of time.

[0017] Moreover, the growth rate of Artemia obtained by this step is significantly improved compared with that of ordinary breeding, producing unexpected technical effects. The inventors conducted transcriptomic sequencing analysis and qRT-PCR verification and believed that nonanoic acid regulated the growth of Artemia and the expression of immune-related genes through the dispersion and sustained release of the carrier system, and comprehensively promoted the growth, development and survival of Artemia. This is the first discovery made by the inventors of this application, which provides a theoretical basis for further optimizing Artemia breeding technology; it not only shortens the breeding cycle, but also ensures the high-density, sustainable and healthy breeding of Artemia, and has important practical application value.

[0018] A method for the application of Artemia in aquaculture, wherein the Artemia is obtained using the aforementioned cultivation method. Compared with the prior art, the Artemia obtained using the aforementioned cultivation method not only carries fewer pathogenic bacteria, but also improves the diversity of the carried bacterial community and the stability of the microecological environment in aquaculture, thus having good ecological safety. Therefore, the application of the Artemia in aquaculture reduces the risk of disease in farmed animals while reducing the use of antibiotics, which is in line with the development trend of green and healthy aquaculture. Moreover, the Artemia obtained using the aforementioned cultivation method not only has a significantly improved hatching rate and survival rate, but also, when used as a biological bait for aquaculture, has the advantage of growing and carrying a healthier bacterial community, making it a high-quality biological bait for aquaculture.

[0019] Beneficial effects of the present invention:

[0020] (1) This application first provides the use of nonanoic acid in Artemia aquaculture. This application achieves both the antibacterial effect of nonanoic acid and the stability of water pH without the addition of acid-base regulators. This overcomes the technical problems of prior art, such as the difficulty in dispersing nonanoic acid when increasing its concentration, the resulting decrease in water pH, insufficient concentration reduction to produce an antibacterial effect, and ecological safety risks.

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

[0022] (3) The cultivation method described in this application promotes the growth and development of Artemia by regulating the expression of genes related to growth and immunity, thereby enhancing Artemia's immunity and ensuring the healthy and sustainable cultivation of Artemia. Compared with the existing technology that only utilizes the antibacterial properties of organic acids, the cultivation method achieves unexpected technical effects and provides a theoretical basis for further optimizing Artemia cultivation technology. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Attachment Figure 2 The emulsion prepared from the nonanoic acid in Examples 1-3 of the present application using SiO2 as a carrier;

[0025] Attachment Figure 3 The powder prepared from nonanoic acid in Examples 4-6 of the present application using chitosan as a carrier;

[0026] Attachment Figure 4 The relative abundance of bacterial genus levels of Artemia carried by the nonanoic acid group (NS) with SiO2 as carrier, the nonanoic acid group (NC) with chitosan as carrier and the blank control group in Example 7;

[0027] Attachment Figure 5 This is the effect of the nonanoic acid group (NS) with SiO2 as the carrier and the nonanoic acid group (NC) with chitosan as the carrier on the number of Vibrio carried by Artemia (Day 7);

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

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

[0030] Attachment Figure 8 The qRT-PCR results of differentially expressed genes in Artemia in Example 10 are as follows: A (left panel). Nonanoic acid group (NS) with SiO2 as carrier and blank control group; B (right panel). Nonanoic acid group (NS) with SiO2 as carrier and blank control group. DETAILED DESCRIPTION

[0031] The present invention is further described below with reference to the following examples. The Bohai Bay Artemia eggs (hatching rate 80%), food-grade SiO2, chitosan, and nonanoic acid used in the examples were all purchased commercially. The pH of the water used for Artemia hatching and culture was 8.23. 1. Effect of Nonanoic Acid on Artemia Hatching and Survival in the Absence of a Carrier

[0032] In order to obtain the safe concentration of nonanoic acid on the hatching rate and survival rate of Artemia, the following experiment (experimental group and control group) was conducted.

[0033] The experimental groups were exposed to different concentrations of nonanoic acid (0, 0.6, 6, 8, 10, 12, 18, 24, 30, 60, 100 μM) in sterile seawater; the control group was not treated and Artemia was hatched and cultured. The specific operation was as follows: Artemia eggs were hatched in 5 mL of sterile seawater at a density of 30 eggs / mL for 24 hours, the aquaculture water temperature was maintained at 28-30 ℃, and the light was continuous (2000 Lux). Chlorella was fed twice a day at 7:00 and 19:00. The total number of Artemia eggs (H), the total number of Artemia hatched (h) and the total number of nauplii surviving for 48 hours (s) were recorded. Three replicates were set up for each group. For detailed results, see Figure 3 Since nonanoic acid is insoluble in water, DMSO should be added in advance to prepare nonanoic acid dilution solution before use.

[0034] like Figure 1 As shown in the results, when the nonanoic acid concentration was lower than 8 μM, there was no significant effect on the hatching rate of Artemia eggs (P>0.05), and when the nonanoic acid concentration was lower than 18 μM, there was no significant effect on the nauplii of Artemia eggs (P>0.05). As the nonanoic acid concentration increased, the hatching rate of Artemia eggs and the survival rate of nauplii gradually decreased. The LC value of nonanoic acid on the hatching rate of Artemia eggs was calculated based on the number of Artemia eggs and nauplii deaths. 50 LC values for nauplii survival 50 values, which were 58.22 μM and 47.89 μM, respectively.

[0035] According to the Turubel safety concentration calculation formula, the safe concentration of nonanoic acid for Artemia is 9.72 μM.

[0036]

[0037] Example 1: Hatching and Culture of Artemia (Nonanoic Acid-SiO2 as an Antibiotic Alternative)

[0038] A simple and healthy method for cultivating Artemia comprises the following steps:

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

[0040] (2) Preparing nonanoic acid aquaculture water: Add the nonanoic acid product described in step (1) to the water used for hatching and aquaculture of Artemia salina, so that the concentration of nonanoic acid in the aquaculture water reaches the required concentration, thereby obtaining nonanoic acid aquaculture water. The nonanoic acid concentration in the water is 150 μM, and the particle size of the silica is 10 μm. Determine the initial pH value of the system.

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

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

[0043] Hatching rate and survival rate of Artemia: Complete the hatching according to step (3) and record the total number of Artemia eggs (H), the total number of Artemia hatched (h), and the total number of Artemia nauplii surviving after 48 h (s). Set up three replicates for each group. The hatching rate is expressed as the percentage of the total number of Artemia hatched (h) to the total number of Artemia eggs (H), and the survival rate is expressed as the percentage of the total number of nauplii surviving (s) to the total number of Artemia eggs (H). The formula is as follows:

[0044]

[0045]

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

[0047] Body length detection: Since the Artemia hatched, 10 Artemia were randomly selected from each experimental group and the control group every day. The body length of Artemia was measured using a stereo microscope once a day. The body length was the distance from the front end of the Artemia head to the bottom end of the tail fork. The body length of Artemia was recorded and the average value was calculated.

[0048] Example 2: Hatching and Culture of Artemia (Nonanoic Acid-SiO2 as an Antibiotic Alternative)

[0049] Different from Example 1, a simple and healthy method for cultivating Artemia comprises the following steps:

[0050] (1) Preparation of nonanoic acid products: The weight-to-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 in the water is 200 μM, and the particle size of the silicon dioxide is 20 μm.

[0052] (3) Artemia hatching and breeding: the hatching time is 24 hours, the density of the Artemia eggs in the water body is 1000 eggs / mL; the continuous illumination 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 Artemia hatching rate, survival rate and body length: Same as Example 1.

[0054] Example 3: Hatching and Culture of Artemia (Nonanoic Acid-SiO2 as an Antibiotic Alternative)

[0055] Different from Example 1, a simple and healthy method for cultivating Artemia comprises the following steps:

[0056] (1) Preparation of nonanoic acid products: The weight-to-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 in the water is 300 μM, and the particle size of the silicon dioxide is 40 μm.

[0058] (3) Artemia hatching and breeding: the hatching time is 24 hours, the density of the Artemia eggs in the water body is 500 eggs / mL; the continuous illumination 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 Artemia hatching rate, survival rate and body length: Same as Example 1.

[0060] Comparative Example 1: Hatching and Culture of Artemia (Nonanoic Acid as an Antibiotic Alternative)

[0061] The difference from Example 2 is that no SiO2 is added.

[0062] Example 4: Hatching and Culture of Artemia (Nonanoic Acid-Chitosan as an Antibiotic Alternative)

[0063] A simple and healthy method for cultivating Artemia comprises the following steps:

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

[0065] (2) Preparing nonanoic acid aquaculture water: Add the nonanoic acid product described in step (1) to the water used for hatching and aquaculture of Artemia salina, so that the concentration of nonanoic acid in the aquaculture water reaches the required concentration, thereby obtaining nonanoic acid aquaculture water. The nonanoic acid concentration in the water is 55 μM, and the chitosan has a degree of deacetylation of 75% and a molecular weight of 180 kDa.

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

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

[0068] Example 5: Hatching and Culture of Artemia (Nonanoic Acid-Chitosan as an Antibiotic Alternative)

[0069] Different from Example 4, a simple and healthy method for cultivating Artemia comprises the following steps:

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

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

[0072] (3) Artemia hatching and breeding: the hatching time is 24 hours, the density of the Artemia eggs in the water body is 1000 eggs / mL; the continuous illumination 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 Artemia hatching rate, survival rate and body length: Same as Example 1.

[0074] Example 6: Hatching and Culture of Artemia (Nonanoic Acid-Chitosan as an Antibiotic Alternative)

[0075] Different from Example 4, a simple and healthy method for cultivating Artemia comprises the following steps:

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

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

[0078] (3) Artemia hatching and breeding: the hatching time is 24 hours, the density of the Artemia eggs in the water body is 500 eggs / mL; the continuous illumination 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 Artemia hatching rate, survival rate and body length: Same as Example 1.

[0080] Comparative Example 2: Hatching and Culture of Artemia (Nonanoic Acid as an Antibiotic Alternative)

[0081] The difference from Example 5 is that chitosan is not added.

[0082] Comparative Example 3: Hatching and Culture of Artemia (Blank Control Group)

[0083] The difference from Example 5 is that no nonanoic acid, SiO2 and chitosan are added.

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

[0085]

[0086] As shown in Table 1, the blank control (no nonanoic acid and carrier added) was compared with Example 3. The pH of the aquaculture water was 8.23, the Artemia hatching rate and 48h survival rate were 81% and 78% respectively, and the average body length on the 7th day was 3.46 mm.

[0087] According to the Turubel safety concentration calculation formula, the safe use concentration of nonanoic acid on the hatching rate and survival rate of Artemia was obtained (9.72μM). In Examples 1-3, which used nonanoic acid-SiO2 as an alternative product, the initial pH of the aquaculture water was 8.23. After adding nonanoic acid-SiO2 at a concentration far exceeding the safe use concentration (150-300μM), the pH of the aquaculture water was very small, ranging from 7.80 to 8.03. The hatching rate of Artemia eggs was 77-80%, the 48h survival rate was 75-77%, and the average body length on the 7th day was 3.63-3.75mm. In Comparative Example 1, which only used nonanoic acid as an alternative product, 200μM nonanoic acid reduced the final pH of the aquaculture water to 7.65. Although the pH of the aquaculture water body is still within the suitable range for Artemia hatching and growth, the nonanoic acid far exceeds the tolerance range of Artemia to nonanoic acid (safe use concentration 9.72 μM), which has a significant negative impact on the hatching of Artemia eggs and the survival rate of Artemia larvae, causing the hatching rate and 48-hour survival rate to drop below 10%. The Artemia hatching rate and survival rate of Examples 1-3 are significantly improved compared with Comparative Example 1, and there is no significant difference with Comparative Example 3. The Artemia body length of Examples 1-3 is significantly promoted compared with Comparative Example 3.

[0088] In Examples 4-6, where nonanoic acid-chitosan was used as an alternative to antibiotics, the initial pH of the aquaculture water was 8.23. After adding nonanoic acid-chitosan exceeding the safe use concentration (55-90 μM), the effect on the pH of the aquaculture water was minimal, and the final pH of the aquaculture water was between 8.05-8.10. The hatching rate of Artemia eggs was 77-80%, the survival rate was 73-76%, and the average body length on the 7th day was 3.77-3.99 mm. In Comparative Example 2, where only nonanoic acid was used as an alternative to antibiotics, 70 μM nonanoic acid only reduced the final pH of the aquaculture water to 7.92, which was within the suitable range for Artemia hatching and growth. However, 70 μM nonanoic acid exceeded the tolerance range of Artemia to nonanoic acid (safe use concentration 9.72 μM), and had a significant negative impact on the hatching of Artemia eggs and the survival rate of larvae, with the hatching rate and 48h survival rate falling to 48 ± 5% and 41 ± 5%. The hatching rate and survival rate of Artemia in Example 4-6 were significantly improved compared with Comparative Example 2, and slightly decreased compared with Comparative Example 3. The growth of Artemia body length in Example 4-6 was more significant than that in Comparative Example 3.

[0089] It can be seen that compared with the carrier-free system, (1) after adding 55-300 μM nonanoic acid-carrier to the aquaculture water of Examples 1-6 of the present application, the final pH is 7.80-8.10, and the pH of the aquaculture water is always stable, which is suitable for the growth and reproduction of Artemia; (2) the hatching rate and survival rate of Artemia obtained by the aquaculture method of Examples 1-6 of the present application are significantly improved (P < 0.05), maintaining good ecological safety; (3) compared with the blank control system (no nonanoic acid and carrier addition), on the basis of not significantly affecting the hatching rate and survival rate of Artemia, not only promotes the growth of Artemia (body length increases significantly), but also increases the safe use concentration of nonanoic acid (i.e., improves the antibacterial effect), achieving an unexpected technical effect. This shows that the nonanoic acid-SiO2 and nonanoic acid-chitosan are alternative antibiotic products. Under the premise of not adding acid-base regulators, they overcome the technical problem of the prior art that increasing the nonanoic acid concentration 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: Analysis of microbial communities in cultured Artemia

[0091] This example analyzes the bacterial flora carried by the cultured Artemia in Examples 1-6 based on 16S rRNA high-throughput sequencing, and the sequencing results are consistent with the actual results. The following is a detailed description of Example 2 (NS group) and Example 5 (NC group) as examples, combined with the blank control group (Control, without nonanoic acid and carrier added):

[0092] (1) Sample preparation

[0093] Starting from the time Artemia hatched, samples were collected on days 1 (nauplii), 3 (pre-adult), and 7 (adult). From each group of three replicates, 200 Artemia were randomly sampled, mixed, rinsed repeatedly with ultrapure water, placed in 1.5 mL EP tubes, and quickly frozen in liquid nitrogen for 30 minutes before being stored at -80°C. After all samples were collected, 16S rRNA-based Artemia microbial flora analysis was performed by Shanghai Meiji Biological Co., Ltd.

[0094] (2) Data analysis

[0095] Paired-end reads generated by Illumina sequencing were spliced based on overlap, and sequence quality was controlled and filtered. After sample differentiation, OTU (Operational Taxonomic Unit) cluster analysis and species taxonomy analysis were performed. Bioinformatics statistical analysis was performed on OTUs at a 97% similarity level. Based on taxonomic information, statistical analysis of community structure was performed at various taxonomic levels.

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

[0097] (3) Verification of the effect of nonanoic acid-carrier on the number of Vibrio carried by Artemia using the dilution plate method

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

[0099] Nonanoic acid with SiO2 as carrier (NS group) and nonanoic acid with chitosan as carrier (NC group) were added to the water for Artemia culture. The relative abundance of the bacterial community carried by Artemia in the nauplii (day 1), quasi-imago (day 3) and adult (day 7) stages of each group was analyzed at the genus level. Figure 4 .like Figure 4 As shown in the figure, the top two dominant bacterial groups in relative abundance were counted, and it was found that: in the nauplii 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. In the nauplii stage, Vibrio was not found as the dominant bacterial group in any of the three groups.

[0100] During the quasi-adult stage, the dominant bacterial communities in the control Artemia were Psychrobacter and Exiguobacterium, with relative abundances of 79.43% and 6.4% respectively. The dominant bacterial communities in the NS Artemia were Marinobacter and Rhodobacter, with relative abundances of 17.4% and 15.1% respectively. The dominant bacterial communities in the NC Artemia were Psychrobacter and Pseudoalteromonas, with relative abundances of 14.4% and 8.0% respectively. Vibrio was not found as the dominant bacterial community in any of the three groups during the quasi-adult stage.

[0101] However, during the adult stage, Vibrio spp. appeared in large numbers in both the blank control and NC groups, becoming the dominant bacterial community. The dominant bacterial communities in the blank control group were Vibrio and Psychrobacter, with relative abundances of 56.5% and 21.7%, respectively. In the NC group, Psychrobacter and Vibrio spp. were dominant, with relative abundances of 79.5% and 10.4%, respectively. In the NS group, Acinetobacter and SM1A02 were dominant, with relative abundances of 11.1% and 9.2%, respectively. Vibrio spp. had a relative abundance of only 4.1%. Studies have reported that Exiguobacterium, Psychrobacter, Oceanobacter, and Rhodobacter are potential probiotics, and their increased abundance can help enhance intestinal health, immunity, and disease resistance in farmed animals.

[0102] As shown in Table 2, the estimated Sobs (155, 94, 116), Chao (156.2, 105.77, 116), and Ace (156.1, 111.33, 126.70) indices of the Artemia microbial community in the NS group at each growth stage were significantly higher than those in the NC group and the blank control group, indicating that SiO2-carrying nonanoic acid enriched the bacterial community carried by Artemia larvae. The Shannon index of Artemia in the NS group (3.17, 2.05, 2.83) at each growth stage was higher than that in the NC group and the blank control group, indicating a more uniform distribution of Artemia species in the NS group. Furthermore, the 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 microbial diversity. Compared with the NC group and the blank control group, the NS group exhibited a more stable and healthy microbial ecosystem.

[0103] Table 2. Alpha diversity analysis of Artemia bacteria at different growth stages in NS, NC and blank control groups

[0104]

[0105] Furthermore, the dilution plate method was used to verify the effect of nonanoic acid on the number of Vibrio carried by Artemia on the 7th day. Figure 5 .like Figure 5The results showed that the number of Vibrio carried by Artemia in the NS group was 1.05×10 3 CFU / tail, the number of Vibrio carried by the NC group was 3.22×10 3 CFU / tail, and both were significantly lower than the number of Vibrio carried by the control group (4.03×10 5 The results of the coating were consistent with those of high-throughput sequencing. On day 7, the survival rates of Artemia in the NS group (77 ± 3%) and the NC group (74 ± 6%) were significantly higher than those in the control group (68 ± 8%).

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

[0107] Example 8: Anti-Vibrio infection experiment

[0108] According to the data of Examples 1-6 and Comparative Examples 1-2, the hatching rate, survival rate and body length of Artemia were significantly improved by using nonanoic acid-SiO2 and nonanoic acid-chitosan as alternative antibiotic products for Artemia cultivation. According to the results of the analysis of the microbial flora carried by cultured Artemia in Example 7, nonanoic acid-SiO2 and nonanoic acid-chitosan significantly reduced the conditional pathogens carried by Artemia, and the nonanoic acid-SiO2-carrying flora was richer in species, with high distribution uniformity and diversity, and the cultured microecology was more stable and healthy.

[0109] Furthermore, Artemia challenge experiments were conducted using Vibrio campbellii BB120 as the pathogen to verify the antibacterial effects of nonanoic acid-SiO2 and nonanoic acid-chitosan against pathogenic Vibrio. The following uses Example 2 (NS group) and Example 5 (NC group) as examples, where V. campbellii BB120 was added during Artemia hatching. This is further described in detail in conjunction with the Vibrio group (adding only V. campbellii BB120) and the blank control group (no antibiotic replacement product or V. campbellii BB120 added).

[0110] Three experimental groups were set up, namely Example 2 (nonanoic acid-SiO2+V. campbellii BB120), Example 5 (nonanoic acid-chitosan+V. campbellii BB120) and Vibrio group (only Vibrio campbellii BB120). When Artemia eggs were hatched, Vibrio campbellii BB120 was added to the aquaculture water of each experimental group to a concentration of 1×10 7The cells were challenged with a final concentration of 10 cells / mL, and the blank control group was not treated. The total number of Artemia eggs (H), the total number of Artemia hatched (h), and the total number of Artemia nauplii surviving at 48 h (s) were recorded. Three replicates were set up for each group. The hatching rate was expressed as the percentage of the total number of Artemia hatched (h) to the total number of Artemia eggs (H), and the survival rate was expressed as the percentage of the total number of surviving Artemia nauplii (s) to the total number of Artemia eggs (H). The calculation formula was the same as in Example 1.

[0111] From Table 3, we can see that in 1×10 7 When challenged with cell / mL Vibrio campbellii BB120, the hatching rate and nauplii survival rate of Artemia eggs added with nonanoic acid-SiO2 or nonanoic acid-chitosan were significantly higher than those of the Vibrio group (P < 0.05), indicating that nonanoic acid-carrier can significantly reduce the adverse effects of Vibrio on Artemia hatching and growth, and improve the safety of nonanoic acid on Artemia. In addition, the hatching rate and nauplii survival rate of Artemia in the NS and NC groups with the addition of nonanoic acid-carrier were not significantly different from those in the blank control group (P > 0.05). The experimental results are consistent with those in Example 7. This further shows that nonanoic acid-carrier can effectively inhibit the pathogenic Vibrio carried by Artemia without affecting the normal growth and development of Artemia, and shows good ecological safety.

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

[0113]

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

[0115] Example 9: Determination of Artemia growth rate

[0116] According to the data of Examples 1-6 and Comparative Examples 1-2, the use of nonanoic acid-SiO2 and nonanoic acid-chitosan as alternative antibiotic products for Artemia cultivation significantly increased the body length of Artemia. The inventors used Example 2 (NS group) and Example 5 (NC group) as examples and combined them with a blank control group to further study the effect of Artemia growth rate. The specific operation is as follows:

[0117] Ten Artemia were randomly selected from each experimental group and the control group. The body length of Artemia was measured using a stereo microscope once a day. The body length was the distance from the front end of the Artemia head to the bottom end of the tail fork. The body length of Artemia was recorded and the average value, average daily growth rate (ADG, Average Daily Growth Rate, 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 is the average body length of Artemia in each group on the 7th day, mm; L0 is the average body length of Artemia in each group on the 1st day, mm; t is time, d.

[0121] As can be seen from Tables 1 and 4, nonanoic acid can increase the average daily growth rate and specific growth rate of Artemia. Among them, nonanoic acid with chitosan as a carrier has the best effect on promoting the growth of Artemia, 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 indicators

[0123]

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

[0125] Example 10: Transcriptome Sequencing Analysis and qRT-PCR Verification of Artemia Cultured in Examples 1-6 and Comparative Examples 1-2

[0126] According to Examples 1-6 and 9, Artemia culture was carried out using nonanoic acid-chitosan or nonanoic acid-SiO2 as an alternative antibiotic product. The Artemia body length and growth rate were significantly improved, achieving unexpected technical effects. To get to the bottom of it, the inventors sequenced and analyzed the transcriptomes of Artemia cultured in Examples 1-6. The transcriptome sequencing results analyzed for the first time the molecular mechanism of nonanoic acid regulating Artemia growth and immune protection at the gene level, revealing the physiological response characteristics of Artemia to nonanoic acid.

[0127] The following takes Example 2 (NS group) and Example 5 (NC group) as examples, and combines the blank control group (no antibiotic replacement product added) as an example to provide a detailed description as follows:

[0128] (1) Transcriptome Sequencing Analysis of Artemia Cultured in Examples 1-6 and Comparative Examples 1-2

[0129] Starting from the hatching of Artemia, each experimental group was sampled on the 7th day. 300 Artemia were randomly selected from the three replicates of each group, mixed and repeatedly rinsed with ultrapure water, placed in a 1.5 mL EP tube, and quickly frozen in liquid nitrogen for 30 minutes and then frozen in a -80 ℃ refrigerator for later use. After all sampling was completed, Shanghai Meiji Biological Co., Ltd. carried out Artemia transcriptome determination. 1) Functional annotation statistics: The obtained transcripts were compared in 6 databases, including NR, Swiss-Prot, Pfam, EggNOG, GO and KEGG; 2) Expression analysis: Based on the expression quantification results, inter-group differential gene analysis was performed to obtain differentially expressed genes between the two groups, and the DEGseq software was used for differential analysis; KEGG enrichment analysis was further performed on the differentially expressed genes, see for details. Figure 6-8 .

[0130] Attachment Figure 6 Figure 2 is the KEGG enrichment analysis of differentially expressed genes between Artemia salina in the nonanoic acid-SiO2 group (NS) and the control group. Figure 6 As shown, differentially expressed genes were annotated into 137 pathways between the NS group and the blank control group. Among the top 30 pathways with the highest enrichment, 27 were related to growth metabolism, with the top three pathways in terms of enrichment being ascorbic acid metabolism, steroid biosynthesis, and cytochrome P450 metabolism. Three pathways were associated with immunity: the cGMP-PKG signaling pathway, the cAMP signaling pathway, and the C-type lectin receptor signaling pathway. Two genes (MRC and GNAO) were significantly upregulated in the NS group, including those involved in the phagosome, melanogenesis, and estrogen signaling pathways (P < 0.05). The MRC gene is involved in regulating innate immunity in animals and plays 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. Upregulated expression of MRC and GNAO genes can enhance Artemia's pathogen recognition and immune response capabilities, promote signal transmission, and help it eliminate foreign pathogens more quickly and maintain a healthy state. This suggests that nonanoic acid, carried by SiO2, can enhance Artemia's immune defense mechanisms, enabling it to better cope with external stress and pathogen infection, thereby increasing its survival rate.

[0131] Attachment Figure 7 Figure 2 is the KEGG enrichment analysis of differentially expressed genes between Artemia salina in the nonanoic acid-chitosan group (NC) and the control group. Figure 7As shown, differentially expressed genes were annotated into 337 pathways between the NC and control groups. Among the top 30 pathways with the highest enrichment, 21 were related to growth metabolism. The top three pathways by enrichment were insulin signaling, carbohydrate digestion and absorption, and starch and sucrose metabolism. Nine immune-related pathways were identified, with the top three pathways by enrichment being EMC receptor interaction, AMPK signaling, and PI3K-Akt signaling. PI3KCA was a significantly upregulated gene in both the insulin signaling and carbohydrate digestion and absorption pathways in the NC group (P < 0.05). MGAM was a significantly upregulated gene in both carbohydrate digestion and absorption and starch and sucrose metabolism (P < 0.05). Research reports indicate that in invertebrates, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3KCA) regulates a variety of physiological processes, including cell growth, metabolism, and proliferation, primarily through its involvement in the PI3K-Akt signaling pathway. PI3KCA upregulates and activates the genes for protein kinase C (PRKCI), tuberin complex 2 (TSC2), and mTOR, further promoting the expression of genes for sterol regulatory element binding protein 1 (SREBP1), glycogen synthase (GYS), glycogen phosphorylase (PYG), phosphoenolpyruvate carboxykinase (PCKA), hexokinase (HK), and acetyl-CoA carboxylase alpha (ACACA), thereby activating downstream pathways for lipogenesis, glycolysis, glucose production, and starch-sucrose metabolism. The mechanistic target of rapamycin (mTOR) is a key regulator of cell signaling pathways. It regulates cell growth in response to nutritional changes and is a central regulator of cell growth and metabolism. The upstream PI3KCA gene regulates the upregulation of the mTOR gene. mTOR binds to the LST8 (MLST8) subunit of the rapamycin complex, phosphorylating and activating the serine / threonine protein kinase (ULK1) and the eukaryotic initiation factor (eIF4B). This phosphorylation 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-digesting enzyme primarily involved in carbohydrate digestion and absorption. Upregulation of MGAM promotes carbohydrate digestion and utilization, enhancing 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 Artemia's energy metabolism and cell growth. This allows Artemia to accelerate growth and development and maintain a high survival rate under conditions of adequate nutrition and high-density culture. The upregulation of these genes enables Artemia to utilize energy more efficiently, support its growth and development, and enhance its immune response.

[0132] Compared with the NS group, the NC group showed significantly more enriched KEGG pathways, consistent with the superior growth-promoting effect of the NC group on experimental animals. This suggests that chitosan-based nonanoic acid triggers 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 Artemia growth. In contrast, the NS group was less effective in promoting growth and development.

[0133] (2) qRT-PCR verification

[0134] In order to verify the reliability of the transcriptome sequencing results, one gene with significant differential expression and 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 tissue using the Trizol method, and the RNA was reverse transcribed into cDNA using the PrimeScript™ FAST RT reagent Kit with gDNA Eraser (TAKARA) kit. 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 Boshang Biotechnology Co., Ltd., using β-actin as the internal reference gene. The primer sequences are shown in Table 5. The relative quantification method (2 -ΔΔCT ) to determine the fold change of the target gene. SPSS 26.0 software was then used for T-test analysis (T-test), and P < 0.05 was considered statistically significant. Figure 8 .

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

[0136]

[0137] Compared with the blank control group, the MRC gene in the NS group was significantly up-regulated (P<0.05) ( Figure 8Left panel); ACACA gene was significantly up-regulated in the NC group (P<0.05) ( Figure 8 This indicates that the PCR verification results are consistent with the differential gene expression upregulation results of transcriptome sequencing analysis, confirming the reliability of the transcriptome sequencing analysis results of this application.

[0138] In summary, the present application first provides the application of nonanoic acid in Artemia cultivation. The application achieves the antibacterial effect of nonanoic acid without adding an acid-base regulator, and achieves both the ecological safety of Artemia and the stability of the pH value of the water body. It overcomes the technical problem in the prior art that increasing the concentration of nonanoic acid leads to the ecological safety risk of Artemia, and the pH of the water body decreases and the concentration of nonanoic acid decreases, which is not enough to produce an antibacterial effect. On this basis, the present application provides a method for cultivating Artemia using nonanoic acid as an alternative to antibiotics. The cultivation method not only significantly improves the hatching rate and survival rate of Artemia and effectively reduces the number of Vibrio carried by Artemia by regulating the expression of growth-related, immune-related genes, but also promotes the growth and development of Artemia, enhances the immunity of Artemia, and ensures the healthy and sustainable cultivation of Artemia. Compared with the prior art that only utilizes the antibacterial properties of organic acids, the cultivation method achieves unexpected technical effects and provides a theoretical basis for further optimizing Artemia cultivation technology.

Claims

1. The application of nonanoic acid in Artemia culture is characterized by: The nonanoic acid is directly added to the aquaculture water body using silicon dioxide or chitosan as a carrier.

2. The use according to claim 1, characterized in that: When the nonanoic acid uses silicon dioxide as a carrier, the concentration of nonanoic acid in water is 130-300 μM; when the nonanoic acid uses chitosan as a carrier, the concentration of nonanoic acid in water is 55-90 μM.

3. The use according to claim 2, characterized in that: When the nonanoic acid uses silica as a carrier, 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 uses chitosan as a carrier, the weight-to-volume ratio of chitosan to nonanoic acid is (0.01-0.1):1, and the deacetylation degree of the chitosan is 75-88% and the molecular weight is 100-180 kDa.

4. The use according to any one of claims 1 to 3, characterized in that: The density of Artemia eggs in the aquaculture water body is no more than 1000 eggs / mL.

5. A simple and healthy method for cultivating Artemia, characterized by: The following steps are involved: (1) The carrier SiO2 or chitosan is mixed evenly with nonanoic acid in a certain weight-to-volume ratio to obtain a nonanoic acid product in the form of an emulsion or powder; (2) adding the nonanoic acid product described in step (1) to the water used for hatching and breeding Artemia to make the concentration of nonanoic acid in the breeding water As described in claim 2, obtaining nonanoic acid aquaculture water; (3) The Artemia salina eggs are placed in the nonanoic acid aquaculture water body described in step (2), and are incubated for 18-24 hours under the conditions of a temperature of 28-30°C, continuous light conditions, and feeding of basic feed to obtain Artemia salina.

6. The Artemia culture method according to claim 4, wherein: In the nonanoic acid emulsion described in step (1), the weight-to-volume ratio of nonanoic acid to the carrier is 0.5-3:1 (g:ml).

7. The Artemia culture method according to claim 4, wherein: In the nonanoic acid powder described in step (1), the weight volume ratio of nonanoic acid to the carrier is 1-10:1 (g:ml).

8. The Artemia culture method according to any one of claims 5 to 7, wherein: The density of the Artemia eggs in the water body in step (3) is no more than 1000 eggs / mL.

9. The Artemia culture method according to any one of claims 5 to 7, wherein: The continuous illumination condition in step (3) is 500-2500 Lux; the basic feed is chlorella, yeast powder, spirulina powder, rice bran, soybean meal or artificial microparticle feed.

10. An application of Artemia in fish and shrimp farming, characterized by: The Artemia is obtained by the cultivation method according to any one of claims 5 to 9.

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