A method for improving the immunity of d-shaped larvae of long oyster seed

Abstract

The application discloses a long oyster seed cultivation method capable of improving immunity of D-type larvae, which is characterized by the following steps: when the long oyster fertilized eggs develop to the initial stage of D-type larvae, the D-type larvae are bred in breeding water containing 0.053 g / L of ammonium chloride for 24 hours, and then the D-type larvae are moved to the breeding water without ammonium chloride for continuous breeding. The application can significantly improve the autophagy level of the D-type larvae, promote the proliferation of the hemocyte of the D-type larvae, and enhance the immune response capacity of the D-type larvae to viruses. In addition, the ammonium chloride can also induce the expression of inflammation-related genes of the D-type larvae, so that the immunity of the D-type larvae of the long oyster is improved comprehensively, the mortality of the larvae is effectively reduced, and the quality of the long oyster seed is improved. Meanwhile, the application also has the advantages of simple operation and low cost.

Description

Technical Field

[0001] This invention relates to the field of shellfish aquaculture, and more particularly to a method for cultivating long oyster seedlings that can improve the immunity of type D larvae. Background Technology

[0002] Oyster ( Crassostrea gigas oysters are an important marine aquaculture species with extremely high economic value. However, during the cultivation of oyster seedlings, many adverse factors such as marine pollution, climate disasters, predatory organism infestations, and pathogen infections can cause large-scale larval mortality. Especially during the critical developmental stage of D-type larvae before the formation of the apical larvae, the larval mortality rate can rise sharply, sometimes exceeding 50%, and in extreme cases even reaching over 80%.

[0003] Ammonium chloride (NH4Cl) is a major byproduct of soda ash production via the combined alkali process. It is a white crystalline solid, characterized by its hygroscopic nature, tendency to clump, and easy decomposition upon heating. It is soluble in water, alcohol, and glycerol, and has been widely applied in various technological fields. In agriculture, ammonium chloride is commonly used as a fertilizer. It not only promotes crop growth and increases yield but also effectively reduces the acidity of fruits while increasing sugar and vitamin C (Vc) content, thus significantly improving the taste and nutritional value of fruits. In animal husbandry, ammonium chloride can be used as a feed additive for ruminants. It helps alleviate heat stress in poultry under high temperatures and prevents diseases such as hypocalcemia, safeguarding the healthy growth of animals. In aquaculture, by adding ammonium chloride to culture media to cultivate algae, and then using these algae as feed additives, the immunity of waterfowl can be significantly improved, providing strong support for the sustainable development of aquaculture. Furthermore, ammonium chloride is used as an expectorant in the medical field.

[0004] Because the Pacific oyster is a mollusc, its digestive system differs significantly from that of ruminants or monogastric animals. It primarily feeds by filtering plankton and organic detritus from the water, relying on its gills and digestive glands for nutrient absorption. Therefore, its nitrogen requirements and utilization mechanisms in its feed differ from those of terrestrial animals. In particular, oysters live in aquatic environments where water quality, such as pH, is crucial for their survival and metabolism. Ammonium chloride's nitrogen form (ammonium nitrogen) is easily converted to ammonia in water, and excessive ammonia is toxic to aquatic organisms, potentially harming the gills and respiratory system of the Pacific oyster, and even causing death. Therefore, to date, there are no reports of using culture water containing a certain concentration of ammonium chloride to treat type D larvae during Pacific oyster seedling cultivation to effectively improve their immunity. Summary of the Invention

[0005] The present invention aims to solve the above-mentioned technical problems existing in the prior art and provide a method for cultivating oyster seedlings that can improve the immunity of type D larvae.

[0006] The technical solution of the present invention is as follows: fertilized eggs of Pacific oyster are obtained by artificial seedling cultivation and cultured. When the fertilized eggs of Pacific oyster develop to the early stage of D-type larvae, they are treated with aquaculture water with a final concentration of ammonium chloride of 0.053 g / L for 24 h. Then, the D-type larvae are transferred to aquaculture water without ammonium chloride for continued cultivation.

[0007] The preferred method of treating type D larvae with a final ammonium chloride concentration of 0.053 g / L in aquaculture water for 24 hours involves splashing an ammonium chloride solution into the aquaculture water during the initial stage of type D larvae rearing, resulting in a final ammonium chloride concentration of 0.053 g / L, and then rearing the type D larvae for 24 hours.

[0008] This invention involves treating the D-type larvae of the Pacific oyster seedlings with ammonium chloride at a final concentration of 0.053 g / L for 24 hours during the seedling cultivation process. The larvae are then transferred to ammonium chloride-free water for further rearing. This invention significantly enhances the autophagy level of D-type larvae, promotes the proliferation of hemolymphocytes, and strengthens their immune response to viruses. Furthermore, ammonium chloride induces the expression of inflammation-related genes in D-type larvae, thereby comprehensively improving the immunity of Pacific oyster D-type larvae, effectively reducing larval mortality, and improving the quality of Pacific oyster seedlings. This invention also boasts advantages such as simple operation and low cost. Attached Figure Description

[0009] Figure 1 This is a statistical chart showing the mortality and deformity rates of type D larvae in the embodiments and control groups of this invention.

[0010] Figure 2 This is a graph showing the changes in the levels of immune-related gene mRNA in type D larvae of the embodiments and control groups of the present invention.

[0011] Figure 3 This is a graph showing the changes in the level of the autophagy-related gene LC3 protein in type D larvae of the embodiments and control groups of the present invention. Detailed Implementation

[0012] This invention discloses a method for cultivating Pacific oyster seedlings that can improve the immunity of type D larvae. The method involves artificially inseminating and culturing Pacific oysters using conventional methods. Once the fertilized eggs develop into type D larvae, the amount of ammonium chloride powder is calculated based on the volume of seawater used for larval rearing. A high-concentration ammonium chloride solution is prepared by mixing ammonium chloride and seawater and then sprinkled into the initial rearing water for type D larvae, achieving a final ammonium chloride concentration of 0.053 g / L. The type D larvae are reared for 24 hours, then filtered through a silk sieve and transferred to a rearing water free of ammonium chloride to continue rearing to seedling stage using conventional methods.

[0013] The ammonium chloride used in this embodiment of the invention is from Beijing Solarbio Technology Co., Ltd., product number A7320.

[0014] experiment: Fertilized eggs of the Pacific oyster were obtained through artificial breeding and divided into two groups, each containing 50 L of water. These groups served as the control group (Blank) and the embodiment group (NH4Cl), respectively. The control group was cultivated using conventional methods of the prior art, while the embodiment group was treated using the method described in this invention.

[0015] In this invention, all experiments were performed in triplicate, and the data were expressed as mean ± standard deviation. One-way ANOVA and LSD multiple comparisons were performed using IBM SPSS Statistics 20 software, and graphs were generated using Origin Graph and GraphPad Prism 8. p Values ​​less than 0.05 indicate a significant difference; p A value less than 0.01 was considered highly significant. Differences between the two groups were analyzed using Student's t-test (“…”). "Indicates p < 0.05;" "express p < 0.01; "express p < 0.001; "express p < 0.0001).

[0016] Experiment 1: Detection of mortality and deformity rate of D-type oyster larvae in each experimental group After treating type D larvae with ammonium chloride for 24 hours in the embodiment group of this invention, type D larvae samples were collected from both the control group and the embodiment group of this invention to calculate the mortality and deformity rates of the larvae. The specific procedure was as follows: 1 mL of seawater was drawn from 50 L of aquaculture water, filtered through a silk sieve to obtain type D larvae samples, and then 1 μL of Gruy's reagent was added to the samples. The treated samples were observed and photographed under a microscope, and the mortality and deformity rates of the seedlings were calculated. The results are as follows: Figure 1 As shown. Figure 1 A: Comparison of mortality rates of type D larvae between the control group and the embodiment group of the present invention; B: Comparison of deformity rate of type D larvae between the control group and the embodiment group of the present invention; C: Normal type D larvae; D: Deformed type D larvae.

[0017] The results showed that after treatment with ammonium chloride for 24 hours, the mortality rate of type D larvae in the embodiment of this invention was significantly reduced, being 0.21 times that of the control group. p < 0.001)( Figure 1A); Compared with the control group, there was no significant difference in the malformation rate in the embodiment group of the present invention ( Figure 1 B). Malformations of type D larvae of the Pacific oyster are classified as follows: convex shell, serrated shell, incomplete shell, abnormal mantle, and embryonic development arrest. Figure 1 C represents the normal type D larvae observed in this experiment. Figure 1 D represents the D-type larvae with abnormal mantle observed in this experiment.

[0018] Experiment 2: Effects of each experimental group on the expression of immune genes related to type D larvae of the Pacific oyster. After treating type D larvae with ammonium chloride for 24 hours in the embodiment group of this invention, type D larvae samples were collected from both the control group and the embodiment group of this invention for RNA extraction. The expression of immune and developmental genes in type D larvae of the Pacific oyster treated with ammonium chloride was detected. RT-qPCR (Real-Time Quantitative Reverse Transcription PCR) was used to detect changes in mRNA expression of genes proven to be related to immunity, such as hematopoiesis, antiviral, and autophagy genes, as well as developmental genes.

[0019] The specific steps are as follows: In this embodiment of the invention, after treating type D larvae with ammonium chloride for 24 h, larval samples from each group were collected into 50 mL tubes using a 300-mesh sieve. The tubes were centrifuged at 8000 rpm for 5 min, the seawater was removed, and the larvae were resuspended in 20 mL of PBS. The resuspending process was repeated three times, centrifuged again at 8000 rpm for 5 min, and the PBS was removed. The samples were then aliquoted into 1.5 mL tubes. RNA samples were treated with 500 μL of Trizol, flash-frozen in liquid nitrogen, and stored at -80°C.

[0020] qRT-PCR was used to detect changes in mRNA expression of genes proven to be related to immunity, including autophagy, hematopoiesis, antiviral activity, and inflammation. Two [units / items] were used. -ΔΔCt Quantitative analysis was performed using this method. The reaction system is shown in Table 1: Table 1 RT-qPCR reaction system Table 1 Reaction system for RT-qPCR

[0021] Note: The reaction program is 95℃, 30 s (1 cycle); 95℃, 5 s, 60℃, 34 s (40 cycles).

[0022] Gene expression results of the control group and the groups in the embodiments of the present invention are as follows: Figure 2 As shown. Figure 2 Middle AE: Autophagy-related genes CgBeclin1 Cg ATG5 Cg ATG16L Cg LC3 and Cg P62; FK: Hematopoietic-related genes Cg Notch1、 Cg TNF-2, Cg BMP-7 Cg CDC-6 Cg CDK-2 and Cg Runx; LN: Antiviral-related genes Cg TRIM, Cg Mx1 and Cg IFI44; OQ: Inflammation-related genes Cg IL17-1 Cg IL17-5 and Cg GSDME.

[0023] Experimental results showed that after 24 h of ammonium chloride treatment of type D oyster larvae, autophagy-related genes... Cg Beclin1 Cg ATG5 Cg ATG16L Cg LC3 and Cg The mRNA expression level of P62 was significantly increased compared with that of the control group. Figure 2 AE), which was 6.01 times that of the control group ( p < 0.01), 3.23 times ( p < 0.01), 4.33 times ( p < 0.0001), 19.97 times ( p < 0.01) and 4.33 times ( p < 0.0001), indicating that ammonium chloride can promote the expression of autophagy-related genes and hematopoietic-related genes during the D-type larval stage. Cg Notch1、 Cg TNF-2, Cg BMP-7 Cg CDC-6 Cg CDK-2 and Cg Runx levels were significantly higher in both groups compared to the control group. Figure 2 FK), which was 4.29 times that of the control group ( p < 0.05), 2.98 times ( p < 0.001), 4.09 times ( p < 0.0001), 2.67 times ( p <0.01), 2.22 times ( p < 0.01) and 3.38 times (p < 0.001), indicating that ammonium chloride treatment of type D oyster larvae enhanced hemolymphocyte proliferation and improved larval immunity; antiviral related genes Cg TRIM, Cg Mx1 and Cg IFI44 was significantly higher than that in the control group. Figure 2 LN was 6.00 times that of the control group (LN). p < 0.001), 4.18 times ( p < 0.0001) and 4.07 times ( p < 0.01), indicating that ammonium chloride treatment can promote the immune response of type D larvae to foreign viruses and improve larval immunity; inflammation-related genes Cg IL17-1 Cg IL17-5 and Cg GSDME levels were significantly higher in both groups compared to the control group, being 2.42 times higher. p < 0.0001), 2.98 times ( p < 0.001) and 2.97 times ( p < 0.05) Figure 2 The OQ indicates that ammonium chloride can help type D larvae quickly identify pathogens, activate antibacterial immune function, and thus improve immunity.

[0024] Experiment 3: Effects of each experimental group on the protein level of LC3, a gene related to autophagy, in D-type larvae of the Pacific oyster. After treating type D larvae with ammonium chloride for 24 h in the embodiment group of the present invention, type D larvae samples from the control group and the embodiment group of the present invention were collected for protein extraction. The protein samples were stored directly at -80℃ to detect the effect of ammonium chloride treatment on autophagy-related protein LC3 in type D larvae of oyster.

[0025] First, the protein samples were quantified using Tubulin. Then, Western blot was used to detect the relative expression level of autophagy-related protein LC3 in D-type larvae after 24 h of ammonium chloride treatment.

[0026] The specific steps are as follows: (1) To ensure the consistency of protein content in each group, the internal reference gene Tubulin was first quantified. (2) Cut out the required SDS-PAGE gel area and cut nitrocellulose membrane (NC membrane) and filter paper to the appropriate size; (3) According to the current direction of the semi-dry electroporation transfer apparatus, place the layers of filter paper, SDS-PAGE gel, NC membrane, and then the remaining filter paper into the transfer apparatus in the following order from bottom to top. The current magnitude is 1.2 × NC membrane area (cm²). 2mA, time 1 h; (4) After the transfer is complete, add 5 mL of Western blot washing buffer and wash the NC membrane 3 times on a shaker with rapid shaking, 5 min each time; (5) Soak the NC membrane in milk solution and shake slowly at room temperature for 2 h. Incubate 5 μL of Tubulin antibody with 5 mL of milk solution at room temperature for 3 h. (6) After adding the colorimetric solution, observe and save the experimental results using a chemiluminescent gel imaging system to ensure that the amount of protein loaded in each group is the same; (7) After determining the amount of protein loaded into each group, the relative expression level of autophagy-related protein LC3 was detected by Western blot. Protein samples stored at -80℃ were thawed on ice and then subjected to SDS-PAGE. (8) Cut the required SDS-PAGE gel area according to the protein size of LC3 and cut the nitrocellulose membrane (NC membrane) and filter paper to the appropriate size; (9) According to the current direction of the semi-dry electroporation transfer apparatus, place the layers of filter paper, SDS-PAGE gel, NC membrane, and then the remaining filter paper into the transfer apparatus in the following order from bottom to top. The current magnitude is 1.2 × NC membrane area (cm²). 2 mA, time 25 min; (10) After the transfer is complete, add 5 mL of Western blot washing buffer and wash the NC membrane 3 times on a shaker with rapid shaking, 5 min each time; (11) Soak the NC membrane in milk solution and shake slowly at room temperature for 2 h. Add 5 μL of LC3 antibody to 5 mL of milk solution and shake slowly at 4°C overnight. (12) Add 5 mL of Western blot washing buffer and wash the NC membrane with rapid shaking 3 times, 5 min each time; (13) Add 5 μL of horseradish peroxidase to rabbit and 5 mL of milk solution and incubate at room temperature with gentle shaking for 1 h; (14) Wash the NC membrane three times with rapid shaking in Western blot washing buffer, 5 min each time; (15) After adding the colorimetric solution, observe the experimental results using a chemiluminescent gel imaging system and save the results.

[0027] Experimental results are as follows Figure 3 As shown. The results indicate that, compared with the control group, the LC3-II band was significantly enhanced in the D-type larvae treated with ammonium chloride for 24 h in the embodiment of this invention, indicating an increase in the protein expression level of the autophagy-related protein LC3, and the protein level was related to the mRNA level ( Figure 2 D) Consistent.

Claims

1. A method for cultivating *Crassostrea gigas* seedlings that can improve the immunity of type D larvae, characterized in that: Fertilized eggs of Pacific oysters were obtained and cultured using artificial seedling methods. Once the fertilized eggs developed into D-type larvae, the D-type larvae were treated with aquaculture water containing ammonium chloride at a final concentration of 0.053 g / L for 24 h. The D-type larvae were then transferred to aquaculture water without ammonium chloride for continued culture.

2. The method for cultivating *Crassostrea gigas* seedlings that can improve the immunity of type D larvae according to claim 1, characterized in that: The treatment of type D larvae with a final ammonium chloride concentration of 0.053 g / L for 24 hours involves splashing an ammonium chloride solution into the aquaculture water during the initial stage of type D larvae rearing, resulting in a final ammonium chloride concentration of 0.053 g / L, and then rearing the type D larvae for 24 hours.

Images