Seedling raising method for improving attachment rate and disease resistance of crassostrea gigas eyespot larvae and application of seedling raising method

Abstract

The invention belongs to the technical field of aquaculture, and particularly relates to a breeding method for improving the attachment rate and disease resistance of crassostrea gigas eyespot larvae and application of the breeding method. According to the method, early-stage crassostrea gigas eyespot larvae are soaked in filtered seawater containing a certain dosage of glutathione, and compared with a control group without glutathione, the growth and development speed of the crassostrea gigas eyespot larvae soaked with glutathione is increased, the adhesion rate is remarkably increased, the lysozyme activity is enhanced, and the vibrio resistance is remarkably improved. The immunopotentiator has remarkable effects of reducing the death rate of crassostrea gigas larvae in the attachment and metamorphosis process and improving the attachment and metamorphosis rate, and can be applied to fry production as the immunopotentiator in the crassostrea gigas attachment and metamorphosis stage.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and in particular relates to a seedling raising method for improving the attachment rate and disease resistance of long-eyed oyster larvae and an application thereof. Background Art

[0002] The long oyster is an important marine aquaculture shellfish in my country, with high aquaculture yield and great economic value. In recent years, problems such as low success rate of oyster breeding have occurred frequently, seriously threatening the high-quality development of the oyster aquaculture industry. During the individual development process, the long oyster undergoes a metamorphosis from floating to fixed, among which the eyed larvae are the key period of attachment metamorphosis, but the larvae during this period are sensitive to environmental changes and have a high mortality rate. Effective methods and ways to solve this problem are extremely scarce. Therefore, improving the quality of eyed larvae and the survival rate of their attachment metamorphosis is the key to ensuring the sustainable development of the oyster seed industry. Summary of the invention

[0003] The invention aims to provide a seedling raising method for improving the attachment rate and disease resistance of eyed larvae of Crassostrea gigas, so as to improve the seedling raising quality of Crassostrea gigas.

[0004] In order to achieve the above object, the specific technical solution adopted by the present invention is:

[0005] A seedling raising method for improving the attachment rate and disease resistance of long oyster eyed larvae, comprising soaking early long oyster eyed larvae in filtered seawater containing a certain dose of glutathione, and transferring the soaked eyed larvae to filtered seawater for cultivation 24 hours later, thereby improving the attachment rate and disease resistance of long oyster eyed larvae.

[0006] Glutathione is a tripeptide composed of glutamic acid, cysteine ​​and glycine, and is the most important low molecular weight antioxidant synthesized in cells. Glutathione S-transferase Kappa 1 (Glutathione STransferase Kappa1, GSTK1) is located in peroxisomes, catalyzing the reaction of glutathione with intracellular free radicals and peroxides, and plays a key role in maintaining the normal physiological function of cells. The inventors found that the transcriptome data of long oysters showed that the expression level of GSTK1 increased significantly in the eyespot larval stage, indicating that glutathione plays a role in the eyespot stage of long oysters and the process of larval attachment. Therefore, the present invention uses eyespot larvae treated with glutathione.

[0007] The glutathione is reduced glutathione, and the final concentration of glutathione in the filtered seawater is 3-7 μmol / L, preferably 7 μmol / L. After being immersed in filtered seawater containing this concentration of glutathione, the eyed larvae develop into juvenile shellfish and have significantly improved resistance to Vibrio splendens, and significantly reduced mortality.

[0008] The second object of the present invention is to apply a method for raising seedlings to improve the attachment rate and disease resistance of eyed larvae of long oysters, and in particular, to apply the method in the long oyster seed breeding industry.

[0009] The third object of the present invention is to provide a method for breeding long oyster seeds.

[0010] To achieve the above-mentioned purpose, the specific technical scheme adopted by the present invention is: a method for breeding long oyster seedlings, including a seedling raising stage, during which the early eyed larvae of long oysters are immersed in filtered seawater containing a certain dose of glutathione, and then transferred to filtered seawater for cultivation after 24 hours.

[0011] The glutathione is reduced glutathione.

[0012] The final concentration of glutathione in the filtered seawater containing glutathione is 3-7 μmol / L.

[0013] The final concentration of glutathione in the filtered seawater containing glutathione is 7 μmol / L. After the treatment, the disease resistance of the larvae is significantly improved and the larvae attachment rate is also significantly improved.

[0014] Compared with the prior art, the present invention has the following characteristics:

[0015] The present invention adopts an oyster seedling raising method with simple operation, low cost and good safety. By soaking the early eyed larvae of long oysters in filtered seawater containing a certain dose of glutathione, compared with the control group without glutathione, the eyed larvae of long oysters soaked in glutathione in the present invention have faster development speed, significantly improved larval attachment rate, enhanced lysozyme activity, and significantly improved anti-vibrio ability. The research results provide a new way to solve the problem of high mortality rate of long oyster larvae during attachment and metamorphosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression changes of glutathione S-transferase Kappa 1 in different developmental stages of Crassostrea gigas in Example 1 are shown.

[0017] Figure 2 This is the development status of the oyster eyed larvae after being soaked in glutathione in Example 1.

[0018] A: Proportion of eyed larvae in different treatment groups; B: Larvae attachment rate in different treatment groups. SW: sea water control group; G3: 3μmol / L glutathione treatment group; G7: 7μmol / L glutathione treatment group. Different letters represent significant differences compared with other groups (p<0.05).

[0019] Figure 3This is the mortality rate of the larvae treated with glutathione in Example 1 after they developed to the juvenile stage and were challenged with Vibrio splendens.

[0020] SW: sea water control group; G3: 3μmol / L glutathione treatment group; G7: 7μmol / L glutathione treatment group. Different letters represent significant differences compared with other groups (p<0.05).

[0021] Figure 4 The lysozyme activity of the oyster eyed larvae after being soaked in glutathione in Example 1.

[0022] SW: sea water control group; G3: 3μmol / L glutathione treatment group; G7: 7μmol / L glutathione treatment group. Different letters represent significant differences compared with other groups (p<0.05). DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and beneficial technical effects of the present invention clearer, the present invention is specifically described below through examples. It should be understood that the examples are only used to further illustrate the present invention, but the content of the present invention is not limited to the contents involved in the examples, and cannot be understood as limiting the scope of protection of the present invention. Technical personnel skilled in the art can make some non-essential improvements and adjustments based on the above-mentioned contents of the present invention.

[0024] Unless otherwise specified, the methods used in the following examples are conventional methods, and are carried out according to the techniques described in the literature in the art or according to the product instructions. The reagents and materials used in the following examples are all commercially available. The Vibrio splendens used in the following examples is a strain preserved by Dalian Ocean University and was isolated from the lesions of dying oysters (Rui Liu et al., Journal of Invertebrate Pathology, 2013).

[0025] Example 1

[0026] (1) Screening of transcriptome data of long oysters: The transcriptome data of long oysters were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov), and then data were screened using Excel. The condition was set that the gene expression level at the eyespot stage was more than twice that of the gene expression at other developmental stages. Glutathione S-transferase Kappa 1 and its expression levels at each stage were screened. Figure 1 As shown, the expression level of glutathione S-transferase Kappa 1 was the highest in the eyespot stage (P1, P2), and then the expression level decreased rapidly in the juvenile stage (S).

[0027] (2) Artificial breeding of long oysters: 20 male and female long oysters with well-developed gonads were selected as breeding broodstock using the drop method and microscope observation method. The eggs were dissected and filtered with 200-mesh and 500-mesh sieves respectively. Then, the filtered eggs were gently poured into the filtered seawater for maturation. Artificial insemination was carried out at a ratio of 5:1 between sperm and eggs. Five minutes after fertilization, the fertilized eggs were gently washed three times with a 500-mesh sieve. The fertilized eggs were then placed in a 200-L incubator for incubation. When the fertilized eggs developed to the D-larval stage, the larval density was controlled at 15-20 / mL, and Isochrysis was fed. The water was changed once a day.

[0028] (2) Glutathione soaking treatment of long-eyed oyster larvae: The early-stage long-eyed oyster larvae were soaked in filtered seawater containing 3μmol / L or 7μmol / L glutathione for 24 hours, and then the larvae were transferred to normal filtered seawater for cultivation, and were recorded as G3 and G7 groups respectively. The control group was normal long-eyed oyster larvae that were not treated with glutathione soaking, recorded as SW group. The mixed feeding of Isochrysis, Platymonas, Chaetoceros, Chlorella, etc. was carried out 3 times a day, and the water was changed once a day.

[0029] (3) Effect of glutathione immersion on the development of oyster larvae: After 24 hours of glutathione immersion, the early eyed larvae of oysters were transferred to normal filtered seawater for cultivation. After 24 hours, the oyster larvae of the experimental group and the control group were sucked with a dropper, and the development of the larvae was observed under a microscope, and the percentage of eyed larvae was counted. The results showed that the proportion of eyed larvae in the oyster larvae treated with 3μmol / L glutathione was 77.28%, and the proportion of eyed larvae in the oyster larvae treated with 7μmol / L glutathione was 73.02%, which were significantly higher than 61.45% in the normal seawater culture group ( Figure 2 A).

[0030] (4) Effect of glutathione immersion treatment on the attachment rate of long-shelled oyster larvae: After 24 hours of glutathione immersion treatment of early-stage eye-spot larvae of long-shelled oysters, the larvae in the experimental and control groups were distributed at 0.2 pcs / mL in a 60L culture bucket containing normal filtered seawater. At the same time, scallop shells were used as the attachment base for the larvae. Each group of 50 sterilized shells was hung in a string in the culture bucket. They were fed with a mixture of Isochrysis algae, Platymonas, and Chlorella three times a day, and the water was changed every two days. After 7 days of attachment, the number of attached larvae in each group was counted, and the attachment rate (attachment rate = the percentage of the average number of attached larvae to the initial total number of larvae) was calculated. Figure 2As shown in B, the attachment rate of oyster larvae in the normal filtered seawater culture group was 35.23%, and the attachment rate of oyster larvae in the 3μmol / L glutathione immersion treatment group was 35.48%. There was no significant difference between the two groups, but the attachment rate of oyster larvae in the 7μmol / L glutathione immersion treatment group was 41.13%, which was significantly higher than that in the normal filtered seawater culture group and the 3μmol / L glutathione immersion treatment group.

[0031] (5) Effect of glutathione immersion on disease resistance of juvenile oysters: When juvenile oysters treated with different concentrations of glutathione developed to the one-month-old juvenile stage, four scallop slices with juvenile oysters attached were selected from the culture bucket in each group, with about 40 juvenile oysters attached to each slice. 8 CFU / mL of Vibrio splendens was temporarily cultured in seawater, and the number of surviving spat was counted every 12 hours. Figure 3 As shown, the survival rate of juvenile spat in the glutathione-treated group was significantly higher than that in the normal seawater culture group, and the survival rate of juvenile spat in the 7 μmol / L glutathione-treated group was significantly higher than that in the 3 μmol / L glutathione-treated group.

[0032] (6) Effect of glutathione immersion on the activity of immune-related enzymes in long oyster larvae: Long oyster larvae that had been treated with glutathione for 24 hours were transferred to normal filtered seawater for incubation for 24 hours, and then larval samples from different treatment groups were collected. The long oyster larvae in each group were ground, centrifuged, and the supernatant was taken. Then, lysozyme (LZM) was detected according to the instructions of the Nanjing Jiancheng kit. Figure 4 As shown, the lysozyme content of the larvae in the 7 μmol / L glutathione treatment group was significantly higher than that in the 3 μmol / L glutathione treatment group and the normal filtered seawater culture group.

[0033] The above results prove that, through the method for treating oyster larvae by soaking with glutathione and the application thereof of the present invention, the development speed of the eyed larvae of oysters is accelerated, the larval attachment rate is significantly improved, the lysozyme activity is enhanced, and the anti-Vibrio ability is significantly improved.

[0034] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for raising seedlings for improving the attachment rate and disease resistance of oyster eyed larvae, characterized in that: The early-stage eyed larvae of Crassostrea gigas were immersed in filtered seawater containing glutathione, and after 24 hours, the immersed eyed larvae were transferred to filtered seawater for cultivation.

2. The method for raising seedlings for improving the attachment rate and disease resistance of long-eyed oyster larvae according to claim 1, characterized in that: The glutathione is reduced glutathione.

3. The method for raising seedlings for improving the attachment rate and disease resistance of long-eyed oyster larvae according to claim 1, characterized in that: The final concentration of glutathione in the filtered seawater containing glutathione is 3-7 μmol / L.

4. The method for raising seedlings for improving the attachment rate and disease resistance of long-eyed oyster larvae according to claim 3, characterized in that: The final concentration of glutathione in the filtered seawater containing glutathione is 7 μmol / L.

5. Use of the method for raising seedlings for improving the attachment rate and disease resistance of eyed larvae of Crassostrea gigas as claimed in any one of claims 1 to 4 in the breeding of Crassostrea gigas seedlings.

6. A method for breeding oyster seed, including a seedling raising stage, characterized in that: In the seedling raising stage, the early-eye larvae of Crassostrea gigas are immersed in filtered seawater containing glutathione, and after 24 hours, they are transferred to filtered seawater for cultivation.

7. The method for breeding oyster seedlings according to claim 6, characterized in that: The glutathione is reduced glutathione.

8. The method for breeding oyster seedlings according to claim 6 or 7, characterized in that: The final concentration of glutathione in the filtered seawater containing glutathione is 3-7 μmol / L.

9. The method for breeding oyster seedlings according to claim 6 or 7, characterized in that: The final concentration of glutathione in the filtered seawater containing glutathione is 7 μmol / L.

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