Breeding method for improving attachment rate and disease resistance of long oyster (crassostrea gigas) nauplii and application thereof

Abstract

The application belongs to the technical field of aquaculture, and particularly relates to a method for improving the attachment rate and disease resistance of long oyster (Crassostrea gigas) nauplii and application thereof. The method is that early nauplii of the long oyster are soaked in filtered seawater containing a certain dose of glutathione. Compared with a control group without adding glutathione, the growth and development speed of the long oyster nauplii soaked in the glutathione is accelerated, the attachment rate is significantly improved, the lysozyme activity is enhanced, and the ability to resist vibrio is significantly increased. The application has a significant effect on reducing the mortality of the long oyster nauplii in the attachment metamorphosis process and improving the attachment metamorphosis rate, and can be used as an immune enhancer for the attachment metamorphosis stage of the long oyster and applied to seed production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquaculture, and particularly relates to a method for improving the attachment rate and disease resistance of long oyster (Crassostrea gigas) eyed larvae and application thereof. BACKGROUND

[0002] Long oyster (Crassostrea gigas) is an important marine aquaculture mollusk in China, with high yield and great economic value. In recent years, the problem of low success rate of oyster seed culture has occurred frequently, which seriously threatens the high-quality development of oyster aquaculture. Long oyster undergoes metamorphosis from planktonic to sessile during individual development, and the eyed larva is the key period of attachment metamorphosis. However, the eyed larva is sensitive to environmental changes during this period, and the mortality rate is high. There are few effective methods and ways to solve this problem. Therefore, improving the quality of eyed larvae and the survival rate of attachment metamorphosis is the key to the sustainable development of oyster seed industry. SUMMARY

[0003] The purpose of the present application is to provide a method for improving the attachment rate and disease resistance of long oyster (Crassostrea gigas) eyed larvae to improve the quality of long oyster (Crassostrea gigas) seed culture.

[0004] To achieve the above-mentioned purpose, the specific technical scheme adopted by the present application is as follows:

[0005] A method for improving the attachment rate and disease resistance of long oyster (Crassostrea gigas) eyed larvae, wherein the early eyed larvae of long oyster (Crassostrea gigas) are soaked in filtered seawater containing a certain dose of glutathione, and after 24 hours, the soaked eyed larvae are transferred to filtered seawater for cultivation, thereby improving the attachment rate and disease resistance of long oyster (Crassostrea gigas) 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 (GSTK1) is located in peroxisomes and catalyzes the reaction of glutathione with intracellular free radicals and peroxides, playing a key role in maintaining the normal physiological function of cells. The inventors found that the transcriptome data of long oyster showed that the expression of GSTK1 increased significantly in the eyed larva stage, indicating that glutathione plays a role in the eyed stage and the attachment process of long oyster larvae. Therefore, the eyed larvae treated with glutathione are used in the present application.

[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 soaking treatment in filtered seawater containing glutathione at this concentration, the resistance of eyed larvae developing to juvenile stage to Vibrio splendidus attack is significantly improved, and the mortality rate is significantly reduced.

[0008] The second object of the present application is to improve the application of the larviculture method for improving the attachment rate and disease resistance of Crassostrea gigas Dall cyprid larvae.

[0009] The third object of the present application is to provide a larviculture method for Crassostrea gigas.

[0010] To achieve the above objects, the present application adopts the following specific technical solutions: a larviculture method for Crassostrea gigas, comprising a larviculture stage, in which early cyprid larvae of Crassostrea gigas are soaked 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, and after treatment, the disease resistance of the larvae is significantly improved, and the attachment rate of the larvae is also significantly improved.

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

[0015] The present application adopts a simple, low-cost and safe oyster larviculture method, in which early cyprid larvae of Crassostrea gigas are soaked in filtered seawater containing a certain dose of glutathione. Compared with the control group without adding glutathione, the cyprid larvae of Crassostrea gigas soaked in glutathione develop faster, the attachment rate of the larvae is significantly improved, the lysozyme activity is enhanced, and the ability to resist Vibrio is significantly improved. The research results provide a new way to solve the problem of high mortality during the attachment metamorphosis of Crassostrea gigas larvae. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 For the expression changes of glutathione S-transferase Kappa 1 in Crassostrea gigas at different development stages in Example 1.

[0017] Figure 2 For the development status of Crassostrea gigas cyprid larvae after glutathione soaking treatment in Example 1.

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

[0019] Figure 3The mortality of the larvae of Example 1 treated with glutathione soaking and developed to the juvenile stage after being attacked by Vibrio splendidus.

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

[0021] Figure 4 The lysozyme activity of the eyed larvae of Crassostrea gigas treated with glutathione soaking in Example 1.

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

[0023] In order to make the purposes, technical solutions and beneficial technical effects of the present application clearer, the present application will be specifically described below through examples. It should be understood that the examples are only used to further illustrate the present application, but the content of the present application is not limited to the content involved in the examples, and should not be understood as a limitation on the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments according to the content of the present application.

[0024] Unless otherwise specified, the methods used in the following examples are conventional methods, which 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 commercially available. The Vibrio splendidus used in the following examples is a strain preserved by Dalian Ocean University, which is isolated from the lesions of dying oysters (Rui Liu et al., Journal of Invertebrate Pathology, 2013).

[0025] Example 1

[0026] (1) Crassostrea gigas transcriptome data screening: Crassostrea gigas transcriptome data were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov), and then data screening was performed using Excel, with the condition set as the gene expression level at the eyed stage being more than 2 times the gene expression level at other development stages, to screen glutathione S-transferase Kappa 1 and its expression level at each stage. As shown in Table 1, the expression level of glutathione S-transferase Kappa 1 was the highest at the eyed stage (P1, P2), and then the expression level rapidly decreased at the juvenile stage (S). Figure 1

[0027] ​(2) Artificial breeding of Pinctada martensii Dunker: 20 Pinctada martensii Dunker individuals with better gonad development were selected as breeding parent oysters by using the drop method and microscope observation method. The eggs were dissected and filtered with 200-mesh and 500-mesh screen gauze in turn. Then, the filtered eggs were gently poured into filtered seawater for maturation. Artificial insemination was carried out according to the ratio of 5:1 of sperm to egg. After 5 minutes of fertilization, the fertilized eggs were gently washed 3 times using 500-mesh screen gauze. Then, the fertilized eggs were placed in a 200-L incubator for hatching. When the fertilized eggs developed to the D-shaped larva stage, the larva density was controlled at 15-20 individuals / mL, and Isochrysis galbana was fed, and the water was changed once a day.

[0028] (2) Glutathione soaking treatment of Pinctada martensii Dunker eye spot larvae: Pinctada martensii Dunker early eye spot larvae were soaked in filtered seawater containing 3 μmol / L or 7 μmol / L glutathione for 24 hours, respectively, 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 development Pinctada martensii Dunker larvae without glutathione soaking treatment, and was recorded as the SW group. Isochrysis galbana, Platymonas sp., Chaetoceros sp., Chlorella sp., etc. were mixed and fed 3 times a day, and the water was changed once a day.

[0029] (3) Effect of glutathione soaking treatment on the development of Pinctada martensii Dunker larvae: After the Pinctada martensii Dunker early eye spot larvae were soaked in glutathione for 24 hours, they were transferred to normal filtered seawater for cultivation. After 24 hours, the larvae in the experimental and control groups were sucked with a dropper, and the development of the larvae was observed under a microscope, and the percentage of eye spot larvae was counted. The results showed that the percentage of eye spot larvae of the Pinctada martensii Dunker larvae soaked in 3 μmol / L glutathione was 77.28%, and the percentage of eye spot larvae of the Pinctada martensii Dunker larvae soaked in 7 μmol / L glutathione was 73.02%, which were significantly higher than 61.45% of the normal seawater cultivation group. Figure 2 A).

[0030] (4) Effect of glutathione soaking treatment on the attachment rate of Pinctada martensii Dunker larvae: After the Pinctada martensii Dunker early eye spot larvae were soaked in glutathione for 24 hours, the larvae in the experimental and control groups were distributed in 60 L of normal filtered seawater at a density of 0.2 individuals / mL, and scallop shells were used as attachment bases for the larvae. Each group consisted of 50 sterilized shells, which were hung in the cultivation tank. Isochrysis galbana, Platymonas sp., Chlorella sp., etc. were mixed and fed 3 times a day, and the water was changed once every 2 days. After 7 days of attachment, the number of attached larvae in each group was counted, and the attachment rate was calculated (attachment rate = percentage of the average number of attached larvae to the total number of initial larvae). Figure 2As shown in Table B, the attachment rate of long oyster larvae in the normal filtered seawater cultivation group was 35.23%, the attachment rate of long oyster larvae in the 3 μmol / L glutathione soaking treatment group was 35.48%, and no significant difference was found between the two groups, but the attachment rate of long oyster larvae in the 7 μmol / L glutathione soaking treatment group was 41.13%, which was significantly higher than that in the normal filtered seawater cultivation group and the 3 μmol / L glutathione soaking treatment group.

[0031] (5) Effect of glutathione soaking treatment on the disease resistance of long oyster juvenile: When the long oyster larvae treated by different concentrations of glutathione soaking treatment developed to 1-month-old juvenile stage, 4 scallop pieces with oyster juveniles attached on each piece, about 40 juveniles, were selected from each group of cultivation barrels, and were respectively placed in 1 L seawater containing 2 x 10 8 CFU / mL Vibrio splendidus for temporary cultivation, and the number of surviving juveniles was counted every 12 hours. As shown in Table C, the survival rate of juveniles in the glutathione treatment group was significantly higher than that in the normal seawater cultivation group, and the survival rate of juveniles in the 7 μmol / L glutathione treatment group was significantly higher than that in the 3 μmol / L glutathione treatment group. Figure 3

[0032] (6) Effect of glutathione soaking treatment on the activity of immune-related enzymes of long oyster larvae: After the long oyster larvae treated by glutathione soaking treatment for 24 hours were transferred to normal filtered seawater for cultivation for 24 hours, the samples of larvae in different treatment groups were collected. The long oyster larvae in each group were ground, centrifuged, and the supernatant was taken, and then the lysozyme (LZM) was detected according to the Nanjing Jiancheng kit instruction. As shown in Table D, the lysozyme content of 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 cultivation group. Figure 4

[0033] The above results prove that, by using the method of soaking long oyster larvae with glutathione and the application thereof, the development speed of the oyster larvae is accelerated, the attachment rate of the larvae is significantly improved, the lysozyme activity is enhanced, and the ability to resist Vibrio is significantly improved.

[0034] The above-described embodiments only express one embodiment of the present application, which is described in a more specific and detailed manner, but it should not be understood as a limitation on the scope of the patent. It should be noted that, for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application.​​

Claims

1. A method for improving the attachment rate and disease resistance of Crassostrea gigas larvae, characterized in that: The early eyed larvae of Pinctada martensii are soaked in filtered seawater containing glutathione, and after 24 hours, the soaked eyed larvae are transferred to filtered seawater for cultivation; the final concentration of glutathione in the filtered seawater containing glutathione is 7 μmol / L.

2. The method for improving the attachment rate and disease resistance of Pinctada martensii larvae according to claim 1, characterized in that: The glutathione is reduced glutathione.

3. Use of the method for improving the attachment rate and disease resistance of eyed larvae of Pinctada martensii according to claim 1 or 2 in the breeding of Pinctada martensii seedlings.

4. A method for breeding Pinctada longisima seedlings, comprising a seedling raising stage, characterized in that: In the seedling stage, the early eyed larvae of Pinctada martensii are soaked in filtered seawater containing glutathione, and after 24 hours, the soaked eyed larvae are transferred to filtered seawater for cultivation; the final concentration of glutathione in the filtered seawater containing glutathione is 7 μmol / L.

5. The Pinctada longisima seed breeding method according to claim 4, characterized in that: The glutathione is reduced glutathione.

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