Method for inducing triploidy in abalone haliotis discus hannai by salinity

Abstract

The application discloses a method for inducing triploid of Haliotis discus hannai Ino by salinity, comprising the following steps: (1) synchronously fertilizing sperm and ovum of Haliotis discus hannai Ino to obtain fertilized eggs; (2) when the proportion of the first polar body appearing in the fertilized eggs obtained in step (1) is 60-70%, the fertilized eggs are subjected to high-salt treatment or low-salt treatment; and (3) the fertilized eggs treated in step (2) are subjected to reperfusion treatment of salinity gradient sand-filtered seawater, so as to gradually adjust the osmotic pressure by the salinity gradient sand-filtered seawater, slow down the damage to the fertilized eggs, and then resuspend in fresh sand-filtered seawater with normal salinity, and perform conventional incubation and cultivation. The application can realize efficient induction of triploid by optimizing the salinity and reperfusion steps, avoids the problems of drug residues and equipment requirements in traditional chemical and physical methods, and has the advantages of safety, simplicity and high efficiency.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of shellfish culture, and particularly relates to a method for inducing triploid of Haliotis discus hannai by salinity. BACKGROUND

[0002] Abalone is an important marine aquaculture economic shellfish, with its abdominal muscle as the main edible part, which is favored in the domestic and foreign consumer market. China is the world's largest abalone producer and consumer, with a total output of 244,000 tons in 2023, of which Haliotis discus hannai is the main breeding species. However, with the rapid development of abalone aquaculture industry, the problems of slow individual growth, degeneration of germplasm and increasing production cost are increasingly prominent. Generally, it takes 2 to 3 years for abalone to reach the market specification, and the continuous rise of labor and infrastructure costs further exacerbates the constraints of its growth characteristics on industry development.

[0003] Triploid bred based on chromosome engineering technology has attracted widespread attention due to its ability to significantly improve the growth rate and individual quality of shellfish. Triploid shellfish, due to its sterile characteristics, can transfer energy for reproduction to individual growth, improving economic benefits during the breeding period. Since Stanley successfully induced triploid of Crassostrea virginica using cytochalasin B in 1956, scholars at home and abroad have conducted triploid breeding research in various shellfish such as oysters, scallops, mussels, clams and abalone. Currently, in addition to some oyster species that have achieved full triploidy through inter-chromosome hybridization, the production of triploid in other shellfish mainly relies on physical and chemical induction methods.

[0004] In the prior art, common methods for inducing triploid of shellfish include:

[0005] 1. Chemical induction: using chemical drugs such as cytochalasin B, 6-dimethylaminopurine (6-DMAP), caffeine, colchicine and nocodazole to induce triploid by inhibiting polar body release of fertilized eggs. However, this method has drug residue problems, which may affect embryo development and shellfish food safety, and also cause potential environmental pollution.

[0006] 2. Physical induction: using physical means such as temperature shock, hydrostatic pressure or electric pulse to interfere with the normal development process of fertilized eggs to induce triploid. This method requires expensive special equipment and has high requirements for operating conditions, with unstable induction efficiency, limiting its application in large-scale production.

[0007] 3. Salinity-induced exploration: In recent years, researchers have tried to use high and low salt to change the way of osmotic pressure to inhibit the release of polar body of fertilized egg to induce triploidy, and have made some progress in shellfish such as oysters and scallops. The genus Haliotis is a narrow-salt shellfish, and the fertilized egg is more sensitive to changes in environmental salinity. The existing salinity induction technology has not yet formed a mature application for this species. For example, Guo Deqiang (2014) showed that the highest induction rate of low salt and high salt to induce triploid of Haliotis discus hannai was only 45.34% and 33.46%, respectively, far from the ideal level of production application, and the induction efficiency and stability still need to be improved. SUMMARY

[0008] The present application aims to overcome the defects of the prior art and provide a method for salinity-induced triploid of Haliotis discus hannai.

[0009] The technical solution of the present application is as follows:

[0010] A method for salinity-induced triploid of Haliotis discus hannai, comprising the following steps:

[0011] (1) synchronously fertilize sperm and eggs of Haliotis discus hannai to obtain fertilized eggs;

[0012] (2) when the proportion of the first polar body of the fertilized eggs obtained in step (1) is 60-70%, the fertilized eggs are treated with high salt or low salt;

[0013] (3) the fertilized eggs treated in step (2) are subjected to salinity gradient sand-filtered seawater rehydration treatment to gradually adjust the osmotic pressure by salinity gradient sand-filtered seawater, slow down the damage to the fertilized eggs, and then resuspended in fresh sand-filtered seawater with normal salinity for conventional incubation and cultivation.

[0014] In a preferred embodiment of the present application, the temperature of the synchronous fertilization is 18-23.5℃.

[0015] In a preferred embodiment of the present application, the high salt treatment comprises placing the fertilized eggs in sand-filtered seawater with a salinity of 41-52ppm for 10-25min.

[0016] Further preferably, the salinity gradient sand-filtered seawater rehydration treatment comprises first placing the fertilized eggs in sand-filtered seawater with a salinity of 37-39ppm for 2-3min, and then placing the fertilized eggs in sand-filtered seawater with a salinity of 32-34ppm for 2-5min.

[0017] In a preferred embodiment of the present application, the low salt treatment comprises placing the fertilized eggs in sand-filtered seawater with a salinity of 12-18ppm for 10-25min.

[0018] Further preferably, the salt gradient sand-filtered seawater reperfusion treatment comprises: first placing in 24-26 ppm sand-filtered seawater for 2-3 min, and then placing in 30-32 ppm sand-filtered seawater for 2-5 min.

[0019] In a preferred embodiment of the present application, the normal salinity fresh sand-filtered seawater has a salinity of 30-35 ppm.

[0020] The present application has the following beneficial effects:

[0021] 1. The present application completely avoids the use of chemical drugs, eliminates the potential impact of drug residues on embryonic development and food safety, and does not pollute the environment, meeting the development requirements of green aquaculture.

[0022] 2. The present application does not require expensive special equipment, and can achieve induction by adjusting the salinity of seawater, which is simple to operate and easy to popularize and apply, significantly reducing production costs.

[0023] 3. By optimizing the salinity (high salinity 41-52 ppm or low salinity 12-18 ppm) and treatment time (10-25 min), and combining with reperfusion treatment, the present application can achieve a triploid induction rate of up to 100%, far exceeding the induction efficiency of traditional physical methods.

[0024] 4. The present application introduces a salt gradient reperfusion step, which effectively slows down the damage of rapid changes in osmotic pressure to fertilized eggs, and can improve the normal development rate of fertilized eggs and the induction efficiency and stability.

[0025] 5. The present application not only meets the technical needs of triploid production of H. discus, but also establishes a safe, simple and stable and efficient triploid induction method for H. discus, which is conducive to the sustainable development of abalone aquaculture industry. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 DNA content analysis chart of face disc larvae of H. discus obtained in Example 1 of the present application (2N: diploid, 3N: triploid).

[0027] Figure 2 DNA content analysis chart of face disc larvae of H. discus obtained in Example 4 of the present application (3N: triploid). DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be further described and explained in the following specific embodiments in conjunction with the accompanying drawings.

[0029] The method for preparing sand-filtered seawater with different salinities in the following examples refers to “Sub-low salinity impact on survival, growth and meat quality of the pacific abalone (Haliotis discus hannai) and hybrids” (Boamah GA, Wang T, Chowdhury IA, Luo X, Huang MQ, Xu CA, Ke CH, You WW. Aquaculture Research, 2020, 51:5184-5193).

[0030] Example 1

[0031] (1) The fertilization water temperature was 21.7°C. Synchronously fertilized Haliotis discus hannai eggs were taken. When the first polar body appeared in 60% of the eggs, the eggs were treated in low-salinity sand-filtered seawater with a salinity of 12 ppm for 15 min.

[0032] (2) The eggs treated in step (1) were sequentially rehydrated in sand-filtered seawater with salinities of 24-25 ppm and 30-31 ppm for 2-3 min, and then resuspended in fresh sand-filtered seawater with a normal salinity (salinity of 33.5 ppm). After conventional incubation and cultivation, Haliotis discus hannai larvae were obtained.

[0033] The Haliotis discus hannai larvae obtained in step (2) were cultured to the face disc larva stage. The ploidy was determined by flow cytometry, and the triploid induction rate was 85.47 ± 1.49%. The DNA content analysis chart is shown in Figure 1

[0034] Example 2

[0035] (1) The fertilization water temperature was 22.4°C. Synchronously fertilized Haliotis discus hannai eggs were taken. When the first polar body appeared in 60% of the eggs, the eggs were treated in high-salinity sand-filtered seawater with a salinity of 52 ppm for 15 min.

[0036] (2) The eggs treated in step (1) were sequentially rehydrated in sand-filtered seawater with salinities of 38-39 ppm and 32-33 ppm for 2-3 min and 3-4 min, and then resuspended in fresh sand-filtered seawater with a normal salinity (salinity of 32.4 ppm). After conventional incubation and cultivation, Haliotis discus hannai larvae were obtained.

[0037] The Haliotis discus hannai larvae obtained in step (2) were cultured to the face disc larva stage. The ploidy was determined by flow cytometry, and the triploid induction rate was 96.94 ± 2.74%.

[0038] Example 3 ​

[0039] (1) The fertilization water temperature is 19.5℃, and the fertilized eggs of H. discus are taken, and when the first polar body appears in 70% of the fertilized eggs, the fertilized eggs are placed in high-salinity sand-filtered seawater with a salinity of 41 ppm for 25 min;

[0040] (2) The fertilized eggs treated in step (1) are sequentially placed in sand-filtered seawater with a salinity of 38-39 ppm and 33-34 ppm for 2-3 min and 3-4 min, and then resuspended in fresh sand-filtered seawater with a normal salinity (salinity of 33.1 ppm), and after conventional hatching and cultivation, H. discus larvae are obtained.

[0041] The H. discus larvae obtained in step (2) are cultured to the face disc larva stage, the ploidy is determined by a flow cytometer, and the triploid induction rate is 93.42±4.22%.

[0042] Example 4

[0043] (1) The fertilization water temperature is 23.0℃, and the fertilized eggs of H. discus are taken, and when the first polar body appears in 60% of the fertilized eggs, the fertilized eggs are placed in high-salinity sand-filtered seawater with a salinity of 52 ppm for 10 min;

[0044] (2) The fertilized eggs treated in step (1) are sequentially placed in sand-filtered seawater with a salinity of 38-39 ppm and 32-33 ppm for 2-3 min and 4-5 min, and then resuspended in fresh sand-filtered seawater with a normal salinity (salinity of 33.5 ppm), and after conventional hatching and cultivation, H. discus larvae are obtained.

[0045] The H. discus larvae obtained in step (2) are cultured to the face disc larva stage, the ploidy is determined by a flow cytometer, and the triploid induction rate is 100.00±0.00%, and the DNA content analysis diagram is shown in Figure 2 .

[0046] The above is only a preferred embodiment of the present application, and therefore cannot limit the scope of the present application, and equivalent changes and modifications made according to the scope and content of the present patent should still be within the scope of the present application.

Claims

1. A method for inducing triploid growth in *Abalone fasciatus* with salinity, characterized in that: Includes the following steps: (1) Synchronize the fertilization of sperm and eggs of abalone with wrinkles to obtain fertilized eggs; (2) When the proportion of the first polar body of the fertilized egg obtained in step (1) is 60-70%, the fertilized egg is subjected to high-salt treatment or low-salt treatment. (3) The fertilized eggs treated in step (2) are subjected to salinity gradient sand-filtered seawater re-infiltration treatment to gradually adjust the osmotic pressure through salinity gradient sand-filtered seawater, thereby reducing the damage to the fertilized eggs. Then, they are resuspended in fresh sand-filtered seawater with normal salinity for conventional incubation and cultivation. High-salt treatment includes placing fertilized eggs in sand-filtered seawater with a salinity of 41-52 for 10-25 minutes; salinity gradient sand-filtered seawater re-infiltration treatment includes placing them first in sand-filtered seawater with a salinity of 37-39 for 2-3 minutes, and then placing them in sand-filtered seawater with a salinity of 32-34 for 2-5 minutes. Low-salt treatment includes placing fertilized eggs in sand-filtered seawater with a salinity of 12-18 for 10-25 minutes; salinity gradient sand-filtered seawater re-infiltration treatment includes placing them first in sand-filtered seawater with a salinity of 24-26 for 2-3 minutes, and then placing them in sand-filtered seawater with a salinity of 30-32 for 2-5 minutes.

2. The method as described in claim 1, characterized in that: The temperature for synchronous fertilization is 18-23.5℃.

3. The method as described in claim 1, characterized in that: The salinity of fresh sand-filtered seawater with normal salinity is 30-35.

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