A method for preparing allopolyploid scallops

Abstract

The present invention provides a method for preparing allodiploid scallops, which is to screen for all-female individuals among the hybrid offspring of Argopecten irradians (♀) and Argopecten purpuratus (♂), fertilize the eggs of the all-female individuals with the sperm of Argopecten irradians, and cultivate the fertilized eggs to obtain allodiploid scallops; the screening is to screen for all-female individuals with a ratio of the nuclear genome derived from Argopecten irradians to the nuclear genome derived from Argopecten purpuratus in the genome higher than 1.5. The method of the present invention is different from the currently mainstream methods of inhibiting the release of polar bodies of diploid fertilized eggs by chemical reagents, salinity adjustment, cold and heat shock, etc., and the method of obtaining triploid offspring by crossing tetraploid and diploid parent scallops, and has opened up a brand-new allodiploid scallop breeding technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of shellfish genetic breeding in marine agriculture, and particularly relates to a method for preparing allogeneic triploid scallops. Background Art

[0002] Scallop farming is an important part of China's seawater aquaculture industry. From 2017 to 2020, China's annual scallop output was 1.828 - 2.008 million tons (China Fisheries Statistical Yearbook, 2018 - 2021), ranking first in the world. Cultivating excellent varieties with fast growth and high yield has always been the central goal of shellfish breeding and an urgent need to promote the healthy and sustainable development of the shellfish industry.

[0003] Triploid shellfish often have production advantages such as fast growth, large size, poor fertility, good quality, and low mortality during the breeding season. Therefore, polyploid breeding technology has attracted much attention and become a research hotspot in shellfish genetic breeding. Currently, methods for inducing polyploid shellfish mostly use chemical inducers such as cytochalasin B (CB) (Stanley et al., 1980; Guo et al., 1994; Li Yongren, 2006), 6-dimethylaminopurine (6-DMAP) (Tian Chuanyuan et al., 1998; Dong Yinghui et al., 2007), salinity regulation (Wang Zhaoping et al., 2009; Zhang Chenchen et al., 2010), heat and cold shock (Quillet E and Panelay P J, 1986), etc. to inhibit the release of polar bodies from diploid fertilized eggs to obtain triploid offspring. Although the induction effects of CB and 6-DMAP are good, the inducers are expensive and highly toxic, which reduces the hatching rate of fertilized eggs and the survival rate of larvae, and the safety of the inducers also needs to be verified; while physical induction methods such as salinity regulation and heat and cold shock also mostly have defects such as low hatching rate or unstable ploidy rate, making large-scale application difficult. Therefore, there is an urgent need in the shellfish industry for a clean, efficient, and large-scale operation suitable triploid direct induction method.

[0004] Since the development of shellfish polyploid induction breeding technology to date, obtaining all-triploid shellfish often requires an indirect method of inducing diploids to become tetraploids and then hybridizing with diploids (Piferrer et al., 2009). Moreover, traditional induction methods still have problems such as low ploidy rate and poor operation stability.

[0005] References:

[0006] Guo X, Allen S K Jr. Viable tetraploids in the Pacific oyster, Crasostrea gigas (Thunberg), produced by inhibiting polar body I in eggs from triploids. Mol Mar Biol Biotechnol, 1994, 3: 42–50.

[0007] Piferrer F, Beaumont A, Jean-Claude Falguière, et al. Polyploid fish and shellfish: Production, biology and applications to aquaculture for performance improvement and genetic containment. Aquaculture, 2009, 293(3–4): 125–156.

[0008] Quillet E, Panelay P J. Triploidy induction by thermal shocks in the Pacific oyster Crasostrea gigas. Aquaculture, 1986, 57: 271–279.

[0009] Stanley J G, Allen S K Jr, Hidu H. Polyploidy induced in the American oyster Crasostrea virginica with Cytochalasin B. Aquaculture, 1981, 23: 1–10.

[0010] Dong Y H, Yang A G, Liu Z H, Zhou L Q. Cytological observation on 6-DMAP-induced triploid of Chlamys farreri. Marine Fisheries Research, 2007, 28(2): 71–75.

[0011] Li Y R. Comparison of the growth of D larvae between diploid, CB-induced triploid and tetraploid groups of Chlamys farreri. Journal of Tianjin Agricultural College, 2006, 13(3): 1–4.

[0012] Tian Chuanyuan, Liang Ying, Wang Rucai, et al. Relationship between hatching rate, D-larvae malformation rate and triploid induction rate of 6-DMAP-induced triploid Pacific oyster. Journal of Ocean University of Qingdao, 1998, 28(3): 421–425.

[0013] Wang Zhaoping, Zhao Ting, Yu Ruihai, et al. Study on a new method - hypotonic induction of triploid Yesso scallop. Journal of Ocean University of China: Natural Science Edition, 2009, 39(2): 193–196.

[0014] Zhang Chenchen, Wang Zhaoping, Yu Ruihai, et al. Hypotonic induction of triploid Chlamys farreri and comparison with other methods.

[0015] Journal of Ocean University of China: Natural Science Edition, 2010, 40(Suppl.): 71–75.

[0016] China Fishery Statistical Yearbook. China Agriculture Press, 2018.

[0017] China Fishery Statistical Yearbook. China Agriculture Press, 2019.

[0018] China Fishery Statistical Yearbook. China Agriculture Press, 2020.

[0019] China Fishery Statistical Yearbook. China Agriculture Press, 2021. Summary of the Invention

[0020] The object of the present invention is to provide a method for preparing allogynogenetic triploid scallops, which can solve the defects existing in the preparation process of triploid shellfish at the present stage, such as the high price and large toxicity of chemical inducers, the cumbersome operation of physical induction methods, the low hatching rate of triploid shellfish and the instability of ploidy rate.

[0021] The method for preparing allogynogenetic triploid scallops provided by the present invention is to screen all-female individuals among the hybrid offspring of bay scallop (♀) and purple scallop fertilize the eggs of the all-female individuals with the sperm of bay scallops, and cultivate the fertilized eggs to obtain allogynogenetic triploid scallops;

[0022] The screening is to screen all-female individuals with a ratio of the nuclear genome derived from bay scallop to the nuclear genome derived from purple scallop in the genome higher than 1.5;

[0023] Furthermore, the eggs of the all-female individuals are matured by the accumulated temperature ripening method. The accumulated temperature ripening method is to cultivate the selected all-female individuals at a constant temperature of 8 °C until the gonads begin to swell, then raise the temperature by 0.5 °C every day. When the ripening water temperature rises to 21 °C and the gonads are nearly mature, keep the temperature constant for 5–8 days, and gradually increase the bait feeding until the gonads of the parent scallops are full;

[0024] As a specific description of the embodiment, the eggs of the all-female individuals are fertilized with the sperm of the bay scallop, where the seawater temperature is 24°C;

[0025] For the cultivation of the fertilized eggs, one condition is that the seawater temperature is controlled at 24 ± 1°C, the salinity is controlled at 25–35 ppt, and the allogynogenetic triploid scallop D-shaped larvae can be obtained after about 24 hours.

[0026] This invention is different from the traditional scallop triploid induction methods, and there are the following innovative points:

[0027] 1) Different methods: Traditional methods mostly use chemical inducers such as CB and 6-DMAP, and physical methods such as hypotonic, cold and heat temperature shock, and hydrostatic pressure are used to treat the fertilized eggs to inhibit the emission of polar bodies, indirectly obtaining triploid offspring; while in this invention, the all-female individuals (diploid) of the offspring of bay scallop (♀) × purple scallop are screened to lay eggs, and are naturally fertilized with the sperm of bay scallop (diploid).

[0028] 2) Different environmental safety and operation feasibility: Traditional chemical inducers are expensive and highly toxic, and the environmental safety of the inducers remains to be verified; physical induction methods such as salinity adjustment and cold and heat shock are cumbersome to operate and difficult to apply on a large scale. While the method of this invention is clean, efficient, and simple to operate, and is suitable for industrial scale seed production.

[0029] 3) Different triploid induction rates: The triploid induction rate of traditional methods is 40–90%, and aneuploids often appear; while the triploid rate of this invention is stable at 100%, and no diploids, tetraploids, or aneuploids appear.

[0030] 4) Different chromosomal compositions of triploid offspring: The triploids obtained by traditional methods are autotriploids of the same species. While the triploids obtained in this invention are allogynogenetic triploids containing the chromosomal groups of bay scallop and purple scallop.

[0031] 5) On the basis of traditional backcross breeding, the temperature conditions for promoting the ripening of the accumulated temperature of the offspring of bay scallop (♀) × purple scallop are optimized, and at the same time, female parents are screened according to the ratio of the nuclear genomes of bay scallop origin and purple scallop origin in the all-female individuals of the hybrid offspring (bay scallop / purple scallop > 1.5 times).

[0032] This invention creates a new breeding method for preparing allogynogenetic triploid scallops through technical links such as screening the all-female individuals (diploid) of the offspring of bay scallop (♀) × purple scallop to lay eggs and naturally fertilizing them with the sperm of bay scallop (diploid). This invention utilizes bay scallop (♀) × purple scallop ​The all-female individuals in the offspring can form diploid eggs, which is different from the current mainstream methods of inhibiting the release of diploid fertilized egg polar bodies through chemical reagents (cytochalasin B, 6-dimethylaminopurine, etc.), salinity regulation, cold and heat shock, and obtaining triploid offspring by hybridization of tetraploid and diploid parent shellfish. It has opened up a new allotriploid scallop breeding technology. The method of the present invention provides feasibility for the future production of 100% scallop triploids in a clean, efficient and large-scale manner, and also provides certain technical support for the sustainable and healthy development of my country's scallop industry and the industrialization of improved varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :Bay scallop (♀)×purple scallop The ratio (a) and proportion (b) of the two sets of nuclear genomes in the offspring all-female individuals (diploid); the black rectangular frame (individuals 15–26) is the all-female parents screened for the preparation of triploid offspring;

[0034] Figure 2 :Flow cytometric detection results of D-shaped larvae of common diploid bay scallop (a) and allotriploid scallop (b);

[0035] Figure 3 :Bay scallop (a), Bay scallop (♀) × Purple scallop Flow cytometry test results of all-female offspring (b) and backcross triploid scallop adults (cl);

[0036] Figure 4 : The bar graph of the triploid detection rate of offspring obtained by this method and the traditional backcrossing method. DETAILED DESCRIPTION

[0037] Bay scallops and purple scallops are hermaphrodites, with gonads confined to the abdomen. The male area (testes) is located on the outer periphery of the abdomen and is milky white when mature; the female area (ovaries) is located inside the male area and is pink / orange-red when mature. There is usually a layer of black film on the surface of the gonad area. As the gonads gradually mature, the black film gradually fades, and the male and female areas can be clearly distinguished. Most of the offspring were of diapause type (the gonads were transparent or undeveloped), but there were a small number of all-female individuals (the entire gonads were pink / orange-red).

[0038] Bay scallop (♀) × purple scallop The identification method of all-female offspring (diploid) is as follows:

[0039] Bay scallop (♀) × purple scallop Select female individuals with all-female gonads in the offspring. Take a small amount of tissue samples in the living state and determine their ploidy by flow cytometry. The specific operations are as follows: Use sterilized forceps to pick 2-3 gill filaments, and use sterilized surgical scissors to cut them into small pieces in 1 mL of 1×PBS. Then filter through a cell sieve with a pore size of 50 μm. Add ice-cold ethanol (-20 °C) to the filtered cell suspension to fix the cells. Centrifuge the fixed cells (300 g, 5 minutes). Resuspend the cells obtained by centrifugation in 1 mL of 1×PBS, and then treat them with RNase (50 μg / mL, 20 μL) and stain them with PI (1 mg / mL, 50 μL) in the dark for 30 minutes. The cells treated as above can be used for ploidy determination by flow cytometry (using ordinary diploid bay scallops as the control group).

[0040] To improve the yield of triploid offspring, for bay scallop (♀) × purple scallop Screen the all-female parent individuals (diploid) of the offspring, and select all-female individuals with a ratio of the nuclear genome derived from bay scallop to the nuclear genome derived from purple scallop in their genomes higher than 1.5. The specific method is as follows:

[0041] Extract the DNA of the female parent of the offspring that has passed the ploidy identification by flow cytometry through a non-lethal method, construct a re-sequencing library and perform high-throughput sequencing on the Illunima HiSeq sequencing platform. After obtaining the sequencing data, count the ratio of the reads of the nuclear genomes derived from bay scallop and purple scallop in the genome of each all-female individual of the hybrid offspring to be screened, and select all-female individuals of the hybrid offspring with a ratio of the reads of the two sets of nuclear genomes of bay scallop (reads ratio > 60%) and purple scallop (reads ratio < 40%) higher than 1.5 times as the female parent for subsequent backcrossing.

[0042] Preferably, an artificial accumulated temperature ripening method is used to promote the gonadal development of the selected all-female individuals of the hybrid offspring.

[0043] After promoting the gonadal development of the selected all-female individuals and ordinary bay scallops, stimulate them to spawn / sperm. One method is to use the method of drying and temperature change stimulation to stimulate the parent scallops to spawn / sperm: Before spawning induction, dry the parent scallops for 30-40 minutes, and then place them in seawater with a temperature increase of 3-6 °C. After the parent scallops are stimulated by drying and then put into seawater at 24 °C, they are active frequently in the first 20-30 minutes, mainly ejaculating sperm; then they ovulate successively, and can lay eggs 4-5 times within 15-20 minutes, exhausting the mature eggs in the female area. The eggs / fertilized eggs can be filtered out with a 500-mesh (pore size 20 μm) silk screen to obtain the sperm of the parent scallops.

[0044] The hybrid progeny parent scallops are cultured at a constant temperature of 8°C for 15 days until the gonads begin to swell, and then the water temperature is increased by 0.5°C per day. When the water temperature for ripening promotion reaches 21°C and the gonads are nearly mature, keep it at a constant temperature for 5 - 8 days, and gradually increase the bait feeding until the gonads of the parent scallops are full. The effective accumulated temperature reaches 290.5 - 333.7 °C·d;

[0045] Then, the sperm filtered from the sperm discharge pool is added to the spawning pool containing seawater at 24°C, and 3 - 5 sperm are controlled around each egg, and the egg density is controlled at 50 - 80 eggs / mL. After fertilization, the heterologous triploid scallop D-shaped larvae are obtained by hatching the fertilized eggs in seawater at 24°C.

[0046] The ploidy of the obtained triploid scallop progeny is detected by flow cytometry.

[0047] In the method of the present invention, the seawater temperature for stimulating the spawning, sperm discharge, fertilization and early hatching of bay scallops is 24 ± 1°C; the cultivation methods of D-shaped and umbo larvae can adopt conventional methods, not limited to the specific records of the embodiments.

[0048] The method of the present invention does not use chemical inducers such as CB and 6-DMAP, and physical methods such as hypotonic, cold and heat temperature shock and hydrostatic pressure to treat fertilized eggs.

[0049] The present invention will be described below in conjunction with embodiments and drawings.

[0050] Example 1

[0051] 1) Obtain the bay scallop (♀) × purple scallop that has been identified and screened The eggs of all-female individuals (diploid) in the progeny

[0052] In early March 2021, at Yantai Haiyi Seedling Co., Ltd. in Shandong Province, the bay scallop (♀) × purple scallop with accumulated temperature for ripening promotion From the progeny population, 52 all-female individuals with all-female gonad type, regular gonad morphology, full development, and pink / orange-red color were selected; the above 52 hybrid progeny individuals of Haima were identified as diploids by flow cytometry; then, by a non-lethal method, 2-3 gill filaments of randomly selected 26 individuals were picked with sterilized forceps and their genomic DNA was extracted, 26 resequencing libraries were constructed and high-throughput sequencing was performed on the Illunima HiSeq sequencing platform; after obtaining the sequencing data, the proportions of nuclear genome reads from Argopecten irradians and Argopecten purpuratus in the genome of each all-female hybrid progeny individual to be screened were counted. The results showed that: in the screening group, the proportions of the two sets of nuclear genome reads of Argopecten irradians (reads proportion 64.08 - 65.81%) and Argopecten purpuratus (reads proportion 33.57 - 35.80%) in 12 all-female individuals were 1.79 - 1.96 (>1.5 times), which was significantly higher than the proportions of the nuclear genome reads of Argopecten irradians and Argopecten purpuratus in the genomes of the other 14 all-female individuals, which were 1.04 - 1.18 (<1.5 times).

[0053] The 12 selected all-female individuals of the hybrid progeny were used as the female parents for subsequent backcrossing ( Figure 1 ). Using the method of air-drying (30 minutes) and temperature-raising (raising 3 - 6°C) stimulation, the above female individuals were made to lay eggs normally in seawater at 24°C (spawning pond, 1 m 3 ). The egg cytoplasm was full and the shape was round, and it was microscopically confirmed that they were not fertilized.

[0054] 2) Obtain sperm of Argopecten irradians (diploid)

[0055] 50 Argopecten irradians (diploid) individuals with the same accumulated temperature and promoted ripening as the female parent were selected. Their gonad type was hermaphrodite, the gonad morphology was regular and fully developed, the color of the female area was pink / orange-red, and the color of the male area was milky white; using the same method of air-drying and temperature-raising stimulation as in step 1), the above Argopecten irradians individuals were made to lay eggs and release sperm normally in seawater at 24°C (sperm discharge pond). The eggs / fertilized eggs of the above Argopecten irradians were filtered out with a 500-mesh silk screen to obtain their sperm; microscopically examined again to ensure that ≥90% of the sperm were active and there was no contamination of the eggs of Argopecten irradians.

[0056] 3) Fertilization:

[0057] The sperm filtered from the sperm discharge pond was added to the spawning pond, controlling 3 - 5 sperm around each egg, and the egg density was 53 ± 3 per mL; after fertilization, the fertilized eggs hatched in normal seawater. During this period, the seawater temperature was 24.2°C and the salinity was 27.2 ppt. After 24 h, the D-shaped larvae of the allopolyploid scallops could be obtained.

[0058] 4) Offspring cultivation: The fertilized eggs of the allotriploid scallop can hatch normally, with a hatching rate of 17.84%. The all-triploid scallop offspring can be obtained according to the conventional cultivation method of bay scallops.

[0059] Detecting the ploidy of offspring: In March 2021, for the D-shaped larvae (n>1000) of the triploid scallop offspring obtained in step 3), ploidy detection was carried out by flow cytometry, and it was found that all the detected individuals were triploid ( Figure 2 ).

[0060] From Figure 2 it can be seen that compared with the D-shaped larvae of ordinary diploid bay scallops, the D-shaped larvae of the allotriploid scallop obtained by the method of the present invention are triploid, and no diploid or aneuploid is produced.

[0061] Example 2

[0062] 1) Obtaining the eggs of all-female individuals (diploid) in the offspring of bay scallop (♀) × purple scallop :

[0063] In late March 2021, in Leping Aquaculture, Laizhou, Yantai, Shandong Province, among the offspring population of bay scallop (♀) × purple scallop promoted by accumulated temperature, 61 individuals with gonad types of all-female, regular gonad morphology, plump development, and pink / orange-red color were selected as female individuals; after being identified by flow cytometry, all 61 individuals of the hybrid offspring of sea and purple scallops were diploid, and they were randomly divided into two groups: a screening group (n = 31) and an unscreened group (n = 30); then by a non-lethal method, that is, using sterilized forceps to pick 2-3 gill filaments of 31 individuals in the screening group in the living state and extract their genomic DNA, 31 resequencing libraries were constructed and high-throughput sequencing was carried out through the Illunima HiSeq sequencing platform; after obtaining the sequencing data, the proportions of nuclear genomic reads from bay scallop and purple scallop in the genome of each all-female individual of the hybrid offspring to be screened were statistically analyzed. The results showed that: in the screening group, the proportions of two sets of nuclear genomic reads of bay scallop (reads proportion 64.12-65.91%) and purple scallop (reads proportion 33.52-35.73%) in the genomes of 15 all-female individuals were 1.79-1.97 (>1.5 times), which was significantly higher than the proportions of nuclear genomic reads from bay scallop and purple scallop in the genomes of the other 16 all-female individuals, which were 1.02-1.16 (<1.5 times). That is, the above 15 all-female individuals of the hybrid offspring were screened as the female parents for subsequent backcrossing. The 30 all-female individuals in the unscreened group were used as controls and also carried out backcrossing experiments at the same time. By using the method of stimulating with air drying (30 minutes) and temperature rising (temperature rising 3-6°C), the above female individuals were placed in seawater at 24°C (spawning pond, 1m 3) spawned normally. The eggs were plump in egg cytoplasm and round in shape, and it was confirmed by microscopic examination that they were unfertilized.

[0064] 2) Obtain sperm of bay scallops (diploid):

[0065] Select 38 bay scallops (diploid) with the same accumulated temperature and promoted ripening as the female parent. Their gonad type is hermaphrodite, the gonad morphology is regular and well-developed, the color of the female area is pink / orange-red, and the color of the male area is milky white; use the same method of drying in the shade and raising the temperature stimulation as in step 1) to make the above bay scallop individuals spawn and ejaculate normally in seawater at 24 °C (sperm ejaculation pool). Use a 500-mesh silk screen to filter out the eggs of the above bay scallops and obtain their sperm; conduct microscopic examination again to ensure that ≥90% of the sperm are active and there is no contamination of bay scallop eggs.

[0066] 3) Fertilization:

[0067] Add the sperm filtered from the sperm ejaculation pool to the spawning pool, control 3 - 5 sperm around each egg, and the egg density is 65 ± 4 per mL; after fertilization, the fertilized eggs are hatched in normal seawater. During this period, the seawater temperature is 24.5 °C and the salinity is 27.3 ppt. After 24 hours, D-shaped larvae of allopolyploid scallops can be obtained.

[0068] 4) Offspring cultivation: The fertilized eggs of allopolyploid scallops can be hatched normally, and the hatching rate is 15.67%. The whole triploid scallop offspring can be obtained according to the conventional cultivation method of bay scallops.

[0069] Detection of offspring ploidy: In August 2021, for the triploid scallop offspring obtained in step 3), 10 individuals were randomly sampled and their ploidy was detected by flow cytometry. It was found that all the detected individuals were triploid, and no diploid or aneuploid individuals were produced ( Figure 3 ).

[0070] From Figure 3 It can be seen that compared with the all-female individuals (diploid) of the offspring of bay scallop (♀) × purple scallop and ordinary diploid bay scallops (diploid), all the scallop adults obtained by the method of the present invention are triploid (triploid rate 100%), and no diploid or aneuploid individuals are produced. In contrast, among the eggs of all-female individuals in the hybrid offspring that have not been screened by this method, after being fertilized with the sperm of bay scallops, the triploid detection rate of the backcross offspring adults is only 12.38 ± 3.97%, which is extremely significant (p < 0.01) compared with 100% in this method ( Figure 4 ).

[0071] In summary, the present invention provides a new method for preparing allogeneic triploid scallops, which has the advantages of being feasible, repeatable, and applicable. It provides the feasibility for the future production of 100% scallop triploids using a clean, efficient, and large-scale applicable backcross technology, and also provides certain technical support for the sustainable and healthy development of the scallop industry in China and the industrialization of improved varieties.

Claims

1. A method for preparing allogynogenetic triploid scallops, characterized in that, the method is to screen for all-female individuals among the hybrid offspring of bay scallop eggs and purple scallop sperm, fertilize the eggs of the all-female individuals with the sperm of bay scallops, and cultivate the fertilized eggs to obtain allogynogenetic triploid scallops; the screening is to screen for all-female individuals with a ratio of the nuclear genome derived from bay scallops to the nuclear genome derived from purple scallops in the genome higher than 1.5; that is, all-female individuals with a bay scallop reads ratio > 60% and a purple scallop reads ratio < 40%.

2. The method according to claim 1, characterized in that, the eggs of the all-female individuals are matured by the method of accumulated temperature promotion.

3. The method according to claim 2, characterized in that, the method of accumulated temperature promotion is to cultivate the screened all-female individuals at a constant temperature of 8 °C until the gonads begin to swell, and then raise the temperature to 21 °C every day until the promotion water temperature reaches 21 °C; keep the temperature constant for 5 - 8 days when the gonads are approaching maturity, and gradually increase the bait feeding until the gonads of the parent scallops are full.

4. The method according to claim 3, characterized in that, the daily temperature increase is 0.5 °C.

5. The method according to claim 1, characterized in that, the eggs of the all-female individuals are made to lay eggs in seawater by the method of air drying and temperature increase stimulation.

6. The method according to claim 5, characterized in that, the air drying time is more than 30 min.

7. The method according to claim 5, characterized in that, the temperature increase is 3 - 6 °C.

8. The method according to claim 1, characterized in that, when fertilizing the eggs of the all-female individuals with the sperm of bay scallops, the seawater temperature is 24 °C.

9. The method according to claim 1, characterized in that, when cultivating the fertilized eggs, the seawater temperature is controlled at 24 ± 1 °C and the salinity is controlled at 25 - 35 ppt.

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