Chemical Induction Method for the Triploid of Apostichopus japonicus 'Huachun No. 1'

Abstract

The present invention discloses a chemical induction method for the triploid of Apostichopus japonicus Huachun No. 1, belonging to the technical field of genetic breeding of marine echinoderms. The chemical induction method comprises the following steps: promoting the maturation of broodstock, artificially inducing ovulation or sperm ejaculation, treating fertilized eggs with staurosporine, hatching the fertilized eggs to obtain juvenile sea cucumbers, and continuing to cultivate the juvenile sea cucumbers. The induction drug used in the present invention is staurosporine, which is a reversible inhibitor related to microfilament assembly and can inhibit the emission of the first polar body of fertilized eggs. Compared with traditional induction drugs such as cytochalasin B and 6-DMAP, staurosporine has the advantages of rapid inhibitory effect, low toxicity and easy cleaning. In addition, the staurosporine induction method provided by the present invention is completely different from the previous physical induction methods, not only reducing the operation difficulty, but also improving the survival rate of larvae and the triploid yield.

Description

Technical Field

[0001] The present invention relates to a method for inducing polyploid sea cucumbers, specifically a chemical induction method for the triploid of Apostichopus japonicus "Huachun No. 1", belonging to the technical field of genetic breeding of marine echinoderms. Background Art

[0002] Apostichopus japonicus is an important mariculture species in the north of China. In 2021, the mariculture area of Apostichopus japonicus in the whole country was 3.6 million mu, with a yield of 160,000 tons, and the total industrial chain output value was 60 billion yuan. The sea cucumber production in Shandong Province ranks first in China. With less than 3% of the fishery output, the sea cucumber has created a high output value of more than 20%. Excellent germplasm is the basis for the high and stable yield of the sea cucumber industry. Cultivating high-yield, stress-resistant and high-quality improved varieties and establishing large-scale seedling breeding technology will provide a sufficient amount of high-quality seedlings for the sea cucumber aquaculture industry, increase the yield of Apostichopus japonicus, and improve economic benefits, which is of great significance for fishermen's employment, production increase and income increase.

[0003] Every year in July and August, after Apostichopus japonicus completes sperm and egg discharge, it enters the "summer sleep" state, stops feeding, and both its weight and immunity will decrease, and it is particularly sensitive to high-temperature weather. In recent years, major coastal Apostichopus japonicus aquaculture areas such as Shandong and Liaoning have generally suffered from continuous high-temperature disasters, and the phenomenon of production reduction and crop failure is serious, which has had a serious impact on Apostichopus japonicus aquaculture.

[0004] Apostichopus japonicus "Huachun No. 1" is a new variety independently developed by the team of Professor Yang Jianmin from the College of Agriculture of Ludong University (Aquatic New Variety Registration Number GS-01-014-2022). It is based on a basic population of 796 wild Apostichopus japonicus individuals collected from Kongtong Island, Haiyang, Rongcheng and Jiaonan waters from 2007 to 2008. With high-temperature tolerance and body weight as target traits, using population breeding technology, it was bred through 4 consecutive generations of selection. Under the same aquaculture conditions, compared with the unselected Apostichopus japonicus and Apostichopus japonicus "Kongtong Island No. 1", the survival rate of Apostichopus japonicus "Huachun No. 1" increased by 33.3% and 30.0% after being cultured at 32°C for 7 days at 12 months old; the survival rate increased by 49.5% and 47.0% respectively at 19 months old, and the body weight increased by 29.0% and 5.7% respectively.

[0005] The diploid of the new variety of Apostichopus japonicus "Huachun No. 1" itself has characteristics such as stronger high-temperature tolerance and shorter summer sleep time than ordinary Apostichopus japonicus. If the characteristics of poor fertility and non-overdeveloped gonads of triploids are added, the difficulty of surviving the summer will be greatly reduced, and the summer survival rate and output rate will be improved. Therefore, the triploid of the new variety of Apostichopus japonicus "Huachun No. 1" has good application prospects.

[0006] The methods for directly inducing triploids include physical methods and chemical methods. Chang Yaqing et al. proposed a method for obtaining triploid sea cucumbers by hydrostatic pressure in the patent CN1586190A (Method for inducing triploid sea cucumbers by hydrostatic pressure). Zhao Ting et al. obtained triploid sea cucumbers by inhibiting the second polar body through salinity changes in the patent CN102100198A (A method for producing triploid sea cucumber seedlings). Both of these two methods are physical methods for directly inducing triploids. Currently, chemical induction methods are widely used in the induction of triploid shellfish, but there is no report on the induction of triploid sea cucumbers. Summary of the Invention

[0007] The object of the present invention is to provide a chemical induction method for triploid sea cucumber "Huachun No. 1" with relatively low operation difficulty, high larval survival rate and high triploid yield.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A chemical induction method for triploid sea cucumber Huachun No. 1, comprising the following steps:

[0010] Step1: Promote the maturation of broodstock;

[0011] Step2: Artificially induce ovulation or sperm ejaculation in broodstock with mature gonads;

[0012] Step3: Treat the fertilized eggs with staurosporine. Specifically, mix the washed and filtered eggs and sperm together. Start timing after the sperm and eggs are mixed. Keep the water temperature at 21°C. After fertilization for 10 - 15 minutes, treat the fertilized eggs with an aqueous solution of staurosporine with a concentration of 10 nM for 25 - 30 minutes, continuously stir and aerate during this period, and then wash off the staurosporine with seawater containing 10 mM DMSO;

[0013] Step4: Hatch the fertilized eggs to obtain juvenile sea cucumbers;

[0014] Step5: Continue to cultivate the collected juvenile sea cucumbers.

[0015] Preferably, in Step1, transfer the broodstock from the outer pond culture area to the seedling rearing workshop 2 months in advance for gonad maturation promotion. The process of gonad maturation promotion is as follows: First, temporarily raise the broodstock in a pool with the same water temperature as the outer seawater for 9 days, then raise the temperature by 0.5°C every day. Stop raising the temperature when the temperature reaches 15°C, and finally make the accumulated temperature value reach more than 800°C. Gradually raise the temperature to 18°C 10 days before spawning. During this period, feed according to the feeding situation of the broodstock. The feeding amount is 4 - 10% of the body weight of the sea cucumber. The daily water exchange rate of the water body is 20 - 30%. Stop feeding the bait 3 days before spawning.

[0016] Preferably, in Step 2, the method for artificially inducing ovulation or sperm ejaculation is as follows: First, the mature gonad sea cucumbers are air-dried for 1 h and then stimulated with running water for 0.5 h. Then, the sea cucumbers are placed in a 5-L container, with only 1 sea cucumber in each container. The container is filled with sand-filtered seawater heated to 20 °C to induce spawning of the sea cucumbers. The stimulated sea cucumbers start to release sperm and eggs at night.

[0017] Preferably, in Step 3, the staurosporine aqueous solution with a concentration of 10 nM is prepared by the following method: Dissolve 100 mmol of staurosporine in 1 mL of DMSO to obtain a stock solution. Then, take 100 μL of the stock solution and dilute it with 1 L of seawater to obtain a staurosporine aqueous solution with a concentration of 10 nM.

[0018] Preferably, in Step 4, the fertilized eggs treated with staurosporine are transferred to a cement pond for hatching. The hatching conditions are as follows: Count the fertilized eggs and control the density of the fertilized eggs at 10 eggs / mL. Stir the pond every 30 min for the first 7 h and every 60 min for the next 7 h. During this period, use the weak aeration method to keep the eggs suspended. Keep the water temperature at 21 °C and the hatching time is 48 h.

[0019] Preferably, in Step 5, the cultivation conditions for juvenile sea cucumbers are as follows: Redistribute the juveniles at a density of 0.5 individuals / mL. The feed includes Dunaliella salina, Dicrateria inornata, Nitzschia closterium, and Chaetoceros. Change the water by 1 / 3 every day and control the water temperature at 21 ± 1 °C. During the attachment stage, use a polyethylene corrugated board as the attachment device and control the attachment density of juvenile sea cucumbers at 0.5 individuals / cm 2 , and the feed includes kelp powder, Sargassum thunbergii powder, Sargassum fusiforme powder, and yeast powder. Keep the water temperature at 19 - 22 °C.

[0020] The advantages of the present invention are as follows:

[0021] (1) The induction drug used in the present invention is staurosporine, which is a reversible inhibitor related to microfilament assembly and can inhibit the emission of the first polar body of fertilized eggs. Compared with traditional induction drugs such as cytochalasin B and 6-DMAP, staurosporine has the advantages of rapid inhibition, low toxicity, and easy cleaning.

[0022] (2) The staurosporine induction method provided by the present invention is completely different from the previous physical induction methods. It not only reduces the operation difficulty but also improves the survival rate of larvae and the triploid yield. Detailed implementation mode

[0023] The chemical induction method of the triploid Apostichopus japonicus "Huachun No. 1" provided by the present invention involves subjecting the diploid Apostichopus japonicus "Huachun No. 1" after gonadal maturation promotion to artificial induced ovulation and sperm ejaculation. After fertilization, staurosporine is used for induction to inhibit the emission of the first polar body of the fertilized eggs. After washing off the drug, the fertilized eggs are transferred to a cultivation container for hatching and larval cultivation. Detection of the larvae and adults by flow cytometry shows that the staurosporine induction method provided by the present invention can directly induce viable triploid Apostichopus japonicus.

[0024] The following specifically introduces the present invention in combination with specific embodiments.

[0025] Example 1

[0026] Step1: Gonadal maturation promotion of broodstock

[0027] Randomly select individuals with plump bodies, regular spine shapes, and weights in the range of 100 - 120 g from the new variety population of Apostichopus japonicus "Huachun No. 1" as broodstock.

[0028] Transfer the broodstock from the outdoor pond culture area to the seedling rearing workshop 2 months in advance for gonadal maturation promotion. The process of gonadal maturation promotion is as follows: First, acclimatize the broodstock in a pool with the same water temperature as the offshore seawater for 9 days, then raise the temperature by 0.5 °C daily. When the temperature reaches 15 °C, stop raising the temperature, and finally make the accumulated temperature value reach more than 800 °C. Gradually raise the temperature to 18 °C 10 days before spawning. During this period, feed according to the feeding situation of the broodstock, and the feeding amount is 4 - 10% of the body weight of the Apostichopus japonicus. The daily water exchange rate of the water body is 20 - 30%. Stop feeding the bait 3 days before spawning.

[0029] During the gonadal maturation promotion period, regularly check the maturity of the gonads of the broodstock by dissection. When it is found that the male gonad is milky white, the female gonad is orange - red, the color becomes darker, and the gonadosomatic index is basically above 10%, and the higher ones can reach 18%, the gonadal maturation promotion ends.

[0030] After the gonadal maturation promotion ends, perform non - lethal ploidy detection on all broodstock by flow cytometry, and the results are all diploid.

[0031] Step2: Artificial induced ovulation or sperm ejaculation

[0032] Air - dry the broodstock with mature gonads for 1 h first, then stimulate with running water for 0.5 h, and then put the broodstock into a 5 - L container. Only put 1 broodstock in each container, and add sand - filtered warmed seawater at 20 °C to the container to induce spawning of the broodstock. The above - stimulated broodstock starts to release sperm and eggs at night.

[0033] During the induction of spawning, record the time of sperm ejaculation or ovulation for each parent sea cucumber. After 0.5 h of egg or sperm discharge, examine the eggs or sperm discharged by each individual under a microscope, and exclude eggs with irregular shapes, uneven yolk colors, and small egg quantities, as well as sperm with slow activation speeds and small active quantities. At the same time, check whether the discharged eggs are contaminated or fertilized in advance, and exclude contaminated or pre-fertilized eggs.

[0034] Wash and filter all the discharged eggs and sperm separately with 500-mesh and 300-mesh silk screens to remove impurities for later use.

[0035] Step3: Treat the fertilized eggs

[0036] Mix the washed and filtered eggs and sperm together. Start timing after the sperm and egg mixture is formed. Keep the water temperature at 21 °C. After 10 min of fertilization, treat the fertilized eggs with an aqueous staurosporine solution at a concentration of 10 nM for 25 min to inhibit the emission of the first polar body of the fertilized eggs, and continuously stir and aerate during this period; then wash the fertilized eggs with seawater containing 10 mM DMSO for 5 min (the purpose is to precipitate the staurosporine in the cells), and wash continuously twice.

[0037] The preparation method of the aqueous staurosporine solution with a concentration of 10 nM is as follows: Dissolve 100 mmol of staurosporine in 1 mL of DMSO to obtain a stock solution, then take 100 μL of the stock solution and dilute it with 1 L of seawater to obtain an aqueous staurosporine solution with a concentration of 10 nM.

[0038] Step4: Incubate the fertilized eggs

[0039] Transfer the fertilized eggs treated with staurosporine to a cement pond for incubation. The specific incubation conditions are as follows: Count the fertilized eggs and control the density of the fertilized eggs at 10 eggs / mL. To prevent the eggs from sinking to the bottom, stir the pond once every 30 min for the first 7 h and once every 60 min for the next 7 h. During this period, use the weak aeration method to keep the eggs suspended. Keep the water temperature at 21 °C, and the incubation time is 48 h.

[0040] After the incubation of the fertilized eggs is completed, filter and collect the auricularia larvae with a 200-mesh silk screen, count the number of larvae, and calculate the larval survival rate as 80% by calculating the ratio of the number of auricularia larvae to the number of fertilized eggs. Then, conduct a mixed inspection of 100 auricularia larvae using a flow cytometer.

[0041] The specific method of the mixed inspection is as follows: Mix all the larvae with PBS, repeatedly pipette them, filter them with a 300-mesh silk screen, add DAPI, and the final concentration of DAPI is 0.1 μg / ml. After 20 min, conduct a flow cytometer detection.

[0042] The detection results showed that the chromosomal ploidy of the mixed larvae varied from 2N to 4N. Among them, 10% had a chromosomal ploidy of 2N, 70% had a chromosomal ploidy of 3N, 5% had a chromosomal ploidy of 4N, and 5% were aneuploid.

[0043] Step5: Cultivate juvenile sea cucumbers

[0044] Continue to cultivate the collected auricularia larvae. The specific cultivation conditions are as follows:

[0045] Redistribute the larvae at a density of 0.5 individuals / mL. The fed baits include Dunaliella salina, Dicrateria inornata, Nitzschia closterium, Chaetoceros spp., etc. Change 1 / 3 of the water every day, and control the water temperature at 21±1°C; during the attachment stage, use a polyethylene corrugated board as the attachment device, and control the attachment density of juvenile sea cucumbers at 0.5 individuals / cm 2 , and feed baits including kelp powder, Sargassum thunbergii powder, Sargassum fusiforme powder, yeast powder, etc., and keep the water temperature at 19 - 22°C.

[0046] During the cultivation of juvenile sea cucumbers, when the juvenile sea cucumbers grow to 2 - 5 mm in length, use a flow cytometer to detect 100 juvenile sea cucumber individuals.

[0047] The specific detection method is as follows: Cut each juvenile sea cucumber individual into pieces with scissors, repeatedly pipette, filter with a 300 - mesh silk screen, add DAPI, and the final concentration of DAPI is 0.1 μg / ml. After 20 min, perform flow cytometer detection.

[0048] The detection results showed that among 100 juvenile sea cucumbers, 24 had a chromosomal ploidy of 2N, 76 had a chromosomal ploidy of 3N, no tetraploid or aneuploid juvenile sea cucumbers were found, and the triploid yield was 76%.

[0049] Example 2

[0050] The operations of Step1 (ripening of broodstock) and Step2 (artificial induction of ovulation or sperm ejaculation) are exactly the same as those in Example 1 and will not be elaborated here.

[0051] Step3: Process the fertilized eggs

[0052] After the eggs and sperm are washed and filtered, fertilize the eggs with an appropriate amount of sperm, with 7 - 8 sperm around each egg being appropriate. Start timing after the sperm and eggs are mixed. The fertilization and hatching temperature is always maintained at 21°C. 15 min after fertilization, treat the fertilized eggs with a 10 nM staurosporine aqueous solution for 30 min to inhibit the release of the first polar body of the fertilized eggs. Continuously stir and aerate during this period, and then wash the fertilized eggs with seawater containing 10 mM DMSO for 20 min to remove staurosporine.

[0053] Step4: Hatch the fertilized eggs

[0054] The fertilized eggs treated with staurosporine were transferred to a cement pond for hatching. The hatching conditions were exactly the same as those in Example 1, and the hatching time was 48 h.

[0055] After the hatching of the fertilized eggs was completed, the larval survival rate was calculated, and the ploidy of the chromosomes of the mixed larval cells was detected.

[0056] After calculation, the larval survival rate was 83%. After detection, the ploidy of the chromosomes of the mixed larval cells varied from 2N to 4N. Among them, the proportion of cells with a chromosome ploidy of 2N was 15%, the proportion of cells with a chromosome ploidy of 3N was 67%, the proportion of cells with a chromosome ploidy of 4N was 3%, and the rest might be aneuploid.

[0057] Step5: Cultivate juvenile sea cucumbers

[0058] After 48 h of hatching of the fertilized eggs, juvenile sea cucumbers were obtained. The juvenile sea cucumbers were continuously cultivated, and the cultivation conditions of the juvenile sea cucumbers were exactly the same as those in Example 1.

[0059] During the cultivation of the juvenile sea cucumbers, when the juvenile sea cucumbers grew to 2 - 5 mm in length, 100 juvenile sea cucumber individuals were detected by a flow cytometer using the same method as in Example 1.

[0060] The detection results showed that among the 100 juvenile sea cucumbers, 28 juvenile sea cucumbers had a chromosome ploidy of 2N, 72 juvenile sea cucumbers had a chromosome ploidy of 3N, no tetraploid or aneuploid juvenile sea cucumbers were found, and the triploid yield was 72%.

[0061] From Example 1 and Example 2, it can be seen that the chemical induction method provided by the present invention successfully obtained viable triploid new varieties of Apostichopus japonicus "Huachun No. 1", and ensured a relatively high larval survival rate and triploid yield, which laid a foundation for the future promotion and use of triploid Apostichopus japonicus "Huachun No. 1" in production.

[0062] It should be noted that the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. Chemical induction method for the triploid of Apostichopus japonicus Huachun No. 1, characterized in that, It includes the following steps: Step1: Promote gonadal maturation of broodstock; Step2: Artificially induce ovulation or sperm ejaculation in broodstock with mature gonads; Step3: Treat the fertilized eggs with staurosporine. Specifically, wash and filter the eggs and sperm and mix them together. Start timing after the sperm and egg mixture. Keep the water temperature at 21°C. 10 - 15 minutes after fertilization, treat the fertilized eggs with an aqueous solution of staurosporine at a concentration of 10 nM for 25 - 30 minutes. Continuously stir and aerate during this period. Then wash off the staurosporine with seawater containing 10 mM DMSO; Step4: Hatch the fertilized eggs to obtain juvenile sea cucumbers; Step5: Continue to cultivate the collected juvenile sea cucumbers.

2. The chemical induction method of the triploid Apostichopus japonicus Huachun No. 1 according to claim 1, characterized in that In Step1, transfer the broodstock from the outdoor pond aquaculture area to the nursery workshop 2 months in advance for gonadal maturation promotion. The process of gonadal maturation promotion is as follows: First, acclimatize the broodstock in a pool with the same water temperature as the offshore seawater for 9 days. Then raise the temperature by 0.5°C per day. Stop raising the temperature when it reaches 15°C. Finally, make the accumulated temperature value reach over 800°C. Gradually raise the temperature to 18°C 10 days before spawning. Feed according to the feeding situation of the broodstock during this period. The feeding amount is 4 - 10% of the weight of the sea cucumber. The daily water exchange rate of the water body is 20 - 30%. Stop feeding the bait 3 days before spawning.

3. The chemical induction method of the triploid Apostichopus japonicus Huachun No. 1 according to claim 1, characterized in that, In Step2, the method of artificially inducing ovulation or sperm ejaculation is as follows: Air-dry the broodstock with mature gonads for 1 h first and then stimulate with running water for 0.5 h. Then put the broodstock into a 5 L container. Only put 1 broodstock in each container. Add pre-warmed seawater filtered through sand at 20°C to the container to induce spawning in the broodstock. The above-stimulated broodstock starts to discharge sperm and eggs at night.

4. The chemical induction method of the triploid Apostichopus japonicus Huachun No. 1 according to claim 1, characterized in that, In Step3, the aqueous solution of staurosporine at a concentration of 10 nM is prepared by the following method: Dissolve 100 mmol of staurosporine with 1 mL of DMSO to obtain the stock solution. Then take 100 μL of the stock solution and dilute it with 1 L of seawater to obtain the aqueous solution of staurosporine at a concentration of 10 nM.

5. The chemical induction method of the triploid of Apostichopus japonicus Huachun No. 1 according to claim 1, characterized in that In Step4, transfer the fertilized eggs treated with staurosporine to a cement pool for hatching. The hatching conditions are as follows: Count the fertilized eggs and control the density of the fertilized eggs at 10 eggs / mL. Stir the pool every 30 minutes in the first 7 h and every 60 minutes in the next 7 h. During this period, use the weak aeration method to keep the eggs suspended. Keep the water temperature at 21°C. The hatching time is 48 h.

6. The chemical induction method of the triploid Apostichopus japonicus Huachun No. 1 according to claim 1, characterized in that, In Step5, the cultivation conditions of the juvenile sea cucumbers are as follows: The larvae were redistributed at a density of 0.5 individuals / mL, and the feed included Dunaliella salina, Dicrateria inornata, Nitzschia closterium, and Chaetoceros spp. One-third of the water was changed daily, and the water temperature was controlled at 21±1°C. During the attachment stage, polyethylene corrugated plates were used as the attachment device, and the attachment density of juvenile sea cucumbers was controlled at 0.5 individuals / cm 2 , and the feed included kelp powder, Sargassum thunbergii powder, Sargassum carpaticum powder, and yeast powder. The water temperature was maintained at 19-22°C.