A method for improving the attachment rate and survival rate of sea urchin larvae

Abstract

The present application relates to the field of aquaculture technology, and more particularly to a method for improving the attachment rate and survival rate of sea urchin larvae, which comprises the following steps: culturing sea urchin fertilized eggs in a culture water body; when the larvae develop to the late prism larvae stage, synchronously adding a chemical induction system and an attachment substrate material to the culture water body; and promoting the sea urchin larvae to complete stable attachment and metamorphic development by maintaining a chemical microenvironment during the attachment and post-attachment stages. In the present application, the chemical induction system and the physical attachment substrate are synchronously added and synergistically act, effectively overcoming the defects of scattered induction signals and unstable microenvironment; the microcapsules can realize the directional slow release of induction components such as gamma-aminobutyric acid, ions and amino acids on the surface area of the attachment base, thereby establishing and maintaining a long-term, stable and concentration-suitable high-efficiency induction microenvironment around the larvae, and realizing the synchronous and synergistic improvement of the attachment rate and survival rate of the sea urchin larvae.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and more specifically, to a method for improving the attachment rate and survival rate of sea urchin larvae. Background Technology

[0002] As an important marine economic species, the attachment and survival rate of sea urchin larvae directly affect the yield and quality of aquaculture. Sea urchin larvae typically attach after reaching a certain developmental stage, but due to factors such as the aquatic environment, attachment substrate, and chemical signals, their attachment and survival rates are often lower than expected. Traditional sea urchin farming methods rely on biological attachment substrates and microbial biofilms in the natural aquatic environment to promote larval attachment. Chinese patent CN113349116A discloses a method to improve the survival rate of purple sea urchin larvae. This method achieves a high survival rate by selecting high-quality eggs for fertilization, setting up the nursery water, selecting superior larvae, adjusting the density of purple sea urchin larvae, and controlling the amount and frequency of feeding, as well as checking for contamination before feeding. Furthermore, it eliminates the need for water changes during the entire nursery process, saving nursery costs and time, and achieving better nursery results.

[0003] However, while direct addition of inducers can increase the active attachment rate of larvae in the short term, the inducers are difficult to form and maintain a long-term, stable, and effective inducing microenvironment on the surface of the substrate. This often leads to problems such as asynchronous attachment, unstable attachment, or failure of metamorphosis and low survival rate due to unsuitable microenvironment after attachment. In view of this, we propose a culture method to improve the attachment rate and survival rate of sea urchin larvae. Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the attachment rate and survival rate of sea urchin larvae in aquaculture, in order to solve the problems mentioned in the background art. Although the direct addition of inducers can improve the active attachment rate of larvae in the short term, the inducers are difficult to form and maintain a long-term, stable and effective inducing microenvironment on the surface of the substrate. As a result, the larvae often attach asynchronously, attach unstable, or fail to metamorphose and have low survival rates due to unsuitable microenvironment after attachment.

[0005] This invention provides a method for improving the attachment rate and survival rate of sea urchin larvae, comprising the following steps: S1.1, cultivating sea urchin fertilized eggs in aquaculture water.

[0006] S1.2 When the larvae develop to the late stage of prism larvae, add 0.1-1.0 g / L of chemical induction system and 50-200 mg / L of substrate material to the aquaculture water at the same time.

[0007] The chemical induction system consists of γ-aminobutyric acid and Ca2+. 2+ / Mg 2+Chitosan-alginate composite microcapsules containing free amino acids have a particle size of 20-80 μm.

[0008] The substrate material is amino-modified polylactic acid / calcium carbonate composite particles.

[0009] S1.3. By maintaining the chemical microenvironment during and after attachment, sea urchin larvae can be promoted to complete stable attachment and metamorphosis.

[0010] Preferably, in step S1.1, the physicochemical environment of the aquaculture water includes: water temperature of 18-22℃, pH of 8.0-8.5, dissolved oxygen concentration of 5-8mg / L, salinity of 30-35‰, and water flow velocity of 0.1-0.5m / s.

[0011] As a preferred embodiment, the preparation method of the chitosan-alginate composite microcapsules is as follows: sodium alginate and chitosan are mixed at a mass ratio of 2-5:1 and dissolved in deionized water at a mass ratio of 1:5 to obtain a mixed solution.

[0012] γ-aminobutyric acid was dissolved in deionized water to obtain a concentration of 1×10⁻⁶. -6 -10 -4 A mol / L solution of γ-aminobutyric acid.

[0013] Calcium chloride and magnesium chloride are dissolved in deionized water to obtain a calcium-magnesium ion mixed solution with a concentration of 0.05-0.10 mol / L.

[0014] Glycine, alanine, glutamic acid, and aspartic acid were mixed in a mass ratio of 1:1:1:1 and dissolved in deionized water to obtain a free amino acid solution with a concentration of 5-10 g / L.

[0015] A solution of γ-aminobutyric acid, a mixed solution of calcium and magnesium ions, and a solution of free amino acids were added sequentially to the mixed solution and stirred until homogeneous to form a microcapsule core material mixture.

[0016] Add 0.1-0.2% by weight of nano-bentonite to the microcapsule core material mixture and disperse it evenly to obtain the microcapsule mixture.

[0017] The microcapsule mixture was added dropwise to a calcium chloride solution with a concentration of 0.5-1.0 mol / L to form microcapsules; the microcapsules were collected, washed with deionized water, and then freeze-dried at -40℃ to -60℃ under vacuum for 12-24 h to obtain chitosan-alginate composite microcapsules.

[0018] Nano-bentonite was pre-stirred in a 1 mol / L calcium chloride solution at 60℃ for 2 hours and then washed to complete calcium ion pre-exchange. This treatment helps to promote the diffusion and exchange of calcium ions between the bentonite layers, enabling it to cross-link more effectively with sodium alginate during the subsequent microcapsule formation process, thereby enhancing the mechanical strength and sustained-release performance of the microcapsule wall.

[0019] Preferably, in the calcium-magnesium ion mixed solution, the molar ratio of calcium ions to magnesium ions is 1-3:1.

[0020] Preferably, the mass ratio of the γ-aminobutyric acid solution to the mixed solution is 0.1-1.0%.

[0021] The mass ratio of calcium and magnesium ion mixed solution to mixed solution is 0.5-2.0%.

[0022] The mass ratio of free amino acid solution to mixed solution is 1-5%.

[0023] Preferably, in S1.2, the preparation method of aminated polylactic acid / calcium carbonate composite particles is as follows: polylactic acid and calcium carbonate are melt-blended at a mass ratio of 100:15-30, and supercritical carbon dioxide is injected under conditions of 180-200℃ and 10-15MPa. After foaming treatment for 30-60s, the pressure is quickly released to obtain porous particles.

[0024] Porous particles were immersed in an ethanol solution of 3-aminopropyltriethoxysilane and reacted at room temperature for 3-5 hours. After the reaction was completed, the product was removed, washed with ethanol, and then freeze-dried at -40°C to -60°C under vacuum for 12-24 hours to obtain aminated polylactic acid / calcium carbonate composite particles.

[0025] Preferably, the mass concentration of the 3-aminopropyltriethoxysilane solution is 2-5%.

[0026] Preferably, the mass ratio of the porous particles to 3-aminopropyltriethoxysilane is 0.5-1.5.

[0027] Preferably, in step S1.3, maintaining the chemical microenvironment involves adjusting the pH of the aquaculture water by adding sodium bicarbonate and sodium carbonate in a mass ratio of 5-10:1, thereby maintaining the pH of the water at 8.1-8.3.

[0028] Preferably, the total dosage of sodium bicarbonate and sodium carbonate is 0.1-0.5 g / L.

[0029] In closed or semi-closed aquaculture systems, the chemical microenvironment of the aquaculture water tends to become acidic due to carbon dioxide produced by the respiration of larvae and microorganisms, organic acids produced by the decomposition of organic matter, and alkalinity consumed by nitrification. The pH is prone to fluctuation and decrease, which affects the stability and metamorphic development of larvae after attachment.

[0030] Compared with existing technologies, the beneficial effects of this invention are as follows: In the aquaculture method for improving the attachment rate and survival rate of sea urchin larvae, this invention effectively overcomes the defects of dispersed induction signals and unstable microenvironment by simultaneously adding and synergistically acting a chemical induction system (chitosan-alginate composite microcapsules) and a physical attachment substrate (aminolactic acid / calcium carbonate composite particles). The microcapsules can achieve targeted and sustained release of inducing components such as γ-aminobutyric acid, ions, and amino acids on the surface of the attachment substrate, thereby establishing and maintaining a long-term, stable, and appropriately concentrated highly efficient induction microenvironment around the larvae. This not only improves the synchronicity and stability of the larval attachment behavior, but also provides a guarantee for the smooth metamorphosis of the larvae after attachment through continuous signal support and a stable pH maintained by the buffer system, ultimately achieving a synchronous and synergistic improvement in the attachment rate and later survival rate of sea urchin larvae. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] γ-aminobutyric acid (CAS No.: 56-12-2, purity: BR, 99%), magnesium chloride (CAS No.: 7786-30-3, purity: 99.9%), aspartic acid (CAS No.: 6899-03-2, purity: 98%), sodium bicarbonate (CAS No.: 144-55-8, purity: AR, 99.8%), and sodium carbonate (CAS No.: 497-19-8, purity: AR, 99.8%) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0033] Calcium chloride (CAS No.: 10043-52-4), alanine (CAS No.: 56-41-7, purity: 99.88%), calcium carbonate (CAS No.: 471-34-1, purity: 99%), and 3-aminopropyltriethoxysilane (CAS No.: 919-30-2) were all purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd.

[0034] Glycine (CAS No.: 56-40-6) was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0035] Glutamic acid (CAS No.: 56-86-0, purity: HPLC ≥ 98%) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0036] Sodium alginate (CAS No.: 9005-38-3, food grade) was purchased from Shenzhen Jinfuyuan Biotechnology Co., Ltd.

[0037] Chitosan (CAS No.: 9012-76-4, degree of deacetylation ≥90%) was purchased from Shandong Haiyihua Biotechnology Co., Ltd.

[0038] Nano-bentonite (cation exchange capacity (CEC) of 95 meq / 100g, average particle size D50 of 20μm) was purchased from Guangzhou Yifeng Chemical Technology Co., Ltd.

[0039] Polylactic acid (CAS No.: 26100-51-6) was purchased from Chengdu Future Elements Biomaterials Co., Ltd. Example 1

[0040] A method for improving the attachment rate and survival rate of sea urchin larvae includes the following steps: S1.1, cultivating sea urchin fertilized eggs in aquaculture water.

[0041] The physicochemical environment of the aquaculture water was as follows: water temperature 20℃, pH 8.3, dissolved oxygen concentration 6mg / L, salinity 32‰, and water flow velocity 0.3m / s.

[0042] S1.2 When the larvae develop to the late stage of prism larvae, 0.5 g / L of chemical induction system (chitosan-alginate composite microcapsules with a particle size of 50 μm) and 120 mg / L of substrate material (amino-lactic acid / calcium carbonate composite particles) are simultaneously added to the culture water.

[0043] S1.3 During the attachment and post-attachment stages, the pH of the water is adjusted by adding sodium bicarbonate and sodium carbonate in a mass ratio of 5:1 to maintain the pH at 8.2, and the total dosage of sodium bicarbonate and sodium carbonate is 0.3 g / L, in order to promote the stable attachment and metamorphosis of sea urchin larvae.

[0044] The preparation method of chitosan-alginate composite microcapsules is as follows: Sodium alginate and chitosan are mixed at a mass ratio of 4:1 and dissolved in deionized water at a mass ratio of 1:5 to obtain a mixed solution.

[0045] γ-aminobutyric acid was dissolved in deionized water to obtain a concentration of 1×10⁻⁶. -5 A mol / L solution of γ-aminobutyric acid.

[0046] Calcium chloride and magnesium chloride were dissolved in deionized water to obtain a calcium-magnesium ion mixed solution with a concentration of 0.07 mol / L (the molar ratio of calcium ions to magnesium ions was 2:1).

[0047] Glycine, alanine, glutamic acid, and aspartic acid were mixed in a mass ratio of 1:1:1:1 and dissolved in deionized water to obtain a free amino acid solution with a concentration of 8 g / L.

[0048] A solution of γ-aminobutyric acid, a mixed solution of calcium and magnesium ions, and a solution of free amino acids were added sequentially to the mixed solution and stirred until homogeneous to form a microcapsule core material mixture.

[0049] The mass ratio of γ-aminobutyric acid solution to mixed solution is 0.5%; the mass ratio of calcium and magnesium ion mixed solution to mixed solution is 1.2%; and the mass ratio of free amino acid solution to mixed solution is 3%.

[0050] Add 0.15% by mass of nano-bentonite to the microcapsule core material mixture and disperse it evenly to obtain the microcapsule mixture.

[0051] The microcapsule mixture was added dropwise to a calcium chloride solution with a concentration of 0.8 mol / L to form microcapsules. The microcapsules were collected, washed with deionized water, and then freeze-dried at -50℃ under vacuum for 16 h to obtain chitosan-alginate composite microcapsules.

[0052] The preparation method of aminated polylactic acid / calcium carbonate composite particles is as follows: polylactic acid and calcium carbonate are melt-blended at a mass ratio of 100:20, and supercritical carbon dioxide is injected at 190℃ and 12MPa. After foaming treatment for 50s, the pressure is quickly released to obtain porous particles.

[0053] Porous particles were immersed in a 4% (w / w) ethanol solution of 3-aminopropyltriethoxysilane, wherein the mass ratio of porous particles to 3-aminopropyltriethoxysilane was 1.0, and the reaction was carried out at room temperature for 4 h. After the reaction was completed, the product was taken out, washed with ethanol, and then freeze-dried at -50℃ under vacuum for 16 h to obtain aminated polylactic acid / calcium carbonate composite particles. Example 2

[0054] The difference between this embodiment and Embodiment 1 is that a chemical induction system of 0.1 g / L is simultaneously added to the aquaculture water. Example 3

[0055] The difference between this embodiment and Embodiment 1 is that a chemical induction system of 1.0 g / L is simultaneously added to the aquaculture water. Example 4

[0056] The difference between this embodiment and embodiment 1 is that 50 mg / L of substrate material is added to the aquaculture water simultaneously. Example 5

[0057] The difference between this embodiment and embodiment 1 is that 200 mg / L of substrate material is added to the aquaculture water simultaneously. Example 6

[0058] The difference between this embodiment and Example 1 is that the total dosage of sodium bicarbonate and sodium carbonate is 0.1 g / L. Example 7

[0059] The difference between this embodiment and Embodiment 1 is that the total dosage of sodium bicarbonate and sodium carbonate is 0.5 g / L. Example 8

[0060] A method for improving the attachment rate and survival rate of sea urchin larvae includes the following steps: S1.1, cultivating sea urchin fertilized eggs in aquaculture water.

[0061] The physicochemical environment of the aquaculture water body is as follows: water temperature 18℃, pH 8.0, dissolved oxygen concentration 5mg / L, salinity 30‰, and water flow velocity 0.1m / s.

[0062] S1.2 When the larvae develop to the late stage of prism larvae, 0.5 g / L of chemical induction system (chitosan-alginate composite microcapsules with a particle size of 20 μm) and 120 mg / L of substrate material (amino-coated polylactic acid / calcium carbonate composite particles) are simultaneously added to the culture water.

[0063] S1.3 During the attachment and post-attachment stages, the pH of the aquaculture water is adjusted by adding sodium bicarbonate and sodium carbonate in a mass ratio of 5:1 to maintain the pH at 8.1, and the total dosage of sodium bicarbonate and sodium carbonate is 0.5 g / L, in order to promote the stable attachment and metamorphosis of sea urchin larvae.

[0064] The preparation method of chitosan-alginate composite microcapsules is as follows: Sodium alginate and chitosan are mixed at a mass ratio of 2:1 and dissolved in deionized water at a mass ratio of 1:5 to obtain a mixed solution.

[0065] γ-aminobutyric acid was dissolved in deionized water to obtain a concentration of 1×10⁻⁶. -6 A mol / L solution of γ-aminobutyric acid.

[0066] Calcium chloride and magnesium chloride were dissolved in deionized water to obtain a calcium-magnesium ion mixed solution with a concentration of 0.05 mol / L (the molar ratio of calcium ions to magnesium ions was 1:1).

[0067] Glycine, alanine, glutamic acid, and aspartic acid were mixed in a mass ratio of 1:1:1:1 and dissolved in deionized water to obtain a free amino acid solution with a concentration of 5 g / L.

[0068] A solution of γ-aminobutyric acid, a mixed solution of calcium and magnesium ions, and a solution of free amino acids were added sequentially to the mixed solution and stirred until homogeneous to form a microcapsule core material mixture.

[0069] The mass ratio of γ-aminobutyric acid solution to mixed solution is 0.1%; the mass ratio of calcium and magnesium ion mixed solution to mixed solution is 0.5%; and the mass ratio of free amino acid solution to mixed solution is 1%.

[0070] Add 0.1% by mass of nano-bentonite to the microcapsule core material mixture and disperse it evenly to obtain the microcapsule mixture.

[0071] The microcapsule mixture was added dropwise to a 0.5 mol / L calcium chloride solution to form microcapsules. The microcapsules were collected, washed with deionized water, and then freeze-dried at -60℃ under vacuum for 12 h to obtain chitosan-alginate composite microcapsules.

[0072] The preparation method of aminated polylactic acid / calcium carbonate composite particles is as follows: polylactic acid and calcium carbonate are melt-blended at a mass ratio of 100:15, and supercritical carbon dioxide is injected at 180℃ and 10MPa. After foaming treatment for 30s, the pressure is quickly released to obtain porous particles.

[0073] Porous particles were immersed in a 2% (w / w) ethanol solution of 3-aminopropyltriethoxysilane, wherein the mass ratio of porous particles to 3-aminopropyltriethoxysilane was 0.5, and the reaction was carried out at room temperature for 3 h. After the reaction was completed, the product was taken out, washed with ethanol, and then freeze-dried at -60℃ under vacuum for 12 h to obtain aminated polylactic acid / calcium carbonate composite particles. Example 9

[0074] A method for improving the attachment rate and survival rate of sea urchin larvae includes the following steps: S1.1, cultivating sea urchin fertilized eggs in aquaculture water.

[0075] The physicochemical environment of the aquaculture water body is as follows: water temperature 22℃, pH 8.5, dissolved oxygen concentration 8mg / L, salinity 35‰, and water flow velocity 0.5m / s.

[0076] S1.2 When the larvae develop to the late stage of prism larvae, 0.5 g / L of chemical induction system (chitosan-alginate composite microcapsules with a particle size of 80 μm) and 120 mg / L of substrate material (amino-lactate / calcium carbonate composite particles) are simultaneously added to the culture water.

[0077] S1.3 During the attachment and post-attachment stages, the pH of the water is adjusted by adding sodium bicarbonate and sodium carbonate in a mass ratio of 5:1 to maintain the pH at 8.3, and the total dosage of sodium bicarbonate and sodium carbonate is 0.5 g / L, in order to promote the stable attachment and metamorphosis of sea urchin larvae.

[0078] The preparation method of chitosan-alginate composite microcapsules is as follows: Sodium alginate and chitosan are mixed at a mass ratio of 5:1 and dissolved in deionized water at a mass ratio of 1:5 to obtain a mixed solution.

[0079] γ-aminobutyric acid was dissolved in deionized water to obtain a concentration of 1×10⁻⁶. -4 A mol / L solution of γ-aminobutyric acid.

[0080] Calcium chloride and magnesium chloride were dissolved in deionized water to obtain a calcium-magnesium ion mixed solution with a concentration of 0.10 mol / L (the molar ratio of calcium ions to magnesium ions was 3:1).

[0081] Glycine, alanine, glutamic acid, and aspartic acid were mixed in a mass ratio of 1:1:1:1 and dissolved in deionized water to obtain a free amino acid solution with a concentration of 10 g / L.

[0082] A solution of γ-aminobutyric acid, a mixed solution of calcium and magnesium ions, and a solution of free amino acids were added sequentially to the mixed solution and stirred until homogeneous to form a microcapsule core material mixture.

[0083] The mass ratio of γ-aminobutyric acid solution to mixed solution is 1.0%; the mass ratio of calcium and magnesium ion mixed solution to mixed solution is 2.0%; and the mass ratio of free amino acid solution to mixed solution is 5%.

[0084] Add 0.2% by mass of nano-bentonite to the microcapsule core material mixture and disperse it evenly to obtain the microcapsule mixture.

[0085] The microcapsule mixture was added dropwise to a 1.0 mol / L calcium chloride solution to form microcapsules. The microcapsules were collected, washed with deionized water, and then freeze-dried at -40℃ under vacuum for 24 h to obtain chitosan-alginate composite microcapsules.

[0086] The preparation method of aminated polylactic acid / calcium carbonate composite particles is as follows: polylactic acid and calcium carbonate are melt-blended at a mass ratio of 100:30, and supercritical carbon dioxide is injected at 200℃ and 15MPa. After foaming treatment for 60s, the pressure is quickly released to obtain porous particles.

[0087] Porous particles were immersed in a 5% (w / w) ethanol solution of 3-aminopropyltriethoxysilane, wherein the mass ratio of porous particles to 3-aminopropyltriethoxysilane was 1.5, and the reaction was carried out at room temperature for 5 h. After the reaction was completed, the product was taken out, washed with ethanol, and then freeze-dried at -40℃ under vacuum for 24 h to obtain aminated polylactic acid / calcium carbonate composite particles.

[0088] The procedure for determining the larval attachment rate is as follows: After simultaneously adding the chemical induction system and the substrate material to the aquaculture water, select a 48-hour observation point; use a quantitative siphon or column sampler to take equal-volume samples at multiple points in different water layers (upper, middle, and lower) and near the substrate layer in the aquaculture container, and mix them to obtain representative samples; gently pour the samples into a petri dish with a fine mesh bottom, let them stand for a while until the larvae are stable, and then observe them under a stereomicroscope; count the number of larvae firmly attached to the substrate particles and the number of planktonic larvae suspended or swimming in the water; the attachment rate is calculated using the formula: Attachment rate (%) = [Number of attached larvae / (Number of attached larvae + Number of planktonic larvae)] × 100%; each experimental group should have no less than three parallel replicates.

[0089] The procedure for determining the metamorphosis success rate after attachment is as follows: After the larvae have completed a large number of attachments and entered the metamorphosis stage (usually 5-7 days after the first addition of the inducer), several substrate samples with attached larvae are randomly selected. Under a stereomicroscope, at least 30 randomly selected attached individuals from each sample are subjected to detailed morphological observation and identification. The key features that mark successful metamorphosis of juvenile sea urchins are identified, including: clear formation and extension of primary tube feet, obvious development and remodeling of calcareous sclerites, and the initial appearance of short and blunt spines. Individuals exhibiting the above typical features are judged to have successfully metamorphosed. The metamorphosis success rate is calculated using the formula: Metamorphosis success rate (%) = (Number of successfully metamorphosed juvenile sea urchins / Total number of observed attached larvae) × 100%. This determination needs to be performed on multiple parallel samples.

[0090] The overall survival rate is determined as follows: This indicator tracks the cumulative survival from the start of the experiment (i.e., when the inducer is added) until the end of the entire attachment and metamorphosis cycle (e.g., when the juvenile sea urchins develop to the stable feeding stage); at the start of the experiment, the initial total number of healthy larvae used is accurately recorded or estimated; at the end of the experimental cycle, all surviving juvenile sea urchins in the culture system are carefully collected; the collection method includes gently rinsing the attached substrate to separate individuals, combined with filtering all water to ensure complete recovery; all recovered live individuals are counted; the overall survival rate is calculated using the formula: Overall survival rate (%) = (total number of surviving juvenile sea urchins / initial total number of larvae) × 100%; this indicator comprehensively reflects the overall effectiveness of the method in inducing attachment, supporting metamorphosis, and maintaining early developmental survival conditions.

[0091] Table 1 Survival results of sea urchin larvae

[0092]

[0093] Data from Examples 1, 2, and 3 show that when the concentration was increased from 0.1 g / L (Example 2) to 0.5 g / L (Example 1), the larval attachment rate increased from 71.5% to 88.6%, indicating that a suitable concentration of sustained-release signal can effectively stimulate and synchronize the attachment behavior of larvae. However, when the concentration was further increased to 1.0 g / L (Example 3), although the attachment rate (85.3%) remained at a high level, the metamorphosis success rate after attachment decreased from 92.1% to 80.2%, resulting in an overall survival rate of 68.4% instead of 81.7%. This indicates that excessively high inducer concentrations can cause continuous overstimulation of attached larvae or lead to local microenvironmental imbalance, interfering with their normal metamorphic development process.

[0094] Data from Examples 1, 4, and 5 show that sufficient attachment substrate is a prerequisite for ensuring a high attachment rate. When the dosage was only 50 mg / L (Example 4), the larval attachment rate was significantly limited to only 75.8%, far lower than that of Example 1 (88.6%). This was because the effective attachment surface area was insufficient, becoming a limiting factor. Although the metamorphosis success rate of successfully attached individuals was still very high (90.5%), the low initial number directly lowered the overall survival rate (68.6%). Increasing the dosage to 200 mg / L (Example 5) provided ample attachment sites, and both the attachment rate (86.2%) and survival rate (79.2%) were close to the optimized level.

[0095] Table 1 shows that the strength of the buffer system affects the stability of the post-attachment stage. Comparing 1, 6, and 7, when the total dosage is only 0.1 g / L (Example 6), the system's buffering capacity is weak and the pH stability of the water is insufficient. This leads to fluctuations in the microenvironment of the attached larvae. Although the attachment rate is still as high as 87.1%, the metamorphosis success rate drops to 84.6%, indicating that the unstable environment hinders the smooth progress of the metamorphosis process. The final overall survival rate is 73.8%. When the dosage is increased to 0.5 g / L (Example 7), the microenvironment stability is enhanced, and the metamorphosis success rate increases to a maximum of 93.5%, thus obtaining the highest overall survival rate (83.1%).

[0096] A comprehensive analysis of the data in Table 1 shows that Example 7 performed best in three key indicators: larval attachment rate (88.9%), post-attachment metamorphosis success rate (93.5%), and overall survival rate (83.1%). Therefore, Example 7 is selected as the optimal example. Comparative Example 1

[0097] Traditional natural attachment was used: no chemical induction system was added, polyethylene plastic sheets were used as the substrate for attachment, and no sodium bicarbonate / sodium carbonate was added. Other cultivation conditions were the same as in Example 7. Comparative Example 2

[0098] Instead of adding chitosan-alginate composite microcapsules, γ-aminobutyric acid, calcium and magnesium ions (calcium chloride, magnesium chloride), and mixed amino acids equivalent to the total amount of contents contained in the microcapsules of Example 7 were directly added to the aquaculture water; polyethylene plastic sheets were used as the substrate; sodium bicarbonate / sodium carbonate was not added; other conditions were the same as in Example 7. Comparative Example 3

[0099] The substrate was replaced with polylactic acid / calcium carbonate composite particles that were not treated with 3-aminopropyltriethoxysilane and were only foamed by supercritical CO2 (i.e., without surface amination), and other conditions were the same as in Example 7.

[0100] Table 2 Survival results of sea urchin larvae

[0101]

[0102] Compared with Example 7, the data in Comparative Example 1 (attachment rate 45.2%, metamorphosis success rate 65.6%, overall survival rate 30.1%) represent the natural level without active intervention; its low attachment rate is due to the lack of targeted chemical induction; while the relatively high metamorphosis success rate (compared to the low number of attachments) indicates that a few individuals that can randomly find suitable attachment points have certain developmental potential; the extremely low overall survival rate reflects the dual disadvantages of difficult attachment and unstable environment in the later stage.

[0103] Comparative Example 2 used a free inducer with the same chemical composition as Example 7 but without microencapsulation; its attachment rate (60.5%) was higher than that of Comparative Example 1, proving that the chemical signal could initially stimulate the attachment behavior of larvae; however, the metamorphosis success rate after attachment dropped sharply to 55.1%, resulting in an overall survival rate of only 33.4%; the reason for this decrease is that the free inducer rapidly diffuses and degrades in the water, and cannot form a continuous and stable effective concentration on the surface of the attachment substrate; although the larvae are attracted and attempt to attach, the attachment behavior is asynchronous and unstable due to the rapid disappearance of the local induction signal and the lack of a stable chemical microenvironment (no buffer system), and most individuals die in the subsequent sensitive metamorphosis stage.

[0104] Comparative Example 3 used sustained-release microcapsules and a buffer system, but the substrate to which it adhered was ordinary porous particles without amination modification. Its data characteristics (adhesion rate 80.9%, metamorphosis success rate 75.4%, and overall survival rate 60.7%) indicate that the persistent chemical signal field provided by the sustained-release microcapsules can effectively attract larvae, making the adhesion rate close to that of Example 7; the buffer system also ensures basic water quality; however, due to the lack of positive charge and biocompatibility brought by amination modification on the substrate surface, the initial adhesion between the larvae and the substrate is insufficient, and the physical bond is not strong; in the complex physiological process of transitioning from attachment to metamorphosis, these weakly attached individuals are easily detached due to their own activity or slight water flow, or their development is hindered due to the poor microenvironment at the interface with the substrate, resulting in a metamorphosis success rate and overall survival rate that are significantly lower than those of Example 7.

[0105] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the attachment rate and survival rate of sea urchin larvae in aquaculture, characterized in that, Includes the following steps: S1.

1. Cultivate sea urchin fertilized eggs in aquaculture water; S1.2 When the larvae develop to the late stage of prism larvae, add 0.1-1.0 g / L of chemical induction system and 50-200 mg / L of substrate material to the aquaculture water at the same time; The chemical induction system consists of γ-aminobutyric acid and Ca2+. 2+ / Mg 2+ Chitosan-alginate composite microcapsules containing free amino acids have a particle size of 20-80 μm. The substrate material is amino-modified polylactic acid / calcium carbonate composite particles; S1.

3. By maintaining the chemical microenvironment during and after attachment, sea urchin larvae can be promoted to complete stable attachment and metamorphosis. In step S1.2, the preparation method of the chitosan-alginate composite microcapsules is as follows: Sodium alginate and chitosan were mixed at a mass ratio of 2-5:1 and dissolved in deionized water at a mass ratio of 1:5 to obtain a mixed solution. γ-aminobutyric acid was dissolved in deionized water to obtain a concentration of 1×10⁻⁶. -6 -10 -4 A mol / L solution of γ-aminobutyric acid; Calcium chloride and magnesium chloride are dissolved in deionized water to obtain a calcium-magnesium ion mixed solution with a concentration of 0.05-0.10 mol / L; Glycine, alanine, glutamic acid, and aspartic acid were mixed in a mass ratio of 1:1:1:1 and dissolved in deionized water to obtain a free amino acid solution with a concentration of 5-10 g / L. A solution of γ-aminobutyric acid, a mixed solution of calcium and magnesium ions, and a solution of free amino acids were added to the mixed solution in sequence and stirred until homogeneous to form a microcapsule core material mixture. Add 0.1-0.2% by weight of nano-bentonite to the microcapsule core material mixture and disperse it evenly to obtain the microcapsule mixture; The microcapsule mixture was added dropwise to a calcium chloride solution with a concentration of 0.5-1.0 mol / L to form microcapsules; the microcapsules were collected, washed with deionized water, and then freeze-dried at -40℃ to -60℃ under vacuum for 12-24 h to obtain chitosan-alginate composite microcapsules. In S1.2, the preparation method of aminated polylactic acid / calcium carbonate composite particles is as follows: polylactic acid and calcium carbonate are melt-blended at a mass ratio of 100:15-30, and supercritical carbon dioxide is injected under the conditions of 180-200℃ and 10-15MPa. After foaming treatment for 30-60s, the pressure is quickly released to obtain porous particles. Porous particles were immersed in an ethanol solution of 3-aminopropyltriethoxysilane and reacted at room temperature for 3-5 hours. After the reaction was completed, the product was removed, washed with ethanol, and then freeze-dried at -40°C to -60°C under vacuum for 12-24 hours to obtain aminated polylactic acid / calcium carbonate composite particles.

2. The method for improving the attachment rate and survival rate of sea urchin larvae according to claim 1, characterized in that, In S1.1, the physicochemical environment of the aquaculture water includes: The water temperature is 18-22℃, the pH is 8.0-8.5, the dissolved oxygen concentration is 5-8mg / L, the salinity is 30-35‰, and the water flow rate is 0.1-0.5m / s.

3. The method for improving the attachment rate and survival rate of sea urchin larvae according to claim 2, characterized in that, In the calcium-magnesium ion mixed solution, the molar ratio of calcium ions to magnesium ions is 1-3:

1.

4. The method for improving the attachment rate and survival rate of sea urchin larvae according to claim 3, characterized in that, The mass ratio of the γ-aminobutyric acid solution to the mixed solution is 0.1-1.0%; The mass ratio of calcium and magnesium ion mixed solution to the total mixed solution is 0.5-2.0%; The mass ratio of free amino acid solution to mixed solution is 1-5%.

5. The method for improving the attachment rate and survival rate of sea urchin larvae according to claim 4, characterized in that, The mass concentration of the 3-aminopropyltriethoxysilane ethanol solution is 2-5%.

6. The method for improving the attachment rate and survival rate of sea urchin larvae according to claim 5, characterized in that, The mass ratio of the porous particles to 3-aminopropyltriethoxysilane is 0.5-1.

5.

7. The method for improving the attachment rate and survival rate of sea urchin larvae according to claim 6, characterized in that, In step S1.3, maintaining the chemical microenvironment involves adjusting the pH of the aquaculture water by adding sodium bicarbonate and sodium carbonate in a mass ratio of 5-10:1, thereby maintaining the pH at 8.1-8.

3.

8. The method for improving the attachment rate and survival rate of sea urchin larvae according to claim 7, characterized in that, The total dosage of sodium bicarbonate and sodium carbonate is 0.1-0.5 g / L.

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