A method for promoting settlement of patinopecten yessoensis larvae

Abstract

This invention provides a method for promoting the attachment of scallop larvae, aiming to increase the larval attachment rate, simplify the operation process, reduce environmental pollution, and is suitable for industrial production. The method involves plasma treatment of polyethylene mesh, using argon, nitrogen, or air as the carrier gas. By controlling the plasma treatment parameters and treating the mesh for a short time, the surface physicochemical properties are improved, increasing larval attachment. This method has minimal impact on larval growth and survival; nitrogen and air plasma treatments also improve metamorphosis success rates. Experiments show that argon, nitrogen, and air plasma treatments increased the yield of marketable seedlings by 133%, 218%, and 126%, respectively. This method is clean, efficient, and reusable, significantly promoting attachment and metamorphosis, providing a high-quality seedling guarantee and important technical support for scallop farming.

Description

Technical Field

[0001] This invention relates to the field of shellfish genetic breeding in marine agriculture, and in particular to a method for promoting the attachment of Yesso scallop larvae. Background Technology

[0002] Ezo scallop ( Patinopecten yessoensis With its delicious meat and high economic value, the scallop is an important marine aquaculture species in my country. Currently, my country has over 200,000 hectares of scallop farming area, with an annual output of up to 300,000 tons, accounting for more than 70% of the world's total scallop farming output. Scallop farming technology is relatively mature, but seedlings almost entirely rely on artificial breeding. In the artificial breeding process of scallops, the attachment and metamorphosis of larvae determine the success or failure of seedling breeding, directly affecting the development of the entire scallop aquaculture industry.

[0003] Currently, polyethylene mesh is the most commonly used substrate for seedling attachment in the industrialized artificial breeding of scallops. Before use, the polyethylene mesh needs to undergo a certain treatment process. The most common treatment method in the industry is: the polyethylene mesh is first soaked in 1% sodium hydroxide solution; before deployment, it is prepared at a rate of 20 meshes / m². 3 The standard method involves soaking the scallop larvae in a solution of 20-30 ppm sodium penicillin for 5-8 hours; after rinsing, it can be used for larval attachment and metamorphosis. In addition, some studies have explored various physical, chemical, and biological treatment methods, such as ultraviolet irradiation, potassium chloride soaking, biofilm covering, and chemical signal induction using acetylcholine or γ-aminobutyric acid (GABA). While these methods have achieved some success in improving the attachment and metamorphosis of bivalves, they also have drawbacks such as complex operation, high technical difficulty, and potential environmental pollution. Therefore, the industry urgently needs a simple, clean, and efficient method to promote the attachment of scallop larvae.

[0004] The physicochemical properties of the substrate surface significantly influence the attachment process of scallop larvae. Plasma treatment is a commonly used material surface modification technique that alters the chemical and morphological properties of materials through the strong interaction between high-energy particles and the material surface. It has been widely applied in biomedical materials, optical materials, and metallic materials. Plasma treatment can achieve rapid surface modification of polyethylene materials, with the modification effect stabilizing in just 3-5 minutes. Under high power (≥300W) and low pressure (<10Pa) conditions, the treatment time can be further reduced to 2-3 minutes. Plasma treatment using inert gases such as argon as carrier gases cleans and modifies the material surface through a physical etching process. When nitrogen or air is selected as the plasma gas source, nitrogen- or oxygen-containing functional groups are introduced into the material surface. Applying this technology to the attachment substrate of Yesso scallops holds promise for increasing larval attachment rates, but further research is needed to validate this technology. Summary of the Invention

[0005] Therefore, this invention proposes a method for promoting the attachment of scallop larvae to solve the above-mentioned problems.

[0006] The technical solution of this invention is achieved as follows: A method for promoting the attachment of scallop larvae, comprising the following steps: (1) Adhesion substrate treatment: Polyethylene mesh is selected as the adhesion substrate, and the mesh is subjected to plasma treatment for a treatment time of ≤5 minutes; (2) Transferring larvae to the original breeding pond: The larvae are transferred to the original breeding pond according to their condition. When transferring larvae, a sieve is used to screen the scallop larvae. (3) Placement of attachment substrate: After the pond is drained, the plasma-treated attachment substrate is rinsed and placed into the new seedling pond. The water is changed daily and mixed feed is provided.

[0007] Furthermore, in step (1), the working gas for ion treatment is at least one of argon, nitrogen, or air with a purity ≥ 99.999%.

[0008] Furthermore, the plasma treatment parameters for step (1) are: vacuum degree 30-35Pa, power supply 250-350W, output frequency 35-45kHz, and treatment time 1-5 minutes.

[0009] Furthermore, the polyethylene mesh in step (1) is woven from polyethylene fine ropes, with a length of 1.0m and a width of 0.2m, containing 3000 woven buckles.

[0010] Furthermore, the larval status determination in step (2) is to transfer the larvae to a new pond when 80% of the larvae have extended their legs and 30% of the larvae have rounded eyes.

[0011] Furthermore, in step (2), the sieve silk has a pore size of 125 μm, and the average shell length of the larvae after screening is 240-260 μm, with a density of 4-6 larvae / mL.

[0012] Furthermore, in step (2), the water temperature of the new seedling pond is controlled at 15-16℃, and continuous aeration is maintained to keep the water in a micro-flow state and the dissolved oxygen is stable at 5-7mg / L.

[0013] Furthermore, in step (3), the substrate is applied at a rate of 25-35 pieces / m². 3 Density deployment and maintenance of a fixed position.

[0014] Furthermore, the feeding in step (3) includes: the initial daily feeding amount is 40,000 cells / mL of water, fed in 4 times, of which golden algae account for ≥70% and flat algae account for ≤30%; the proportion of flat algae is gradually increased during the development period, and the final daily feeding amount is increased to 60,000 cells / mL of water.

[0015] Furthermore, in step (3), the water is changed twice a day, and the amount of water changed each time is 1 / 2.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) It can significantly improve the attachment rate of scallop larvae and the quantity of commercial seedlings. By treating polyethylene mesh with plasma with specific parameters, the physical and chemical properties of the mesh surface are improved. Combined with scientific management of pond turning and attachment substrate placement, experiments show that argon, nitrogen and air plasma treatment can increase the quantity of commercial seedlings by 133%, 218% and 126% respectively, significantly improving the seedling production efficiency. (2) It has little impact on larval growth and survival, and can improve the success rate of metamorphosis. Nitrogen and air plasma treatment promotes attachment while ensuring normal larval development. The larvae after screening are of uniform size, which is conducive to cultivation and management and shortens the attachment time. (3) Simplify the operation process and reduce costs. No complicated process is required, the plasma treatment time is short, the substrate can be reused, which can reduce costs by 30-50%, and the daily water change and feeding operations are standardized and easy to implement. (4) Green and environmentally friendly, reducing environmental pollution. No harmful chemicals are used in the whole process, and plasma treatment is clean and efficient, which meets the requirements of ecological farming. (5) Applicable to industrial production. The method is stable and controllable, with clear parameters for each step. The substrate density, water temperature and other conditions are suitable for large-scale seedling cultivation, providing high-quality seedlings and important technical support for the scallop farming industry, and promoting industrial development. Attached Figure Description

[0017] Figure 1 The diagram shows the placement of the substrate, where (A) the placement locations of the substrate, (a) the inlet, (b) the outlet, and (c) the substrate, which are placed upstream, midstream, and downstream of the water flow direction, respectively; (B) the binding method of the mesh for the plasma treatment group and the control group, (d) the polyethylene mesh, and (e) the stone weight; (C) a schematic diagram of the polyethylene mesh, each with 3,000 knots, with the sampling locations within the red boxes, located in the upper, middle, and lower layers of the mesh.

[0018] Figure 2 The attachment status of plasma-treated and control groups was compared 16 days after the attachment substrate was applied. (A) Attachment substrate; (B) Scallop juveniles.

[0019] Figure 3The effects of plasma treatment on the attachment, metamorphosis, and growth of scallop larvae during substrate deployment were investigated, including (A) the number of attached larvae 10 days after substrate deployment, the number of juvenile scallops 16 days after substrate deployment, and the metamorphosis rate. Significance between groups with the same gas source but different treatment times was indicated by different lowercase letters (a, b); significance between groups with the same treatment time but different gas sources was indicated by different uppercase letters (A, B).

[0020] Figure 4 The effects of plasma treatment on the attachment, growth, and survival of juvenile scallops at the end of the intermediate rearing period were investigated, including (A) attachment substrate; (B) commercial scallop seedlings; (C) quantity and shell length of commercial seedlings; and (D) survival rate. Significance between groups with the same gas source but different treatment times was represented by different lowercase letters (a, b); significance between groups with the same treatment time but different gas sources was represented by different uppercase letters (A, B).

[0021] Figure 5 The plasma-treated and control groups of scallop seedlings were commercially viable after the intermediate rearing period ended. Detailed Implementation

[0022] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0023] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0025] Example 1 (1) Adhesion substrate treatment: The mesh is woven from polyethylene fine rope, with a length of 1.0m × width of 0.2m, including 3000 braided buckles; The experimental groups were treated with argon, nitrogen, and air plasma, respectively.

[0026] Argon group: First, the reaction chamber of the plasma equipment is evacuated. Then, argon with a purity of 99.999% or higher is selected as the working gas, the vacuum degree is 35Pa, the power supply is controlled at 300W, and the output frequency is 40kHz. The mesh is subjected to short-time plasma treatment, with treatment times of 1min (Ar-1min; n=9), 2min (Ar-2min; n=9), and 4min (Ar-4min; n=9). Nitrogen group: The working gas is nitrogen with a purity of 99.999% or higher. Other conditions are the same as those of the argon group. The mesh is subjected to short-time plasma treatment for 1 min (N2-1 min; n=9), 2 min (N2-2 min; n=9), and 4 min (N2-4 min; n=9). Air group: The working gas is air, and other conditions are the same as those of the argon group. The mesh is subjected to short-time plasma treatment, with treatment times of 1 min (Air-1 min; n=9), 2 min (Air-2 min; n=9), and 4 min (Air-4 min; n=9). The control group (n=9) was treated using the traditional method: the mesh was soaked in 20ppm sodium penicillin for 5 hours before application, with a mesh density of 20 meshes / m². 3 .

[0027] (2) Pond transfer: On February 22, 2025, at Shandong Laizhou Haiyi Seedling Industry Co., Ltd., when about 80% of the scallop larvae in the nursery pond had extended their legs and 30% of the larvae had rounded eyes, the scallop larvae were screened with a 120-mesh sieve (pore size 125μm). The average shell length of the larvae was 250μm. The larvae were transferred to the new nursery pond. The water temperature in the new nursery pond was controlled at 16℃. Continuous aeration was maintained to keep the water in a micro-flow state and the dissolved oxygen was stabilized at 6mg / L. The larval rearing density was adjusted to 5 larvae / mL.

[0028] (3) Placement of the substrate: The mesh sheets used in the plasma treatment group (Ar-1min, Ar-2min, Ar-4min, N2-1min, N2-2min, N2-4min, Air-1min, Air-2min, Air-4min) and the control group, as well as the mesh sheets soaked in sodium penicillin (widely used in seedling farms), were simultaneously placed in three seedling ponds (seedling pond 1, seedling pond 2, and seedling pond 3). In each seedling pond, one set of experimental mesh sheets was placed upstream, midstream, and downstream according to the water flow direction during water changes. Figure 1 A). Mesh sheets treated with the same gas source plasma for different times and control mesh sheets were tied from the bottom to the same weight. Figure 1 B). The volume of water in each nursery pond is 20m³. 3 600 net panels were placed in the system, and rinsed thoroughly with sand-filtered seawater before placement. After adding the substrate, the water was changed twice daily, with half the water replaced each time; golden algae (…) were used. I. galbana ) and flat algae ( P. subcordiformis Mixed feeding, mainly with golden algae, gradually increasing the proportion of flat algae, with a daily feed amount of 40,000 cells per milliliter of seawater, divided into 4 feedings, gradually increasing to 60,000 cells per milliliter of seawater as the development stage progresses.

[0029] Ten days after the attachment substrate was applied, all the scallop larvae had attached to the polyethylene mesh. Five loops were cut from each of the top, middle, and bottom layers of each mesh. Figure 1 C), the number of scallop larvae attached to different groups of mesh panels was counted. Sixteen days after the attachment substrate was applied, all scallop larvae on the polyethylene mesh panels had completed metamorphosis. Figure 2 Five snaps were cut from each of the top, middle, and bottom layers of each net sheet, and the attachment, metamorphosis, and growth indicators of juvenile scallops on different groups of net sheets were statistically analyzed. Figure 3 ), Calculation formula: Attachment quantity: (Number of larvae at 10 days + Number of juveniles at 10 days) / deduct; Chick count: Number of chicks at 16 days / dec; Metamorphosis rate: Number of juvenile shells at 16 days / Number of attached larvae at 10 days × 100%.

[0030] The number of larvae attached to the nets in the plasma-treated group was significantly higher than that in the control group (13.48±3.41 larvae / set). Nitrogen plasma treatment had the most significant promoting effect on the attachment of scallop larvae, increasing the attachment density by 143.77~161.13%; the argon and air groups increased by 124.26~133.38% and 45.92~137.76%, respectively. The number of juvenile scallops attached in the plasma-treated group also continued to be superior, with the nitrogen group maintaining a juvenile scallop count of 24.17~26.53 larvae / set, an increase of 170.36~196.76% compared to the control group. The effects of plasma treatment of the substrate with different gas sources on the metamorphosis process of scallop larvae varied: the metamorphosis rate of the nitrogen plasma treatment group was 73.55-77.42%, significantly higher than that of the control group (66.35±2.48%); in the argon group, the metamorphosis rate of the Ar-1min group was similar to that of the control group, but the metamorphosis rates of the Ar-2min and Ar-4min groups were significantly reduced (53.30-60.75%); the air group showed a certain degree of improvement (66.74-72.57%).

[0031] In the early morning of March 9, 2025, these attachment sites were transferred to the open sea area (37.59°N, 121.33°E) of Taozi Bay in Yantai Economic Development Zone to complete the seedling protection period. 65 days later, these nets were retrieved by Shandong Yantai Haiyi Seedling Industry Co., Ltd., and the commercial scallop seedlings were brushed off and transferred to large-mesh collection bags (0.425mm aperture). During this process, the attachment, growth, and seedling protection status of each net were recorded. Figure 4 ).

[0032] The number of marketable scallop seedlings in the control group was 3.01±0.83, while the argon-treated and air-treated groups had 5.93~7.02 seedlings / set and 5.87~6.79 seedlings / set, respectively. The nitrogen plasma-treated mesh still maintained a significant advantage in the number of marketable seedlings, at 8.97±2.93, 9.56±1.83, and 8.85±1.62 seedlings / set, respectively, representing an increase of 194.02~217.61% compared to the control group. There was no significant difference in shell length between the plasma-treated and control groups for scallop seedlings. Figure 5 In terms of seedling survival rate, only the Air-4min group (40.25±4.03%) showed a significant improvement, indicating that plasma treatment does not affect the growth and seedling survival of Yesso scallop larvae.

[0033] The attachment, metamorphosis, and intermediate rearing processes of scallop larvae have a crucial impact on the yield of marketable seedlings. Treatment of polyethylene mesh with argon, nitrogen, and air plasma significantly promoted the attachment process of scallop larvae, with this process contributing overwhelmingly to the increase in marketable seedling yield (Table 1). In the argon plasma treatment group, the majority of the increase in marketable seedling yield came from improved attachment (89.55–96.41%). In the Ar-2min and Ar-4min groups, the metamorphosis process did not contribute to the increase in marketable seedling yield due to the reduced metamorphosis rate. Nitrogen plasma treatment promoted all three processes, showing a more balanced contribution, with the increase in larval attachment rate still contributing 71.16–83.32%. The contribution of air plasma treatment to the increase in commercial seedling yield showed a time-dependent effect. The Air-2min group was almost entirely dependent on the improvement of the attachment process (99.58%), while the contributions of the metamorphosis (10.41%) and intermediate temporary rearing (21.18%) processes to the increase in commercial seedling yield in the Air-4min group were not negligible.

[0034] Table 1: Contribution of different processes to the increase in commercial seedling yield

[0035] Compared to the control group treated with conventional methods, polyethylene mesh treated with argon, nitrogen, and air plasma showed a significantly higher yield of marketable scallop larvae, with the N2-2min group exhibiting the best performance among all plasma treatment groups. These results demonstrate that nitrogen plasma treatment can effectively promote the attachment and metamorphosis of scallop larvae without affecting their growth or scallop survival rates.

[0036] This invention provides an innovative method for treating attachment substrates, which significantly improves the attachment rate of scallop larvae and the yield of commercial seedlings compared to traditional techniques.

[0037] Experimental data show that: 1) After treatment with argon, nitrogen and air plasma, the larval attachment rate increased by 127%, 154% and 119% respectively, and the yield of commercial seedlings increased by 133%, 218% and 126% respectively, showing significant treatment effects; 2) From an economic perspective, this technology achieves cost savings of 30-50%, significantly reducing the input of human and material resources; 3) More importantly, this method produces zero pollution throughout the entire process, meeting green environmental protection requirements and solving the environmental pollution problems associated with traditional methods. It is a clean and efficient new treatment process (Table 2). This technological breakthrough not only significantly improves seedling production efficiency, but its environmental characteristics also provide an innovative solution for the sustainable development of aquaculture, making it of significant value for widespread application.

[0038] Table 2: Comparison of Nitrogen Plasma Treatment with Traditional Methods

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of promoting settlement of Patinopecten yessoensis larvae, characterized by: Includes the following steps: (1) Adhesion substrate treatment: Select polyethylene mesh as the adhesion substrate and perform plasma treatment on the mesh for a treatment time of ≤5 minutes; The working gas of the plasma treatment is at least one of argon, nitrogen or air; The plasma treatment parameters are: vacuum degree 30-35Pa, power supply 250-350W, output frequency 35-45kHz, and treatment time 1-5 minutes. (2) Transferring larvae to the original breeding pond: The larvae are transferred to the original breeding pond according to their condition. When transferring larvae, a sieve is used to screen the scallop larvae. (3) Placement of attachment substrate: After the pond is drained, the plasma-treated attachment substrate is rinsed and placed into the new seedling pond. The water is changed daily and mixed feed is provided.

2. The method of claim 1, wherein the method is characterized by: The polyethylene mesh in step (1) is woven from polyethylene fine rope, with a length of 1.0m and a width of 0.2m, and includes 3000 woven buckles.

3. The method for promoting the attachment of scallop larvae as described in claim 1, characterized in that: The step (2) larval status determination is to transfer the larvae to a new nursery when 80% of the larvae in the original nursery have extended their legs and 30% of the larvae have rounded eyes.

4. The method for promoting the attachment of scallop larvae as described in claim 1, characterized in that: In step (2), the sieve silk has a pore size of 125 μm, and the average shell length of the larvae after screening is 240-260 μm, with a density of 4-6 larvae / mL.

5. The method for promoting the attachment of scallop larvae as described in claim 1, characterized in that: In step (3), the water temperature in the new seedling pond is controlled at 15-16℃.

6. The method for promoting the attachment of scallop larvae as described in claim 1, characterized in that: The step (3) adheres the base at 25-35 pieces / m 3 The density is laid out and maintained fixed.

7. The method for promoting the attachment of scallop larvae as described in claim 1, characterized in that: The feeding in step (3) includes: the initial daily feeding amount is 40,000 cells / mL of water, fed in 4 times, of which golden algae account for ≥70% and flat algae account for ≤30%; the proportion of flat algae is gradually increased during the development period, and the final daily feeding amount is increased to 60,000 cells / mL of water.

8. The method for promoting the attachment of scallop larvae as described in claim 1, characterized in that: In step (3), the water is changed twice a day, and half of the water is changed each time.

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