Perinereis aibuhitensis triploid induction method

Abstract

The invention discloses a perinereis aibuhitensis triploid induction method, which comprises the following steps: placing perinereis aibuhitensis fertilized eggs in ice water for cold shock treatment, then carrying out gradient heating to room temperature, selecting sexually mature perinereis aibuhitensis for artificial insemination, cutting female and male perinereis aibuhitensis into pieces in a stainless steel metal filtering cylindrical net in sequence, and carrying out freeze drying to obtain the perinereis aibuhitensis triploid. According to the method, the stainless steel metal filtering cylindrical net is used for assisting the artificial insemination of the perinereis aibuhitensis, the triploid of the perinereis aibuhitensis is induced through cold shock treatment, the perinereis aibuhitensis induction rate is identified to be 80% or above by means of a flow cytometry and tissue slices, the operation is simple, the cost is low, and the method is suitable for large-scale popularization and application. The cost is saved, and the method is effective and can be used for inducing the marine annelids triploid.

Description

Technical Field

[0001] This invention relates to the field of sandworm culture, and more particularly to a method for inducing triploid sandworms with two teeth. Background Technology

[0002] Sandworms, commonly known as "sea centipedes" (or "liuqi" in Fujian), are low-lying invertebrates found in marine tidal flats with extremely high nutritional value. They are also used as raw materials for high-end seafood, fishing bait, live feed for aquatic seedlings, health products, and medicines. Sandworms have a wide range of applications and a large market capacity. However, currently, most sandworm seedlings on the market come from natural harvesting or a small number are self-bred and raised in outdoor cement ponds, resulting in a huge market gap.

[0003] For example, patent CN119678873A discloses a method for cultivating sandworms using artificial perches, including: measuring the body size of sandworms, constructing U-shaped artificial perches, placing sandworms in a water tank, and rearing and harvesting. By using a modified PVC transparent flexible tube, an artificial perch suitable for sandworm growth is simulated. The inner ring of the tube enables the sandworms to adapt to the perch environment, allowing them to grow in the perch. This facilitates laboratory observation of the sandworms' growth status and ecological behavior, and makes manual harvesting easier. It greatly reduces the need for sediments such as marine mud in polychaete farming, providing a simple and quick method for polychaete farming and scientific experiments.

[0004] Patent CN102349474A discloses a method for artificially cultivating sandworms in a biological filter. The method mainly involves laying a gravel layer, a coarse sand layer, a fine sand layer, and a soil layer sequentially from bottom to top within the filter, which is equipped with an inlet pipe and an outlet pipe. Before releasing the sandworm seedlings, the entire filter is filled with fresh water and soaked for one week. After one week, the fresh water is drained, and fresh seawater is injected. The sandworm seedlings are artificially sprinkled, with a size of 3-10 segments, and a stocking density of 600-2000 seedlings / square meter. The amount of feed is 8-12% of the total weight of the sandworms in the filter. The feed consists of artificially cultured marine fish waste and excrement collected, concentrated, and dried after being filtered through a 50-100 mesh silk screen. The water temperature for raising sandworms in the filter bed is 4-40℃. For the first 1-2 weeks after stocking, water is added without draining. After 1-2 weeks, the water can be manually changed twice daily, each time replacing 1 / 3 of the filter bed's water volume. This invention enables the artificial, large-scale cultivation of sandworms with a high survival rate.

[0005] Artificial breeding has revealed that sexually mature sandworms can lay eggs on their own, but after egg-laying, the mothers die, severely hindering aquaculture development and causing economic losses. Germplasm is the "chip" of aquaculture; in response to the national call to build a maritime power and to revitalize the seed industry, creating superior sterile sandworm germplasm is therefore urgently needed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for inducing triploid *Pteris vittata* to solve the above-mentioned technical problems.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for inducing triploidy of *Nereidum didentum* involves placing fertilized *Nereidum didentum* eggs in ice water for cold shock treatment, followed by a gradual increase in temperature to room temperature.

[0008] Preferably, sexually mature *Pterygium bispinipes* are selected for artificial insemination. Female and male *Pterygium bispinipes* are cut into pieces sequentially in a stainless steel metal filter cylinder. The cylinder is then immersed in seawater at room temperature, and the sperm and eggs are released from the cut gonadal tissue and fertilized under the activation of seawater.

[0009] Preferably, the male sandworm is shredded for 1-2 minutes, and the time when the shredding of the male tissue ends is the time when fertilization begins. This time is recorded for subsequent cold shock treatment of the fertilized eggs.

[0010] Preferably, the cold shock treatment of the fertilized egg is performed by placing the fertilized egg in ice water (0-4℃) for 45 minutes after fertilization, and then gradually raising the temperature to room temperature (each time by 8℃, in 3 times).

[0011] Preferably, ploidy identification is performed 20 days after the sandworm larvae hatch. The cells are classified and counted using flow cytometry. The DNA content of the experimental sandworms is measured using a normally artificially raised diploid sandworm sample as a reference. The ploidy of the cells is detected based on the DNA content and fluorescence intensity. After flow cytometry detection, the gonadal development is further observed through gonadal tissue sections.

[0012] Preferably, the gonadal tissue sections were randomly sampled from the control and induction group rearing ponds at 1 month, 2 months, and 3 months of age, respectively. The sections were fixed with Born's reagent, dehydrated overnight with graded ethanol, cleared with xylene, embedded in paraffin, and sectioned to a thickness of 4 μm. The sections were then stained with hematoxylin and eosin (H&E) and the gonadal development was observed using an optical microscope. The gonads of triploid sandworms atrophied, and the germ cells remained in the early stage of development.

[0013] Preferably, the stainless steel metal filter cylindrical mesh includes a cylindrical body with an open top and a closed bottom. An air bladder is fitted on the top of the cylindrical body and is fixedly connected to the cylindrical body. Several seawater inlet and outlet components are provided on the side of the cylindrical body.

[0014] Preferably, a sealing cap is provided at the top of the cylinder, and the sealing cap is threadedly connected to the cylinder.

[0015] Preferably, a counterweight column is provided at the bottom of the cylinder, and the counterweight column is fixedly connected to the cylinder.

[0016] Preferably, the seawater inlet / outlet assembly includes a circular tube that penetrates the cylindrical body and is fixedly connected to the cylindrical body. A filter disc is provided at one end of the circular tube outside the cylindrical body and is fixedly connected to the circular tube. Several through holes are provided through the filter disc. A guide rod is provided through the center of the filter disc. A limiting plate is fixedly provided at one end of the guide rod outside the circular tube, and a sealing ball is fixedly provided at one end of the guide rod inside the circular tube. A spring is sleeved on the guide rod, with one end of the spring fixedly connected to the limiting plate and the other end of the spring fixedly connected to the filter disc.

[0017] The beneficial effects of this invention are: This invention utilizes a stainless steel cylindrical filter mesh to assist in the artificial insemination of *Nereidum spp.* (a type of sandworm). Through cold shock treatment, triploidy is induced in the sandworm. Flow cytometry and tissue section analysis confirm an induction rate of over 80%. The method is simple, cost-effective, and efficient, and can be used to induce triploidy in marine annelids. Specifically: 1. Simple and fast: In actual operation, this invention only requires placing the fertilized eggs of *Pteris vittata* in ice water for 45 minutes after fertilization and then gradually rewarming them to induce triploidy. The induction operation is simple, the time is short, and the induction rate is high.

[0018] 2. Cost-saving: The invention induces triploid *Pteris vittata* using temperature shock, requiring no special equipment. This method is suitable for rapid processing in farms and large-scale breeding, saving on consumable and instrument costs.

[0019] 3. High induction rate: The triploid *Nematocystis didentata* induced by the operation of this invention has an induction rate of over 80%, as identified by flow cytometry and tissue sections.

[0020] 4. High survival rate: The survival rate of triploid sandworms with double toothed circumference induced by the operation of this invention is over 60%, which is relatively high compared to the control (91%). Attached Figure Description

[0021] Figure 1 This invention relates to the detection of ploidy in *Pteris didentata*. In this embodiment, A represents the control group (2n); B represents the triploid induction group (3n).

[0022] Figure 2 This invention provides an embodiment for identifying the gonadal tissue of *Pterocarya didentata*. A1 and A2 are controls, and A2 is a magnified view of the location indicated by the arrow in A1; B1 and B2 are triploid induction groups, and B2 is a magnified view of the location indicated by the arrow in B1; the arrows indicate the location of the gonads. Figure 3 This is a schematic diagram of the structure of a stainless steel metal filter cylindrical mesh for inducing triploid polychaete in a double-toothed perichaete worm according to the present invention; Figure 4This is a schematic diagram of the seawater inlet / outlet component of a double-toothed periseri triploid induction method according to the present invention; Reference numerals: 1. Sealing cap; 2. Airbag; 3. Counterweight column; 4. Seawater inlet / outlet assembly; 5. Limiting plate; 6. Spring; 7. Through hole; 8. Circular tube; 9. Sealing ball; 10. Filter disc; 11. Guide rod; 12. Cylinder. Detailed Implementation

[0023] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0024] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," "front end," "rear end," "both ends," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0025] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. Example

[0026] Reference Figure 1-2 (1) On March 26, 2024, a triploid induction experiment of *Pterygodium diploidum* was conducted at Fujian Yangze Marine Biotechnology Co., Ltd. Healthy sexually mature *Pterygodium diploidum* (body length > 15 cm, weight > 2 g, 40 females and 10 males) were selected for artificial insemination. Female and male *Pterygodium diploidum* were cut into pieces in a stainless steel metal filter cylinder with a pore size of 2 mm. The cylinder was immersed in a tank of seawater at room temperature. The sperm and eggs were released from the cut gonadal tissue and fertilized under the activation of seawater. The cutting time of male *Pterygodium diploidum* was strictly controlled (1-2 min). The time when the male tissue was cut was the start time of fertilization. This time was recorded for subsequent cold shock treatment of fertilized eggs.

[0027] (2) Four minutes after fertilization, the fertilized eggs sank to the bottom of the tank. The seawater was discarded and ice seawater was added back. The temperature was controlled at 0-4℃. The fertilized eggs were subjected to cold shock treatment in ice water for 45 minutes. Then the temperature was gradually increased to room temperature (8℃ each time, in 3 times). They were cultured normally in sync with the untreated control. The water temperature was 20℃-25℃ and the salinity was 25‰-30‰.

[0028] (3) After the sandworm larvae hatched for 20 days, their cells were classified and counted using flow cytometry. Using a normally artificially cultured diploid *Pterygodon spp.* as a reference, the DNA content of the experimental sandworms was determined. Cell ploidy was detected based on the DNA content and fluorescence intensity of the cells. Ten *Pterygodon spp.* individuals were taken from each of the induction group and the control group. Buffer solution was added, the samples were chopped, filtered, and DAPI staining solution was added. The samples were then analyzed. Finally, the DNA content of 8 cells was found to be 3n, and the triploidy induction rate was 80%.

[0029] (4) After flow cytometry analysis, the induction rate was further observed and statistically analyzed through gonadal tissue sections. Samples were randomly taken from the control and induction groups at 1, 2, and 3 months of age and fixed with Born's reagent. The tissues fixed overnight were dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, sectioned to a thickness of 4 μm, and stained with hematoxylin and eosin (H&E). Finally, gonadal development was observed using an optical microscope. The results showed that the gonads of the 3-month-old control group were of normal size, and germ cells could proliferate and differentiate, while the gonads of the induction group were atrophied, and germ cells remained at an early stage. This further histologically confirms that they are triploid. Example

[0030] like Figure 3-4 As shown, while all other parts are the same as in Example 1, the difference between this embodiment and Example 1 is that: the stainless steel metal filter cylindrical mesh includes a cylindrical body 12, which is open at the top and closed at the bottom. An air bladder 2 is fitted onto the top of the cylindrical body 12 and is fixedly connected to the cylindrical body 12. Several seawater inlet / outlet components 4 are provided on the side of the cylindrical body 12. A sealing cap 1 is provided at the top of the cylindrical body 12 and is threadedly connected to the cylindrical body 12. A counterweight column 3 is provided at the bottom of the cylindrical body 12 and is fixedly connected to the cylindrical body 12. The seawater inlet / outlet assembly 4 includes a circular pipe 8 that passes through a cylindrical body 12 and is fixedly connected to the cylindrical body 12. A filter disc 10 is provided at one end of the circular pipe 8 outside the cylindrical body 12 and is fixedly connected to the circular pipe 8. Several through holes 7 are provided through the filter disc 10. A guide rod 11 is provided through the center of the filter disc 10. A limiting plate 5 is fixedly provided at one end of the guide rod 11 outside the circular pipe 8 and a sealing ball 9 is fixedly provided at one end of the guide rod 11 inside the circular pipe 8. A spring 6 is sleeved on the guide rod 11. One end of the spring 6 is fixedly connected to the limiting plate 5 and the other end of the spring 6 is fixedly connected to the filter disc 10.

[0031] Remove the sealing cap 1, cut the female and male sandworms into pieces, and put them into the cylinder 12. After the stainless steel metal filter cylindrical mesh is placed in the seawater, the cylinder 12 floats on the water surface under the buoyancy of the airbag 2. The water flows into the round tube 8 through the through hole 7, and then the water flows open the sealing ball 9, allowing seawater to enter the cylinder 12. When the cylinder 12 is full of seawater, the guide rod 11 is pulled back and the sealing ball 9 is reset under the elastic force of the spring 6, so that the sealing ball 9 is inserted into the round tube 8 and the round tube 8 is sealed, thereby preventing the sandworms in the cylinder 12 from drifting out of the cylinder 12.

[0032] 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 illustrative of the principles of 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 this invention is defined by the appended claims and their equivalents.

Claims

1. A method for inducing triploidity in *Pteris vittata*, characterized in that: This involves placing the fertilized eggs of *Pteris vittata* in ice water for cold shock treatment, followed by a gradual warming process to room temperature.

2. The method for inducing triploidity in *Nematocystis jirovecii* according to claim 1, characterized in that: Sexually mature *Pterygodon spp.* were selected for artificial insemination. Female and male *Pterygodon spp.* were cut into pieces sequentially in a stainless steel metal filter cylinder. The cylinder was then immersed in seawater at room temperature. The sperm and eggs were released from the cut gonadal tissue and fertilized under the activation of seawater.

3. The method for inducing triploidity in *Pteris vittata* according to claim 2, characterized in that: The time for cutting male sandworm tissue into pieces is 1-2 min. The time when the male tissue is cut off is the time when fertilization begins. This time is recorded for subsequent cold shock treatment of fertilized eggs.

4. The method for inducing triploidity in *Nematocystis jirovecii* according to claim 1, characterized in that: The cold shock treatment of the fertilized eggs was carried out by placing the fertilized eggs in ice water (0-4℃) for 45 minutes after fertilization, and then gradually raising the temperature to room temperature (each time by 8℃, in 3 times).

5. The method for inducing triploidity in *Nematocystis jirovecii* according to claim 4, characterized in that: After the sandworm larvae hatched for 20 days, ploidy was determined. Flow cytometry was used to classify and count the cells. Using a normally artificially bred diploid sandworm sample as a reference, the DNA content of the experimental sandworms was measured. Cell ploidy was detected based on the DNA content and fluorescence intensity of the cells. After flow cytometry analysis, gonadal development was further observed through gonadal tissue sections.

6. The method for inducing triploidity in *Nematocystis jirovecii* according to claim 5, characterized in that: The gonadal tissue sections were randomly sampled from the control and induction groups at 1-month, 2-month, and 3-month-old rearing ponds. The sections were fixed with Born's reagent, dehydrated overnight with graded ethanol, cleared with xylene, embedded in paraffin, and sectioned to a thickness of 4 μm. The sections were then stained with hematoxylin and eosin (H&E). Finally, the gonadal development was observed using an optical microscope. The gonads of triploid sandworms atrophied, and the germ cells remained in the early stages of development.

7. The method for inducing triploidity in *Nematocystis jirovecii* according to claim 6, characterized in that: The stainless steel metal filter cylindrical mesh includes a cylindrical body (12), the top of the cylindrical body (12) is open and the bottom is closed. An air bag (2) is fitted on the top of the cylindrical body (12), and the air bag (2) is fixed to the cylindrical body (12). Several seawater inlet and outlet components (4) are provided on the side of the cylindrical body (12).

8. The method for inducing triploidity in *Nematocystis jirovecii* according to claim 7, characterized in that: The top of the cylinder (12) is provided with a sealing cap (1), which is threadedly connected to the cylinder (12).

9. The method for inducing triploidy in *Nematocystis jirovecii* according to claim 8, characterized in that: The bottom of the cylinder (12) is provided with a counterweight column (3), which is fixedly connected to the cylinder (12).

10. The method for inducing triploidy in *Pteris vittata* according to claim 9, characterized in that: The seawater inlet / outlet assembly (4) includes a circular tube (8) that penetrates the cylinder (12) and is fixedly connected to the cylinder (12). A filter disc (10) is provided at one end of the circular tube (8) outside the cylinder (12) and is fixedly connected to the circular tube (8). Several through holes (7) are provided through the filter disc (10). A guide rod (11) is provided through the center of the filter disc (10). A limiting plate (5) is fixedly provided at one end of the guide rod (11) outside the circular tube (8) and a sealing ball (9) is fixedly provided at one end of the guide rod (11) inside the circular tube (8). A spring (6) is sleeved on the guide rod (11). One end of the spring (6) is fixedly connected to the limiting plate (5) and the other end of the spring (6) is fixedly connected to the filter disc (10).

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