Method for inducing oviposition of mesna and method for collecting and preserving ova of mesna

Abstract

The invention discloses a method for inducing oviposition of mesna and a method for collecting and storing mesna eggs, and relates to the technical field of water ecological restoration, and the method comprises the following steps: S1, collecting local mesna in an external pond, and temporarily culturing the local mesna in an indoor container; s2, filtering and sterilizing natural river water or lake water, and then adding calcium chloride, dry yeast powder and mixed algae liquid to serve as a special culture solution for the meson; s3, soaking a plurality of water-absorbing porous materials into green algae, and taking out after a period of time; and S4, adding a certain amount of special culture solution for the meson into the culture cylinder, adding a plurality of pieces of water-absorbing porous materials soaked by green algae, introducing a plurality of white meson larvae in a good state into the culture cylinder, and then sequentially carrying out three-stage culture. The method can efficiently induce the oviposition of the meson, shortens the culture time, improves the egg yield, and improves the economic benefit; the porous quartz nano material is used for attracting the meson to spawn directionally.

Description

Technical Field

[0001] The present invention relates to the technical field of water ecological restoration, and in particular to a method for inducing spawning of ostracods and a method for collecting and preserving ostracod eggs. Background Art

[0002] Ostracods are a class of tiny crustaceans widely distributed in aquatic ecosystems, including freshwater, oceans, and wetlands. They are widely used in water quality monitoring, ecotoxicology, biological research, and environmental assessment. Ostracods are highly adaptable, able to survive in varying water quality conditions and filter-feed on plankton (especially algae) in the water. By filter-feeding, ostracods ingest large amounts of phytoplankton, cyanobacteria, and organic matter from the water, thereby inhibiting the excessive growth of cyanobacteria and other algae. Their excreta also provide nutrition to other microorganisms in the water, helping to maintain the balance of aquatic ecosystems. Therefore, they have important application value in the management of eutrophication, the prevention and control of cyanobacteria, ecological restoration, and water quality monitoring.

[0003] In natural water bodies, ostracod eggs are generally laid outside the body and attached to stones in the water or stems and leaves of aquatic plants for hatching. Since ostracods lay eggs in many places and they are not fixed, it is very difficult to collect the eggs. In laboratory research and ecological applications of ostracods, the production, collection and preservation methods of ostracod eggs are crucial to the research and production process. At present, although there have been some studies on ostracods, there are still certain technical gaps in how to induce them to lay eggs, increase the egg-laying rate, and how to efficiently collect and preserve eggs. In particular, in practical applications, how to improve the egg-inducing rate, ensure the quality and hatching rate of eggs, and increase the shelf life of eggs are all issues that need to be solved urgently. Summary of the Invention

[0004] In order to address the deficiencies in the prior art, the present invention provides a method for inducing ostracod egg laying and a method for collecting and preserving ostracod eggs, which can induce ostracods to lay eggs in a targeted and efficient manner.

[0005] In order to achieve the purpose of the present invention, the following scheme is proposed: A method for inducing spawning of ostracods, comprising the following steps: S1. Collect local ostracods from external ponds and temporarily raise them in containers indoors; S2. Filtering and sterilizing natural river or lake water, adding calcium chloride, dry yeast powder, and mixed algae solution to prepare a special culture medium for ostracods, wherein the mixed algae solution includes one or more of Chlorella, Scenedesmus, Microcystis, Dinoflagellate, and Diatom; S3, soaking several pieces of water-absorbent porous materials in green algae and taking them out after a period of time; S4. Add a certain amount of ostracod culture medium to the culture tank, add several pieces of water-absorbing porous material soaked in green algae, introduce a number of white ostracod larvae in good condition into the culture tank, and then go through three stages of cultivation in sequence: Stage 1: Place the culture tank in an artificial climate chamber at a temperature of 18°C ​​to 22°C, a light intensity of 2500 lux, and a light-dark cycle of 16h:8h for 14 days. Stage 2: Drain one-third of the culture medium and bottom sediment and replenish with specialized culture medium for ostracods. Adjust the artificial climate chamber conditions to 20°C to 25°C, 2500 lux of light intensity, and a 12h:12h light-dark cycle for seven days. After the first and second stages of cultivation, the ostracods gradually grow larger and their color deepens to brownish-yellow. The third stage: drain 1 / 3 of the culture medium and bottom sediment, and replenish the special culture medium for ostracods; adjust the conditions of the artificial climate box to 20℃~25℃, light intensity of 2000lux, and light and dark time of 8h:16h. Orange insect egg clusters appear on the surface of the water-absorbing porous material, and egg laying lasts for 21 days.

[0006] Furthermore, in step S2, the concentration of calcium chloride is 0.05 g / L~0.2 g / L, the amount of high-activity dry yeast powder added is about 0.01%, and the amount of mixed algae liquid added is about 2%, wherein the ratio of Chlorella: Scenedesmus: Microcystis: Dinoflagellate: Diatom is 5:5:1:1:1.

[0007] Furthermore, in step S3, the water-absorbing porous material is taken out after soaking for 24 hours.

[0008] Furthermore, in step S4, a certain amount of ostracod culture medium is added into the culture tank, wherein the amount of water-absorbing porous material is 2 pieces / liter, and the amount of ostracod larvae is 50 / liter.

[0009] Furthermore, in the second stage of cultivation, about 0.1% concentrated algae liquid and about 0.002% highly active dry yeast powder were added to the culture tank every 3 days. The concentrated algae liquid was a mixed algae liquid concentrated by centrifugation, and the volume was 1 / 5 of the original volume.

[0010] Furthermore, in the third stage of cultivation, about 0.1% concentrated algae solution and about 0.002% highly active dry yeast powder were added to the culture tank every three days.

[0011] Furthermore, in the third stage of cultivation, if white newborn ostracod larvae appear, the white newborn ostracod larvae are transferred to other cultivation tanks for expanded cultivation.

[0012] A method for collecting and preserving ostracod eggs is used for collecting and preserving ostracods obtained by the method for inducing spawning of ostracods, comprising the following steps: taking out a water-absorbing porous material, rinsing attachments on the surface of the material with a small amount of clean water, transferring the water-absorbing porous material into a bucket, adding pure water to submerge the material, and placing the material in a refrigerator at 4°C to preserve the ostracod eggs for a long time.

[0013] The beneficial effects of the present invention are: 1. The present invention utilizes the combined stimulation of light and temperature to efficiently induce ostracods to lay eggs, shortening the culture time, increasing the egg yield, and improving economic benefits.

[0014] 2. By soaking the water-absorbing porous material with green algae, the ostracods are attracted to lay eggs in a targeted manner, solving the problem of difficult egg collection caused by the multiple and irregular egg-laying locations in natural water bodies. The attached green algae provide ample food for the larvae, while the porous structure provides shelter for the ostracods.

[0015] 3. It fills the gap in the existing technology of indoor ostracod culture technology, has low requirements for the culture environment, is relatively simple to operate, and has controllable culture conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A flow chart of the method for inducing spawning of ostracods is shown. DETAILED DESCRIPTION

[0017] Example 1 like Figure 1 As shown, this embodiment provides a method for inducing spawning of ostracods, comprising the following steps: S1. Collect local ostracods from external ponds and temporarily raise them in containers indoors.

[0018] S2. After filtering and sterilizing natural river or lake water, add 0.1g / L calcium chloride, 0.01% highly active dry yeast powder, and 2% mixed algae liquid as a special culture medium for ostracods. Calcium chloride can provide the calcium required for the ostracod shells. The mixed algae liquid contains Chlorella, Scenedesmus, Microcystis, Dinoflagellate, and Diatom in a ratio of 5:5:1:1:1.

[0019] Among them, 0.1g / L calcium chloride means adding 0.1g calcium chloride to 1L water, 0.01% high-activity dry yeast powder means adding 0.1g high-activity dry yeast powder to 1L water, and 2% mixed algae liquid means adding 20mL mixed algae liquid to 1L water.

[0020] S3. Soak several pieces of water-absorbing porous materials in the green algae and take them out after soaking for 24 hours. The water-absorbing porous materials are porous quartz nanomaterials.

[0021] S4. Add 7 L of ostracod culture medium to the culture tank, add 14 pieces of porous quartz nanomaterials soaked in green algae, introduce 350 healthy white ostracod larvae into the culture tank, and then culture them in the following three stages: Stage 1: Place the culture tank in an artificial climate chamber at a temperature of 18°C ​​to 22°C, a light intensity of 2500 lux, and a light-dark cycle of 16h:8h for about 14 days; Phase II: Drain 1 / 3 of the culture medium and bottom sediment, and add special culture medium for ostracods; adjust the conditions of the artificial climate chamber to 20℃~25℃, 2500lux light intensity, and 12h:12h light-dark cycle for about 7 days. Add 7mL of concentrated algae solution and 0.14g of highly active dry yeast powder to the culture tank every 3 days. The ostracods gradually grow and their color gradually deepens to brown-yellow, indicating that the ostracods are mature at this time; the concentrated algae solution is the mixed algae solution concentrated by centrifugation, and the volume is 1 / 5 of the original volume.

[0022] Stage 3: Drain one-third of the culture medium and bottom sediment and replenish with specialized ostracod culture medium. Adjust the artificial climate chamber conditions to 20°C–25°C, 2000 lux of light intensity, and an 8:16 light-dark cycle. Add 7 mL of concentrated algae solution and 0.14 g of highly active dry yeast powder to the culture tank every three days. Orange egg masses will appear on the surface of the nanomaterial, and egg laying will continue for approximately 21 days. If white newborn ostracod larvae appear, transfer them to other culture tanks for expansion.

[0023] This invention utilizes a combination of light and temperature stimulation to efficiently induce ostracod egg laying, shortening incubation time, increasing egg yield, and boosting economic benefits. The invention utilizes a porous quartz nanomaterial as a habitat and egg-laying platform for ostracods. This material possesses a honeycomb microporous structure, excellent surface adhesion, and strong adsorption capacity, attracting ostracods to rest and lay eggs on its surface. This solves the problem of difficult egg collection in natural water bodies, where ostracods lay eggs in multiple and irregular locations.

[0024] The conditions for inducing spawning of ostracods were determined as follows: Experiment 1 (1) Experimental purpose Determine the effects of potassium, calcium, sodium, and magnesium on the growth and reproduction of ostracods.

[0025] (2) Experimental steps ①Select ostracods at the end of the first stage; ② Take 10L of pure water, add 1g of high-activity yeast powder and 200mL of mixed algae solution, mix and dissolve completely; take 22 tissue culture bottles, add 300mL of the above culture solution and 15 ostracods to each bottle; ③Add the corresponding amount of reagents according to the numbers in Table 1; Table 1 Reagents in each experimental group Table 1 ④ Place in an artificial climate chamber with a temperature of 25°C (12 hours) and 20°C (12 hours); a humidity of 50%; a light intensity of 2000 lux, and a lighting time of 12 hours; ⑤Observe the experimental results, take photos and record them, and add concentrated algae liquid and highly active dry yeast powder every week.

[0026] (3) Experimental results Table 2 Egg laying time and egg laying amount of each experimental group Conclusion: Excessive concentration can easily cause the death of ostracods; the first appearance time of ostracod eggs is similar, and the calcium chloride experimental group has relatively more ostracod eggs; when the calcium chloride concentration is 0.05g / L~0.2g / L, the ostracod eggs are relatively more.

[0027] Experiment 2 (1) Experimental purpose Determine the effects of potassium, calcium, sodium, and magnesium on the growth and reproduction of ostracods; explore the effects of light duration on the growth and reproduction of ostracods.

[0028] (2) Experimental steps ①Select the second-stage ostracods; ② Take 20L of pure water, add 2g of highly active yeast powder and 400mL of mixed algae solution, mix and dissolve completely; take 20 wide-mouth bottles, add the above culture solution respectively, and add 50 ostracods to each bottle; ③ Add the corresponding amount of reagents according to the numbers in Table 3; Table 3 Reagents of each experimental group Table 2 ④ Place in an artificial climate chamber with a temperature of 25°C (12h) and 20°C (12h); humidity of 50%; light intensity of 2000 lux, and set the corresponding lighting time according to the table above; ⑤Observe the experimental results, take photos and record them, and add concentrated algae liquid and highly active dry yeast powder every week.

[0029] (3) Experimental results Table 4. Egg laying time and egg laying amount of each experimental group Table 2 Conclusion: Ostracods in some experimental groups could not adapt and died; the time for the first obvious appearance of ostracod eggs in the 8-h light experimental group was shorter; after 21 days, the amount of ostracod eggs in the calcium chloride experimental group was relatively greater.

[0030] Experiment 3 (1) Experimental purpose Determine the effects of Chlorella, Scenedesmus, Microcystis, Dinoflagellates, and Diatoms on the growth and reproduction of ostracods.

[0031] (2) Experimental steps ①Select the first-stage newly born ostracods; ② Take 20L of pure water, add 2g of highly active dry yeast powder, mix and dissolve completely; take 18 wide-mouth bottles, add 800mL of the above culture medium and 40 newborn ostracods to each bottle; ③ According to the number in Table 5, add the corresponding amount of laboratory cultured algae solution (Chlorella, Scenedesmus, Microcystis, Dinoflagellate, Diatom); Table 5 Reagents of each experimental group Table 3 ④ Place the culture tank in an artificial climate chamber and culture for about 14 days under the conditions of 20°C, 2500 lux of light intensity, and 16h:8h of light and dark cycle; ⑤Afterwards, adjust the conditions of the artificial climate chamber to 25℃ (12h) and 20℃ (12h); the light intensity is 2500lux, and the lighting time is 12h, which lasts for about 7 days; ⑥ Adjust the conditions of the artificial climate chamber to 20℃~25℃, light intensity to 2000lux, and light-dark time to 8h:16h until the end of the experiment.

[0032] ⑦Observe the experimental results, take photos and record them. Every three days, add 0.002% highly active dry yeast powder to the blank group and 16 mL of the corresponding algae solution to the other groups.

[0033] (3) Experimental results Table 6 Spawning time and egg production of each experimental group Table 3 Conclusion: The first appearance of ostracod eggs in the Microcystis, Dinoflagellate and Diatom experimental groups was slightly earlier than that in the other experimental groups; the egg production in the Chlorella and Scenedesmus experimental groups was higher than that in the other experimental groups.

[0034] Experiment 4 (1) Experimental purpose To determine the effects of different proportions of mixed algae solutions (Chlorella, Scenedesmus, Microcystis, Dinoflagellates, Diatoms) on the growth and reproduction of ostracods.

[0035] (2) Experimental steps ①Select the first-stage newly born ostracods; ② Take 20L of pure water, add 2g of highly active dry yeast powder, mix and dissolve completely; take 18 wide-mouth bottles, add 800mL of the above culture medium and 40 newborn ostracods to each bottle; ③ According to the number in Table 7, add 16mL of mixed algae solution according to the proportion in the table below; Table 7 Reagents of each experimental group Table 4 ④ Place the culture tank in an artificial climate chamber and culture for about 14 days at a temperature of 20°C, a light intensity of 2500 lux, and a light-dark cycle of 16h:8h; ⑤Afterwards, adjust the conditions of the artificial climate chamber to 25℃ (12h) and 20℃ (12h); the light intensity is 2500lux, and the lighting time is 12h, which lasts for about 7 days; ⑥ Adjust the conditions of the artificial climate chamber to 20℃~25℃, light intensity to 2000lux, and light-dark time to 8h:16h until the end of the experiment.

[0036] ⑦Observe the experimental results, take photos and record them, and add 16mL of the corresponding algae solution every three days.

[0037] (3) Experimental results Table 8 Egg laying time and egg laying amount of each experimental group Table 4 Conclusion: The higher the proportion of Chlorella and Scenedesmus in the culture medium, the greater the amount of ostracod eggs laid; among them, the maximum amount of ostracod eggs was achieved when the ratio of Chlorella: Scenedesmus: Microcystis: Dinoflagellate: Diatom was 5:5:1:1:1; the higher the proportion of Microcystis, Dinoflagellate and Diatom, the shorter the time for the first spawning of ostracods.

[0038] Experiment 5 (1) Experimental purpose Determine the effects of porous quartz nanomaterials, volcanic rocks, calcified rocks, and porous sponges on induced spawning of ostracods.

[0039] (2) Experimental steps ①Select mature ostracods that have passed the first and second stages of cultivation; ② Take three pieces of porous quartz nanomaterials and approximately equal volumes of volcanic rock, calcified fossils, and porous sponges and soak them in green algae for about 24 hours; ③ In a 7L glass jar, add 7L of pure water, 0.7g of highly active dry yeast powder, and 140mL of mixed algae solution (Chlorella: Scenedesmus: Microcystis: Dinoflagellate: Diatom = 5:5:1:1:1). After mixing thoroughly, add approximately 350 mature ostracods. Add the ingredients from ② to the glass jar. ④ Place in an artificial climate chamber with a temperature of 25°C (12 hours) and 20°C (12 hours); a humidity of 50%; a light intensity of 2000 lux, and a lighting time of 12 hours; ⑤Observe the experimental results, take photos and record them, and add corresponding concentrated mixed algae liquid and highly active dry yeast powder every 3 days.

[0040] (3) Experimental results Table 9 Spawning time and egg production of each experimental group Table 5 Conclusion: Porous quartz nanomaterials can better attract ostracods to stay and lay eggs.

[0041] Example 2 This embodiment provides a method for collecting and preserving ostracod eggs, which is used to collect and preserve ostracods obtained using the method for inducing ostracod egg laying described in Example 1, comprising the following steps: removing the nanomaterial, rinsing the attachments on the surface of the nanomaterial with a small amount of clean water, transferring the nanomaterial to a 25L bucket, adding pure water to submerge the nanomaterial, and placing it in a 4°C refrigerator to preserve the ostracod eggs for a long time.

[0042] The above embodiments are only used to illustrate the technical ideas and features of the present invention and are not intended to be exclusive or limit the present invention. It should be understood by those skilled in the art that various changes or equivalent substitutions made to the present invention without departing from the scope of the present invention are within the scope of protection of the present invention.

Claims

1. A method for inducing spawning of ostracods, characterized in that: The following steps are involved: S1. Collect local ostracods from external ponds and temporarily raise them in containers indoors; S2. Filtering and sterilizing natural river or lake water, adding calcium chloride, dry yeast powder, and mixed algae solution to prepare a special culture medium for ostracods, wherein the mixed algae solution includes one or more of Chlorella, Scenedesmus, Microcystis, Dinoflagellate, and Diatom; S3, soaking several pieces of water-absorbent porous materials in green algae and taking them out after a period of time; S4. Add a certain amount of ostracod culture medium to the culture tank, add several pieces of water-absorbing porous material soaked in green algae, introduce a number of white ostracod larvae in good condition into the culture tank, and then go through three stages of cultivation in sequence: Stage 1: Place the culture tank in an artificial climate chamber at a temperature of 18°C ​​to 22°C, a light intensity of 2500 lux, and a light-dark cycle of 16h:8h for 14 days. Stage 2: Drain one-third of the culture medium and bottom sediment and replenish with specialized culture medium for ostracods. Adjust the artificial climate chamber conditions to 20°C to 25°C, 2500 lux of light intensity, and a 12h:12h light-dark cycle for seven days. After the first and second stages of cultivation, the ostracods gradually grow larger and their color deepens to brownish-yellow. The third stage: drain 1 / 3 of the culture medium and bottom sediment, and replenish the special culture medium for ostracods; adjust the conditions of the artificial climate box to 20℃~25℃, light intensity of 2000lux, and light and dark time of 8h:16h. Orange insect egg clusters appear on the surface of the water-absorbing porous material, and egg laying lasts for 21 days.

2. The method for inducing spawning of ostracods according to claim 1, characterized in that: In step S2, the concentration of calcium chloride is 0.05 g / L to 0.2 g / L, the amount of dry yeast powder added is 0.01%, and the amount of mixed algae liquid added is about 2%, wherein the ratio of Chlorella: Scenedesmus: Microcystis: Dinoflagellate: Diatom is 5:5:1:1:

1.

3. The method for inducing spawning of ostracods according to claim 1, characterized in that: In step S3, the water-absorbing porous material is taken out after soaking for 24 hours.

4. The method for inducing spawning of ostracods according to claim 1, characterized in that: In step S4, a certain amount of ostracod culture medium is added into the culture tank, wherein the amount of the water-absorbing porous material is 2 pieces / liter, and the amount of the ostracod larvae is 50 / liter.

5. The method for inducing spawning of ostracods according to claim 1, characterized in that: In the second stage of cultivation, about 0.1% concentrated algae liquid and about 0.002% highly active dry yeast powder were added to the culture tank every 3 days. The concentrated algae liquid was a mixed algae liquid concentrated by centrifugation, and the volume was 1 / 5 of the original volume.

6. The method for inducing spawning of ostracods according to claim 1, characterized in that: In the third stage of cultivation, about 0.1% concentrated algae solution and about 0.002% highly active dry yeast powder were added to the culture tank every 3 days.

7. The method for inducing spawning of ostracods according to claim 1, characterized in that: In the third stage of cultivation, if white newborn ostracod larvae appear, the white newborn ostracod larvae are transferred to other culture tanks for expanded cultivation.

8. A method for collecting and preserving ostracod eggs, characterized in that: The method is used for collecting and preserving ostracods obtained by the method for inducing spawning of ostracods as claimed in any one of claims 1 to 7, comprising the following steps: taking out a water-absorbing porous material, rinsing attachments on the surface of the material with a small amount of clean water, transferring the water-absorbing porous material into a bucket, adding pure water to submerge the material, and placing the material in a refrigerator at 4°C to preserve ostracod eggs for a long time.

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