A segmented culture method of Pseudodaphnia andersonii and its application

Abstract

The present invention provides a segmented culture method of Pseudo-dart Daphnia andersonii and its application, and relates to the technical field of aquaculture. The present invention provides a segmented culture method of Pseudo-dart Daphnia andersonii, wherein the parent of Pseudo-dart Daphnia andersonii is subjected to nutritional fortification and environmental regulation to promote its mother to hang eggs, the parents to mate, the mother to give birth to young, the parents are separated from the young, the young produced are collected, and different baits are fed to them for nutritional fortification, so that the nutrition is more comprehensive while the number is continuously expanded. It has been confirmed by experiments that the large-scale culture of Pseudo-dart Daphnia andersonii can be realized by the segmented culture method of the present invention, and the density of Pseudo-dart Daphnia andersonii can be increased to 5-8 / mL, which can more effectively improve the survival rate and growth rate of aquatic animal seedlings, and lay the foundation for the development of high-quality biological baits in the aquaculture industry.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a segmented culture method of Pseudodaphnia andersonii and an application thereof. Background Art

[0002] In recent years, with the increase in people's demand for aquatic products, the aquaculture industry has developed rapidly, which has greatly promoted the development of artificial breeding of aquatic animals. In the process of aquatic animal breeding, in addition to using microalgae, rotifers, and Artemia as starter baits, copepods have attracted people's attention because of their many advantages.

[0003] During the growth process, copepods need to go through 11-12 periods of metamorphosis before they can develop into adults. Their size is 100-1000μm, which can provide different calibers of starter bait for aquatic animal larvae. Copepods are rich in nutrients, rich in EPA and DHA. DHA is more than 10 times that of fortified Artemia. High content of DHA plays an important role in the normal pigmentation and nerves of some marine fish larvae during their development stage. Copepod nauplii also contain high phospholipids to meet the growth needs of fish and shrimp larvae. They are not only easy to digest, but also promote the absorption and transport of other lipids. Due to its many advantages, copepods are considered to be excellent live baits. In view of this, it is urgent to realize large-scale cultivation and nutritional fortification of copepods to meet the needs of the aquaculture market for animal starter baits.

[0004] At present, the main methods for culturing copepods are outdoor extensive farming and indoor intensive farming. Outdoor extensive farming generally uses outdoor ponds to cultivate copepods. As early as 1986 to 1994, in Norway, cod larvae were placed in ponds to cultivate copepods and achieved good results. However, this cultivation method is greatly affected by the abundance of microalgae in the water body and weather changes, so there are problems such as extremely unstable yields, short duration, and easy contamination by viruses and bacteria. Indoor intensive farming began in the 1990s, and people aimed to replace Artemia nauplii through large-scale cultivation of copepods. Some species of copepods, such as C. clarkii, C. tangnii, and C. true wide, have become the objects of intensive cultivation due to their strong adaptability to the environment. These species are characterized by small individuals, fast reproduction speed, and a wide range of suitable temperatures. They are suitable for laboratory cultivation and are very easy to form dominant populations outdoors. Compared with outdoor pond cultivation, intensive cultivation has stable yields, is less affected by nature, and has guaranteed quality, but the production cost is high. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a segmented culture method of Pseudodaphnia andersonii, which can realize large-scale culture of Pseudodaphnia andersonii and provide high-quality biological bait of different calibers for marine organisms at different developmental stages.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a segmented culture method of Pseudodaphnia andersonii, the method comprising the following steps:

[0008] (1) Collection and selection of adult Pseudodaphnia annae;

[0009] (2) Primary culture: inoculating the selected adult Daphnia Andrews into seawater, feeding one or more microalgae selected from Thalassiosira salina, Chaetoceros hornwort, and Chrysophyte until larvae are produced, and then filtering out the larvae using a 125-150 μm silk screen;

[0010] (3) Secondary culture: collecting the larvae filtered out in step (2) for culture, feeding one or more of golden algae, Chaetoceros hornwort and Thalassiosira to adults, supplementing with photosynthetic bacteria after the larvae develop to adults until they lay eggs, and collecting the produced larvae;

[0011] (4) Tertiary culture: The larvae obtained from the secondary culture are placed in a column photobioreactor for culture, and fed with one or more species of Chrysophyll, Chaetoceros and Thalassiosira until they reach the copepod larval stage, and then fed with scrubbed shrimp flakes until they reach adulthood.

[0012] Preferably, a plankton net with a pore size of 0.112 mm is used to filter the surface of seawater in an inverted 8-way manner for 1-3 minutes to collect Pseudodaphnia andersonii.

[0013] Preferably, the adult Pseudodaphnia andersonii are selected based on the characteristics of the collected Pseudodaphnia andersonii.

[0014] Preferably, in step (2), the temperature of the seawater is 25-30° C., the salinity is 23, the light intensity is 1000 lux, and the light cycle is 12h:12h.

[0015] Preferably, the culture conditions in step (3) are: salinity 20-30, pH 7.5-8.5, light intensity 8000 lux, and light cycle 12h:12h.

[0016] Preferably, the density of adults hanging eggs in step (3) is 3000-5000 eggs / L.

[0017] Preferably, the culture method in step (4) is an open semi-aerated semi-continuous method.

[0018] Preferably, the column-type photobioreactor comprises a fixed support, support legs, support footrests, a reactor body, a connecting device, a valve, and replaceable sieves with different apertures.

[0019] Preferably, the culture conditions in step (4) are: temperature 30-35°C, salinity 23-30.

[0020] The present invention also provides application of the segmented culture method in aquaculture.

[0021] The present invention provides a segmented culture method of Pseudo-dart Daphnia andersonii, wherein the parent of Pseudo-dart Daphnia andersonii is subjected to nutritional fortification and environmental regulation to promote the mother to lay eggs, the parents to mate, the mother to give birth to young, the parents are separated from the young, the young are collected, and different baits are fed to them for nutritional fortification, so that the nutrition is more comprehensive and the number is continuously expanded. It is confirmed by experiments that the segmented culture method of the present invention can realize large-scale culture of Pseudo-dart Daphnia andersonii, and the density of Pseudo-dart Daphnia andersonii can be increased to 5-8 / mL, and the survival rate and growth rate of aquatic animal seedlings are more effectively improved, laying a foundation for the development of high-quality biological baits in the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the structural diagram of the column photobioreactor.

[0023] Figure 2 is the number of nauplii under different parental densities.

[0024] Figure 3 Figure 3 is the effect of feeding different microalgae baits on the larvae of Pseudodendron andersoni, where A is the difference in developmental time of the larvae of Pseudodendron andersoni fed with different microalgae baits, and B is the difference in survival rate of the larvae of Pseudodendron andersoni fed with different microalgae baits.

[0025] Figure 4 The effects of adding photosynthetic bacteria on the larvae of Pseudodendron andersonii, where A is the effect of adding photosynthetic bacteria on the development time of Pseudodendron andersonii larvae, and B is the effect of adding photosynthetic bacteria on the survival rate of Pseudodendron andersonii larvae. DETAILED DESCRIPTION

[0026] The present invention provides a segmented culture method of Pseudodaphnia andersonii, the method comprising the following steps:

[0027] (1) Collection and selection of adult Pseudodaphnia annae;

[0028] (2) Primary culture: inoculating the selected adult Daphnia Andrews into seawater, feeding one or more microalgae selected from Thalassiosira salina, Chaetoceros hornwort, and Chrysophyte until larvae are produced, and then filtering out the larvae using a 125-150 μm silk screen;

[0029] (3) Secondary culture: collecting the larvae filtered out in step (2) for culture, feeding one or more of golden algae, Chaetoceros hornwort and Thalassiosira to adults, supplementing with photosynthetic bacteria after the larvae develop to adults until they lay eggs, and collecting the produced larvae;

[0030] (4) Tertiary culture: The larvae obtained from the secondary culture are placed in a column photobioreactor for culture, and fed with one or more species of Chrysophyll, Chaetoceros and Thalassiosira until they reach the copepod larval stage, and then fed with scrubbed shrimp flakes until they reach adulthood.

[0031] In the present invention, preferably, a plankton net with a pore size of 0.112 mm is used to filter the surface of the seawater for 1-3 minutes in an inverted 8 manner to collect Pseudodaphnia andersonii; the plankton net is preferably a No. 13 plankton net. The present invention selects and obtains adult Pseudodaphnia andersonii according to the characteristics of the collected Pseudodaphnia andersonii; the characteristics of the Pseudodaphnia andersonii are preferably observed under a dissecting microscope, and the adult Pseudodaphnia andersonii are preferably male and female developed to sexual maturity. In the present invention, the characteristics preferably include: female: body length 1.1-1.3mm, head and first thoracic segment fused; posterior thoracic angle blunt, distal margin with a row of small teeth; abdominal reproductive segment base with a hook-shaped spine on both sides, ventral reproductive aperture with a barbed spine; tail fork length is about 2.5 times the width, tail setae short, the base of the third tail setae on the outside is swollen; the fifth pair of thoracic legs is monopodial and symmetrical, the third segment is long and has an outer distal spine, the fourth segment is wide and short, the inner distal corner extends into a feather-like coarse spine, the outer distal margin also has a small spine, the distal segment is narrow and thorn-like, its length is about 4 times the length of the previous segment, and its inner margin base also has a small spine; male: body 1.0-1.1mm long, with blunt-rounded posterior corners of the chest and a pair of small spines on the dorsal distal edge; the tail setae are relatively long and not swollen at the base; the fifth pair of thoracic legs are asymmetrical, with the left leg having no inner limb, the inner edge of the second segment having a very wide protrusion with a blunt, beak-shaped tip, the inner edge of the third segment having a triangular protrusion with two small denticles on the tip, the distal segment being nearly rectangular, with a thorn at the proximal end of the outer edge, and a small denticle at the inner distal edge; the inner edge of the second coxae of the right leg having a coarse spiny inner limb with a small spine near the distal end, the outer distal spine of the first segment of the external limb being as long as the proximal end of the next segment, the inner edge protruding with a spine, the second segment being thick, the distal segment being narrow and curved like a claw, and the base of its inner edge being raised with a small spine.

[0032] In the present invention, the selected adult Pseudo-dart Daphnia andersonii is inoculated into seawater, and one or more microalgae selected from Thalassiosira, Chaetoceros, and Chrysophyte are fed until larvae are produced, and then the larvae are filtered out using a 125-150 μm sieve. In the present invention, the temperature of the seawater is preferably 25-30°C, the salinity is preferably 23, the light intensity is preferably 1000 lux, and the photoperiod is 12h:12h. In the present invention, the seawater is preferably placed in a transparent container to facilitate the observation of the development of Pseudo-dart Daphnia andersonii. In a specific embodiment of the present invention, the transparent container is preferably a transparent plastic barrel, and the volume of the transparent plastic barrel is preferably 50-100L. In the present invention, the inoculation density of the adult Pseudo-dart Daphnia andersonii is preferably 1 / mL, and the density of the mixed microalgae is preferably 1×10 4 -1×10 5 cell / mL.

[0033] In the collecting step (2) of the present invention, the larvae are filtered out for cultivation, and one or more of golden algae, horn algae and sea serrata are fed to adults. After the larvae develop to adults, photosynthetic bacteria are supplemented until eggs are hung, and the produced larvae are collected. In the present invention, the culture container is preferably a transparent container. In a specific embodiment of the present invention, the transparent container is preferably a transparent plastic barrel, and the volume of the transparent plastic barrel is preferably 50-100L. In the present invention, the density of the larvae culture is preferably 1 / mL; the culture conditions are preferably: salinity 20-30, pH 7.5-8.5, light intensity 8000lux, and light cycle 12h:12h. In the present invention, after the larvae develop to adults, photosynthetic bacteria are supplemented while feeding microalgae. The photosynthetic bacteria are rich in protein and can not only supplement the nutrition of the larvae but also purify the water quality. In the present invention, the density of the adults hanging eggs is preferably 3000-5000 / L, and the number of larvae produced is preferably 7000-8000 / L.

[0034] The present invention places the larvae obtained from the secondary culture in a column photobioreactor for culture, feeds one or more of Zhanjiang Chrysophyte, Chaetoceros and Thalassiosira to the copepod larvae stage, and then feeds the rubbed shrimp slices to the adult. In the present invention, the culture method is preferably an open semi-inflated semi-continuous method; the culture conditions are preferably: temperature 30-35°C, salinity 23-30. In the present invention, the column photobioreactor includes a fixed bracket, bracket legs, bracket footrests, a reactor body, a connecting device, a valve, and replaceable screens of different apertures. The photobioreactor is made of fully transparent acrylic material, with a screen at the bottom, and the culture water can be switched to pass through the screen to obtain a large number of larvae at different growth stages, and then the sterilized water and bait are added to the reactor for continued culture. In the present invention, the density of flea bodies in the culture stage is preferably 1 / mL. The entire growth and development process of the water flea of ​​the present invention includes nauplii, copepod larvae and adults. Under the three-stage culture conditions, the nauplii develop into copepod larvae after 60-80 hours, and develop into adults after 150-180 hours. Then, the nauplii of suitable caliber can be harvested in time according to production needs.

[0035] The present invention also provides application of the segmented culture method in aquaculture.

[0036] In the present invention, oxygenation is required in all culture stages, and preferably the water body is ensured to be in a microwave state.

[0037] In the present invention, all containers are disinfected with iodine solution, the first-level culture seawater is boiled for disinfection before use, and the second-level and third-level culture seawater is disinfected with 50% strong chlorine in an amount of 20 grams per ton of water, and after disinfection for 12 hours, 10 grams of sodium thiosulfate per ton of water is used for neutralization, and the water can be used after aeration for 24 hours. Before use, the water needs to be tested, and a certain amount of individuals are placed in the disinfected and neutralized water, and the vital signs are observed to be normal before use.

[0038] In the present invention, the primary culture feed is microalgae, and the concentration of the microalgae is 1×10 4 -1×10 5cell / L, the microalgae include one or more of Thalassiosira, Chaetoceros, and Chrysophyte. In the present invention, the method for culturing the microalgae preferably includes the following steps: inoculating the microalgae species into a microalgae culture solution for culturing, the culture temperature is 22-30°C, and the culture light intensity is 1200-2500lux. In the present invention, the method for culturing the microalgae species preferably includes the following steps: inoculating the required microalgae species into a microalgae culture solution for culturing: algae species quality: select algae that are not contaminated by pests, have vigorous growth, and have strong vitality for inoculation; the number of algae: it is better to be large rather than small. The more the inoculation number, the more dominant the algae can be in the culture process and the shorter the culture cycle; inoculation time: the inoculation time is selected between 8 and 10 am, and algae with strong floating movement ability are selected as algae species according to their phototropism characteristics. In the present invention, the temperature of the microalgae culture is preferably 22-30°C, more preferably 25-28°C; the light intensity of the microalgae culture is preferably 1200-2500 lux, more preferably 1500-2000 lux, and most preferably 1800 lux. The present invention does not specifically limit the time for the microalgae culture, and the time for the microalgae culture routinely used by those skilled in the art can be used. In the present invention, the microalgae culture solution uses the dechlorinated seawater used for secondary culture and tertiary culture as a solvent, and includes components with the following concentrations: NaNO3 75.0 mg / L, NaH2PO4·H2O 5.0 mg / L, Na2SiO3·9H2O 30.0 mg / L, and trace metals 1 ml / L; the trace metals include the following components: FeCl3·6H2O 3.15 g / L, Na2EDTA·2H2O 4.36 g / L, CuSO4·5H2O 9.8 mg / L, Na2MOO4·2H2O 6.3 mg / L, ZnSo4·7H2O 22.0 mg / L, COCl2·6H2O 10.0 mg / L, and MnCl2·H2O 180.0 mg / L.

[0039] In the present invention, the algae species are provided by the algae resources and aquaculture environment repair team of Guangdong Ocean University.

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] Example 1

[0044] The body lengths of Pseudodaphnia andersoni at different developmental stages are: nauplii: 121-320μm, copepod larvae: 325-860μm, and adults: 900-1200μm.

[0045] Segmented culture method:

[0046] (1) Field collection of zooplankton: According to the collection method for qualitative detection of zooplankton, use a 13# plankton net to slowly drag "∞" on the water surface for about 3 minutes. Put the collected water sample into a container disinfected with iodine solution and bring it back to the laboratory for separation as soon as possible.

[0047] (2) Selection of adult Pseudodaphnia andersonii: Select adult Pseudodaphnia andersonii with strong mobility under a dissecting microscope, and select male and female Pseudodaphnia andersonii that have developed to sexual maturity.

[0048] (3) Primary culture (seed preservation): The isolated Pseudo-dart Daphnia Andrews was placed in a 1000 mL conical flask, the water temperature was kept at 25 degrees, the salinity was 23, the light intensity was 1000 lux, the photoperiod was 12h:12h, and oxygenation was ensured, with the water flow showing a microwave state. The inoculation density was 1 cell / ml, and the feeding microalgae was a mixture of three microalgae: Thalassiosira, Chaetoceros, and Chrysophyta, and the density was kept at 1×10 5 cell / ml. When larvae are observed in the water, filter them out promptly with a 125μm mesh. The reproductive activity cycle of the parent is about 40 days. If the female's reproductive capacity is significantly weakened or it no longer lays eggs, it should be eliminated and no longer used for production.

[0049] (4) Secondary culture (parent culture): A 50L transparent plastic bucket is often used as a container for culturing larvae. The larvae produced in the same batch of primary culture are collected to make the larvae density 1 / ml. The environmental conditions are salinity 23, pH 8.0, light intensity 700lx, and light-dark ratio 12h:12h. During the larval stage, one or more mixed algae liquids of golden algae, Chaetoceros algae and Thalassiosira are fed. After the larvae develop into adults, microalgae are fed and photosynthetic bacteria are added. After the larvae develop into adults and lay eggs, the larvae produced are collected daily and the water is completely replaced.

[0050] (5) Tertiary culture (larval culture): A 2t column photobioreactor is used as the culture container. The photobioreactor used to realize Figure 1 The culture conditions were as follows: temperature 30-35℃, salinity 23-30, flea density controlled at 1 flea / mL, multiple meals per day feeding method, nauplii stage feed was one or more of Zhanjiang Isometric Chrysophyll, Chaetoceros and Thalassiosira, feed concentration was 30×10 4 / mL, and feed the rubbed shrimp chips after the copepod larvae. Under this condition, the nauplii develop into copepod larvae after about 60-80 hours, and develop into adults after 150-180 hours. The larvae of suitable caliber can be harvested in time according to production needs.

[0051] Among them, the specific mating and selection criteria of Pseudodaphnia andersonii in the segmented culture method are as follows:

[0052] (1) Active individuals with intact limbs were selected from wild Pseudodaphnia Andrews and placed in a suitable environment: temperature 20-35℃, salinity 20-30, pH 7.5-8.5. Under a microscope, it can be observed that the difference between male and female individuals is obvious: mature male individuals have grasping limbs and spinous processes on both sides of the male abdomen.

[0053] (2) Under suitable conditions, the female Daphnia Andrews will lay eggs quickly, and the eggs are stored in the egg sac. When mating, the two abdomens face each other, the head and thorax are facing each other, and the body is in a straight line. At this time, the environment should be kept quiet to avoid disturbing the mating activity. During mating, the male places the sperm capsule on the abdomen of the female. One mating can fertilize and develop several batches of eggs. After the eggs are fertilized, they will develop into nauplii through embryonic development. Starting from the nauplii stage I after hatching, each molt enters a new development stage.

[0054] Using artificial culture technology, it takes 7-9 days for Pseudo-dart Daphnia Andrews to develop from nauplii to the first sexual maturity. By culturing it in stages, the density can reach 5-10 / mL, significantly increasing the content of EPA, DHA and other ingredients in the flea body, making it more nutritious, and thus effectively improving the survival rate and growth rate of aquatic animal seedlings.

[0055] Example 2

[0056] According to the method steps described in Example 1, Daphnia cyprinidae was collected from an abandoned pond in Zhanjiang (E110°31′46.46″N20°56′10.48″) and cultured in stages. In the step (3) of staged culture, different parent densities were used for staged culture during the first stage culture. Specifically, the parent densities of Daphnia cyprinidae were set at 200 / L, 800 / L, 2000 / L, 5000 / L, 7000 / L, and 9000 / L in 1L of water. The experimental time was set to 7 days, the number of nauplii was counted continuously, the water was changed every day, and the algae feeding amount was 1 mgC / L. The experimental results are shown in Tables 1 and Figure 2 As shown:

[0057] Table 1 Effect of different parental densities on the number of larvae

[0058]

[0059]

[0060] It can be seen that when the density was 5000 / mL, the number of nauplii produced was the largest, at 7833 / mL. During the 7-day experimental period, when the density was 2000 / mL, the number of nauplii produced on the 6th day was second only to the highest number of nauplii produced. When the parent density was controlled at 2-5 / mL, the number of nauplii could reach a maximum of 7.8 / mL.

[0061] Example 3

[0062] 1. Effects of microalgae diets on the development and reproduction of Pseudodaphnia andersonii

[0063] (1) Developmental experiments

[0064] Under the dissecting microscope, 10 healthy and lively newly hatched NII stage nauplii were selected and placed in a 15mL test tube. 10mL of fresh seawater was added to each test tube and marked. The culture conditions were: temperature 28℃, salinity 27, and light 1200lx. Fresh bait microalgae were fed daily to make the bait concentration in the water body 1mgC / L (calculated by carbon content). Feeding was done once a day, and the water was completely changed before feeding. Four experimental groups were set up in the experiment, feeding Zhanjiang Isochrysis, Mu's Ceratocystis, Thalassiosira and Chlorella, respectively. Each experimental group was set up with two groups, A and B. Group A was used to observe the development of nauplii, and Group B was used to observe the development of copepod larvae. Each group had 3 parallels. At the beginning, the development of the larvae was observed every 6 hours. When the larvae developed to N5 stage nauplii and C4 copepod larvae, it was changed to observation every 2 hours. Each time, 3-4 larvae were randomly selected for observation. After observation, the larvae were immediately returned to the original test tubes. The development time and survival rate of nauplii and copepod larvae were recorded respectively. The results were as follows: Figure 3 .

[0065] It can be seen that different microalgae feeds have a significant effect on the development time of Pseudo-dart Daphnia andersonii (P<0.05). In the nauplii to copepod larvae stage, the Pseudo-dart Daphnia andersonii fed with Isochrysis Zhanjiangensis had the shortest development time from nauplii to copepod larvae, which was 70h. The second was the Chaetoceros group, with a development time of 74h, and then the Thalassiosira group, with a development time of 84h. The Pseudo-dart Daphnia andersonii fed with Chlorella had the longest development time from nauplii to copepod larvae, which required 93h. From the perspective of the entire development stage, the development time of the larvae fed with Isochrysis Zhanjiangensis was also the shortest, at 193h. The second was the Chaetoceros group and the Thalassiosira group, with development times of 212h and 239h, respectively. The time for the larvae to develop into adults of the Pseudo-dart Daphnia andersonii fed with Chlorella was the longest among the four groups, requiring 258h.

[0066] From the individual survival data, in the nauplii to copepod stage, the survival rate of the larvae of Pseudo-dart Daphnia andersonii fed with Isochrysis Zhanjiangensis was the highest, at 90%, followed by the Chaetoceros group and the Thalassiosira group, with larval survival rates of 83% and 80%, respectively. The larval survival rate of Pseudo-dart Daphnia andersonii fed with Chlorella was the lowest, at 67%. From the perspective of the entire development stage, the larval survival rate of Pseudo-dart Daphnia andersonii fed with Isochrysis Zhanjiangensis was significantly higher than that of the other three groups, reaching 60%. The copepod survival rates of Pseudo-dart Daphnia andersonii fed with Thalassiosira, Chaetoceros and Chlorella were all lower than 50%, at 47%, 43% and 40%, respectively. The above results show that feeding Isochrysis Zhanjiangensis is beneficial to the growth and survival of Pseudo-dart Daphnia andersonii from nauplii to copepods and from copepods to adults.

[0067] (2) Reproduction experiment

[0068] Under the dissecting microscope, one mature male and one female Pseudo-dart Daphnia Andrews were selected and placed in a 15 mL test tube, and 10 mL of fresh seawater was added to each test tube. Four experimental groups were set up in the experiment, and they were fed with Zhanjiang Isochrysis, Mou's Ceratocystis, Thalassiosira and Chlorella, respectively. Each experimental group had three parallels, and was fed once a day. The concentration of the microalgae in the bait (in terms of carbon content) was controlled to be 1.0 mgC / L. The larval situation of Pseudo-dart Daphnia Andrews was observed daily. When the larvae were found, they were counted in time, and the parents were selected and placed in fresh algae solution for further cultivation. The experiment was carried out for 10 consecutive days, and the number of larvae and the number of larvae were recorded. The results are shown in Table 2.

[0069] Table 2 Differences in total larvae, egg production per time and reproductive times of Pseudodaphnia andersonii fed with different microalgae baits

[0070]

[0071] It can be seen that there are obvious differences in the total number of larvae produced, the number of eggs laid each time and the number of reproduction times of Pseudo-dart Daphnia Andrews fed with different microalgae baits.

[0072] 2. Effects of adding photosynthetic bacteria to microalgae feed on the development and reproduction of Pseudodaphnia andersonii

[0073] (1) Developmental experiments

[0074] Ten healthy and lively newly hatched NII stage nauplii were selected under a dissecting microscope and placed in 15 mL test tubes. 10 mL of fresh seawater was added to each test tube and marked. The microalgae bait was Isochrysis Zhanjiangensis. The photosynthetic bacteria were purchased from Guangdong Green Hundred Biological Development Co., Ltd., and the number of viable bacteria was >0.5×10 8cfu / mL. A total of 5 experimental groups were set up in the experiment. In addition to making the concentration of microalgae in the water body reach 1mgC / L each time, each group was fed with photosynthetic bacteria in different proportions. The method of feeding photosynthetic bacteria: the photosynthetic bacteria were centrifuged at a speed of 8000rpm to remove the culture medium, and then diluted with distilled water and fed to copepods, so that the concentrations of photosynthetic bacteria in the water body were: 0mgC / L, 0.25mgC / L, 0.5mgC / L, 1mgC / L and 2mgC / L. Each experimental group was divided into two groups, A and B. Group A was used to observe the development of nauplii, and group B was used to observe the development of copepod larvae. There were 3 parallels in each group. The culture conditions and observation methods were the same as step 1. The experimental results are shown in the following table. Figure 4 .

[0075] It can be seen that the addition of photosynthetic bacteria has a significant effect on the development time of the larvae of Pseudo-dart Daphnia andersonii (P<0.05). In the development stage from nauplii to copepod larvae, under the condition of feeding the same amount of microalgae bait, the added photosynthetic bacteria can shorten the development time of the larvae. Among them, when the concentration of the added photosynthetic bacteria reached 1 mgC / L, the time required for the larvae was the shortest, which was 56±0.00h. Compared with the control group (0 mgC / L), the development time was shortened by about 20%. When the concentration of the added photosynthetic bacteria was 0.25 mgC / L, 0.5 mgC / L and 2 mgC / L, the larval development time was slightly shortened compared with the control group, which were 67.33±0.47h, 67.33±0.47h and 66±0.00h, respectively. In the entire development stage, under the condition of feeding the same amount of microalgae bait, the addition of photosynthetic bacteria can significantly affect the development time of the larvae. When the concentration of photosynthetic bacteria was 0.25mgC / L and 0.5mgC / L, the larval development time of the two experimental groups reached 184±3.30h and 183±4.24h, respectively, which was slightly shortened compared with the control group. When the concentration of photosynthetic bacteria added was 1mgC / L, the time required for the larvae to develop into adults was the shortest, which was 165±3.56h. Compared with the control group (0mgC / L), the development time was shortened by about 14.5%. When the concentration of photosynthetic bacteria added was 2mgC / L, the time required for the larvae to develop into adults was the longest, which was 210±0.00h.

[0076] In terms of individual survival rate, except for the 1mgC / L experimental group, which reached the level of the control group (90%), the survival rates of the other experimental groups were significantly lower than those of the control group. When the concentration of photosynthetic bacteria was 0.5mgC / L, the survival rate of nauplii was the lowest, which was 63%±4.71%. During the entire development stage, the survival rates of copepod larvae in the two groups with the concentration of photosynthetic bacteria added of 0.5mgC / L and 1mgC / L reached 67±4.16% and 73±1.57%, respectively, which were increased by about 12% and 22% respectively compared with the control group. The survival rates of copepod larvae in the two experimental groups with the concentration of photosynthetic bacteria added of 0.25mgC / L and 2mgC / L were significantly lower than those of the control group. Among them, when the concentration of photosynthetic bacteria added was 2mgC / L, the survival rate of copepod larvae was the lowest, which was 47%±3.14%.

[0077] (2) Reproduction experiment

[0078] Under the dissecting microscope, select one mature male and one female Daphnia pulex with eggs and place them in a 15mL test tube, and add 10mL of fresh seawater to each test tube. A total of 5 experimental groups were set up in the experiment. In addition to making the microalgae in the water body 1mgC / L, each group was also fed with photosynthetic bacteria in different proportions. The feeding method was the same as the development experiment in step 1. Each group was set up with 3 parallels. The larval situation of Daphnia pulex was observed every day. If the larvae were found, they were counted in time, and the parents were selected and placed in fresh algae solution for further cultivation. The experiment was carried out for 10 consecutive days, and the number of larvae and the number of larvae were recorded. The results are shown in Table 3.

[0079] Table 3 Effects of adding photosynthetic bacteria on the total number of larvae, egg production per time and reproduction times of Pseudodaphnia andersonii

[0080]

[0081] It can be seen that under the condition of feeding the same amount of microalgae bait, adding different concentrations of photosynthetic bacteria had a significant effect on the total number of larvae produced, the number of eggs laid each time and the number of reproduction times of Pseudo-dart Daphnia Andrews.

[0082] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for segmented cultivation of Pseudodaphnia andersonii, characterized in that: The method comprises the following steps: (1) Collection and selection of adult Pseudodaphnia annae; (2) Primary culture: inoculating the selected adult Daphnia Andrews into seawater, feeding one or more microalgae selected from Thalassiosira, Chaetoceros, and Chrysophyte until larvae are produced, and then filtering out the larvae using a 125-150 μm screen; in step (2), the seawater temperature is 25-30° C., the salinity is 23, the light intensity is 1000 lux, and the photoperiod is 12h:12h; (3) Secondary culture: collecting the larvae filtered out in step (2) for culture, feeding one or more of golden algae, Chaetoceros hornwort and Thalassiosira to adults, supplementing with photosynthetic bacteria after the adults develop to adults until eggs are laid, and collecting the produced larvae; the culture conditions in step (3) are: salinity 20-30, pH 7.5-8.5, light intensity 8000 lux, and photoperiod of 12h:12h; (4) Tertiary culture: The larvae obtained from the secondary culture are placed in a column photobioreactor for culture, and are fed with any one of Isochrysis Zhanjiangensis and Thalassiosira marinum until they reach the copepod larval stage, and then are fed with rubbed and washed shrimp flakes until they become adults; the culture conditions in step (4) are: temperature 30-35° C., salinity 23-30.

2. The method according to claim 1, characterized in that A plankton net with a pore size of 0.112 mm was used to collect Pseudodaphnia Andrews by towing the surface of the seawater in an inverted 8-way pattern for 1-3 minutes.

3. The method according to claim 1, characterized in that Adult Pseudodaphnia andersonii were selected based on the characteristics of the collected Pseudodaphnia andersonii.

4. The method according to claim 1, characterized in that The density of the eggs in step (3) is 3000-5000 eggs / L.

5. The method according to claim 1, characterized in that The culture method in step (4) is an open semi-aerated semi-continuous method.

6. The method according to claim 1, characterized in that The column-type photobioreactor comprises a fixed support, support legs, support foot supports, a reactor body, a connecting device, a valve, and replaceable screens with different apertures.

7. Use of the segmented culture method according to any one of claims 1 to 6 in aquaculture.

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