A method for domesticating freshwater daphnia using marine bacteria

Abstract

The application discloses a method for domesticating freshwater daphnia by using marine bacteria and belongs to the technical field of daphnia breeding. The method comprises the following steps: expanding the captured freshwater daphnia; adding bacillus velezensis freeze-dried powder into freshwater to activate, then adding the bacillus velezensis freeze-dried powder into seawater culture medium to expand, finally centrifuging to collect bacterial bodies and storing the bacterial bodies at low temperature for standby; when the freshwater daphnia is expanded to small individuals, the collected bacillus velezensis bacterial bodies are used as food to feed the freshwater daphnia until the freshwater daphnia adapts to target salinity seawater. The domestication method changes the food source of daphnia, improves the salt tolerance of the freshwater daphnia, and effectively solves the problem of poor domestication result in the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of Daphnia cultivation, and in particular relates to a method for domesticating freshwater Daphnia by utilizing marine bacteria. Background Art

[0002] Daphnia, also known as cladocerans, are an important group of zooplankton belonging to the order Cladocera, class Crustacea, phylum Arthropoda. Their laterally flattened bodies allow them to frequently make short leaps through the water, hence the name "water flea" or simply "daphnia." Daphnia play a crucial role in ecosystems, particularly as primary consumers in the food chain and a source of live food for young fish and shrimp.

[0003] Most species of Daphnia exist in freshwater bodies, and only a very small number of species, such as the Mongolian Moina, can adapt to seawater breeding. They also have problems such as harsh breeding conditions and low yields, and are often affected by the environment, leading to breeding failure.

[0004] Freshwater daphnia have a large production volume, but cannot survive in seawater. Daphnia cultured in freshwater will only survive for 30 minutes in 1% salinity seawater before all of them die. The dead daphnia have a poor effect on attracting fish, and if the dead daphnia are not cleaned up in time, the water quality will deteriorate and cause mass death of fish fry.

[0005] In the past, there have been many methods of gradually acclimating daphnia by changing the salinity of water bodies. These methods are to change the external environment, cause stress to daphnia, and force daphnia to adapt to the external environment. However, they all have problems such as slow acclimation, low breeding salinity, and low total lethal salinity. Taking the method of acclimation using a slow salinity change method disclosed in the prior art as an example, the freshwater daphnia are collected from a small Na2SO4 water body with a salinity of 0.334%. Through the slow salinity change method of acclimation, the freshwater daphnia can be improved to a breeding salinity of 0.88% and a total lethal salinity of 1.3%. The average salinity of seawater is about 3% throughout the year, and it can only drop to 0.6% in the rainy season. However, the rainy season is not the fry breeding season, and the market demand for daphnia is extremely low. Obviously, this acclimation result is not satisfactory and cannot be used in production. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for acclimating freshwater daphnia using marine bacteria. This acclimation method improves the freshwater daphnia's tolerance to salinity by changing the daphnia's food source, effectively solving the problem of poor acclimation results in the prior art.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0008] A method for acclimating freshwater daphnia using marine bacteria comprises the following steps:

[0009] (1) Cultivate the captured freshwater daphnia;

[0010] (2) adding the freeze-dried powder of Bacillus velezensis into fresh water to activate, then adding into seawater medium to expand, and finally centrifuging to collect the bacterial bodies and storing at low temperature for standby;

[0011] (3) when the expansion in step (1) appears small Daphnia individuals, the bacterial bodies of Bacillus velezensis collected in step (2) are fed as food to the Daphnia until the Daphnia adapts to the target salinity seawater.

[0012] Further, the Daphnia in step (1) includes any one of the genus Ceriodaphnia, the genus Scapholeberis and the genus Diaphanosoma.

[0013] Further, the expansion method in step (1) is that the captured Daphnia is placed in clean fresh water to prepare Daphnia liquid, and then the expansion is fed with yeast powder particles and concentrated green algae as feed, and the feeding is performed once every 10-12 hours.

[0014] Further, the concentration of Daphnia in the Daphnia liquid is 0.3-0.7 per ml; the feeding amount of yeast powder particles is 1 per 200-300 ml of Daphnia liquid; and the feeding amount of concentrated green algae is 200-400 w cells / ml.

[0015] Further, an animal calcium source is added to the water body in the process of expansion and feeding.

[0016] Further, the animal calcium source is crushed shells or animal bones.

[0017] Further, the inoculation concentration of Bacillus velezensis in seawater medium in step (2) is 0.2-2 mg / ml.

[0018] Further, the seawater medium in step (2) is seawater beef extract peptone medium or seawater potato medium, and the salinity of the seawater medium is 2.6-4%; and the expansion time is 6-8 days.

[0019] Further, the centrifugation parameters in step (2) are 2000-4000 rpm, and the centrifugation time is 3-5 min, and after centrifugation, the same salinity of sterilized seawater is added to the collected bacterial bodies for preservation.

[0020] Further, in step (3), when small individuals appear again in the Daphnia after eating the bacterial bodies of Bacillus velezensis, natural seawater is added to the system to increase the salinity of the system by 0.2-0.4%, and the above steps are repeated until the system reaches the target salinity.

[0021] Further, in step (3), the bacterial bodies of Bacillus velezensis are added as food, and at the same time, concentrated green algae are also added as food, and the feeding amount of Bacillus velezensis bacterial bodies and concentrated green algae is 200-400 w cells / ml.

[0022] The beneficial effects produced by the present invention are:

[0023] 1. The effect of a substance on an animal's body is primarily determined by the amount of the substance in the animal's internal environment. If it is not absorbed, there is no effect. Besides injection, direct ingestion through food can rapidly increase the amount of the substance absorbed by the body. Due to the inherent characteristics of marine bacteria, they can significantly increase the amount of salt absorbed by freshwater daphnia, thereby increasing the salt content in their bodies, stimulating the expression of genes that adapt to salinity changes, increasing the number of corresponding receptors and transporters on the daphnia cell surface, and enhancing the activity of corresponding enzymes, thereby gradually improving the daphnia's salinity tolerance.

[0024] 2. By feeding marine bacteria to freshwater daphnia, the salinity is acclimated, allowing the daphnia to spontaneously adapt to the environment from the inside out. There is no need to accurately grasp the salinity changes, and the stress on the daphnia is small. It has the advantages of high success rate and simple operation.

[0025] 3. In the present invention, the addition of an animal calcium source during the expansion of freshwater daphnia can replenish the calcium source for freshwater daphnia in a timely manner, promote the rapid calcification of the freshwater daphnia exoskeleton, promote the molting of freshwater daphnia, shorten the soft shell period of freshwater daphnia, reduce the risk of infection during the soft shell period, and thus avoid the death of freshwater daphnia due to calcium deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 图1 This is a statistical chart showing the survival time of Daphnia in seawater of different salinity. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. That is, the embodiments described are only part of the embodiments of the present invention, rather than all embodiments.

[0028] Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention as claimed, but is merely intended to represent selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0029] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0030] The features and performance of the present invention are further described in detail below with reference to the embodiments and drawings. Example

[0031] A method for acclimating freshwater daphnia using marine bacteria comprises the following steps:

[0032] (1) The captured Daphnia genus ( Bosmina ) were cultured in the following manner: freshwater daphnia captured from rice paddies were placed in mineral water to prepare a daphnia solution, wherein the concentration of freshwater daphnia in the daphnia solution was 0.3 daphnia / ml, and crushed shells were added to the daphnia solution; yeast powder granules and concentrated chlorella were then used as feed for culture, wherein the yeast powder granules were fed at a rate of 1 granule per 200ml of daphnia solution; and concentrated chlorella was fed at a rate of 200w cells / ml, and was fed once every 10 hours;

[0033] (2) 0.1g of Bacillus velezensis ( Bacillus velezensis ) The freeze-dried powder was activated by adding fresh water, and then expanded in 500 ml of seawater beef extract peptone medium. The salinity of the seawater medium was 2.6%, and the inoculation concentration of Bacillus velezensis in the seawater medium was 0.2 mg / ml. The culture was carried out for 6 days. Finally, the culture was centrifuged at 2000 rpm for 3 minutes, the supernatant was removed, and the cells were collected. Sterile seawater of the same salinity was added to the cells for elution, and the centrifugation was repeated. The cells were then collected and sterile seawater of the same salinity (1 times the weight of the cells) was added to the cells for low-temperature storage until use.

[0034] (3) When the culture is expanded in step (1) to the point where young freshwater daphnia appear, the Bacillus Velez subtilis cells collected in step (2) are fed to the freshwater daphnia at a feeding rate of 200w cells / ml. At the same time, concentrated Chlorella vulgaris is also added as food at a feeding rate of 200w cells / ml, and the feeding is carried out once every 10 hours. When young individuals appear again in the freshwater daphnia after consuming the Bacillus Velez subtilis cells, it means that the freshwater daphnia have reproduced a new generation. At this time, natural seawater is added to the system to increase the salinity of the system by 0.2%. The above steps are repeated, that is, natural seawater is added to the system once for each generation of freshwater daphnia, until the system reaches the target salinity.

[0035] In the above operation, adding natural seawater to the system can speed up the acclimation time of freshwater Daphnia. However, if no seawater is added, the acclimation purpose can be achieved by feeding only Bacillus Velezii bacteria, but the acclimation time is relatively long. Example

[0036] A method for taming freshwater daphnia using marine bacteria comprises the following steps:

[0037] (1) The captured Daphnia genus ( Ceriodaphnia ) were cultured in the following manner: freshwater daphnia captured from rice paddies were placed in clean fresh water to prepare a slurry containing 0.7 freshwater daphnia per ml, and crushed animal bones were added to the slurry. Yeast powder pellets and concentrated Nannochloropsis algae were then used as feed for culture, with one yeast powder pellet added per 300 ml of slurry and 400w cells / ml of concentrated Nannochloropsis algae, fed every 12 hours.

[0038] (2) 1g of Bacillus velezensis ( Bacillus velezensis ) The freeze-dried powder was activated by adding fresh water, and then expanded in 500 ml of seawater beef extract peptone medium. The salinity of the seawater medium was 4%, and the inoculation concentration of Bacillus Velez in the seawater potato medium was 2 mg / ml. The culture was carried out for 8 days. Finally, the culture was centrifuged at 4000 rpm for 5 minutes, the supernatant was removed, and the cells were collected. Sterile seawater of the same salinity was added to the cells for elution. The centrifugation was repeated, and the cells were collected. Sterile seawater of the same salinity was added to the cells at 3 times the weight of the cells for low-temperature storage.

[0039] (3) When the culture is expanded to the point where young freshwater daphnia appear in step (1), the Bacillus Velez subtilis cells collected in step (2) are fed to the freshwater daphnia at a feeding rate of 400w cells / ml. At the same time, concentrated Nannochloropsis algae are also added as food at a feeding rate of 400w cells / ml, and the feeding is carried out once every 10 hours. When young individuals appear again in the freshwater daphnia after consuming the Bacillus Velez subtilis cells, it means that the freshwater daphnia have reproduced a new generation. At this time, natural seawater is added to the system to increase the salinity of the system by 0.4%. The above steps are repeated, that is, natural seawater is added to the system once for each generation of freshwater daphnia until the system reaches the target salinity.

[0040] In the above operation, adding natural seawater to the system can speed up the acclimation time of freshwater Daphnia. However, if no seawater is added, the acclimation purpose can be achieved by feeding only Bacillus Velezii bacteria, but the acclimation time is relatively long. Example

[0041] A method for taming freshwater daphnia using marine bacteria comprises the following steps:

[0042] (1) The captured Daphnia genus ( Chydorus ) were cultured in the following manner: freshwater daphnia captured from rice paddies were placed in mineral water to prepare a daphnia solution, wherein the concentration of freshwater daphnia in the daphnia solution was 0.4 daphnia / ml, and crushed shells were added to the daphnia solution; yeast powder granules and concentrated chlorella were then used as feed for culture, with one yeast powder granule added for every 250ml of daphnia solution; and concentrated chlorella was fed at a rate of 300w cells / ml, once every 11 hours;

[0043] (2) Add 0.5g of Bacillus velezensis ( Bacillus velezensis ) The freeze-dried powder was activated by adding fresh water, and then expanded in 500 ml of seawater beef extract peptone medium. The salinity of the seawater medium was 3.5%, and the inoculum concentration of Bacillus velezensis in the seawater medium was 1 mg / ml. The culture was carried out for 7 days. Finally, the culture was centrifuged at 3000 rpm for 4 minutes, the supernatant was removed, and the cells were collected. Sterile seawater of the same salinity was added to the cells for elution, and the centrifugation was repeated. The cells were then collected and sterile seawater of the same salinity was added to the cells at twice their weight for low-temperature storage.

[0044] (3) When the culture is expanded in step (1) to the point where young freshwater daphnia appear, the Bacillus Velez subtilis cells collected in step (2) are fed to the freshwater daphnia at a feeding rate of 300w cells / ml. At the same time, concentrated Chlorella vulgaris is also added as food at a feeding rate of 300w cells / ml, and the feeding is performed once every 10 hours. When young individuals appear again in the freshwater daphnia after consuming the Bacillus Velez subtilis cells, it means that the freshwater daphnia have reproduced a new generation. At this time, natural seawater is added to the system to increase the salinity of the system by 0.3%. The above steps are repeated, that is, natural seawater is added to the system once for each generation of freshwater daphnia, until the system reaches the target salinity.

[0045] In the above operation, adding natural seawater to the system can speed up the acclimation time of freshwater Daphnia. However, if no seawater is added, the acclimation purpose can be achieved by feeding only Bacillus Velezii bacteria, but the acclimation time is relatively long. Example

[0046] A method for acclimating freshwater daphnia using marine bacteria comprises the following steps:

[0047] (1) The captured Daphnia genus ( Bosmina ) were cultured in the following manner: freshwater daphnia captured from rice paddies were placed in mineral water to prepare a daphnia solution, wherein the concentration of freshwater daphnia in the daphnia solution was 0.0.6 daphnia / ml, and crushed shells were added to the daphnia solution; yeast powder granules and concentrated chlorella were then used as feed for culture, wherein the yeast powder granules were fed at a rate of 1 granule per 250 ml of daphnia solution; and concentrated chlorella was fed at a rate of 250w cells / ml, and was fed once every 10 hours;

[0048] (2) 0.7g of Bacillus velezensis ( Bacillus velezensis ) The freeze-dried powder was activated by adding fresh water, and then expanded in 500 ml of seawater beef extract peptone medium. The salinity of the seawater medium was 3%, and the inoculation concentration of Bacillus velezensis in the seawater medium was 1.5 mg / ml. The expansion time was 8 days. Finally, the culture was centrifuged at 3800 rpm for 4 minutes, the supernatant was removed, and the cells were collected. Sterile seawater of the same salinity was added to the cells for elution, and the centrifugation was repeated. The cells were then collected and three times the weight of the cells were added to the cells for low-temperature storage.

[0049] (3) when step (1) is expanded to appear small daphnia individuals, the bacillus bacteria collected in step (2) are fed to daphnia as food, the feeding amount is 400w cells / ml, and at the same time, concentrated chlorella is also added as food, the feeding amount is 300w cells / ml, and feeding is carried out every 10h; when the daphnia after eating bacillus bacteria appears small individuals again, that is, the daphnia has reproduced a new generation, at this time, natural seawater is added to the system, so that the salinity of the system increases by 0.3%, and the above steps are repeated, that is, the daphnia adds natural seawater to the system once every generation, until the system reaches the target salinity.

[0050] In the above operation, the addition of natural seawater to the system can accelerate the acclimation time of daphnia, but if no seawater is added and only bacillus bacteria are fed, the purpose of acclimation can also be achieved, but the acclimation time is relatively long.

[0051] Comparative example 1

[0052] A daphnia acclimation method, comprising the following steps:

[0053] The obtained daphnia of the genus daphnia Bosmina ) is placed in water for 12h, concentrated chlorella is added as food, the feeding amount is 300w cells / ml, the slow salinity acclimation method with high daphnia survival rate is adopted, natural seawater is added to the system on the third day to increase the salinity of the system by 0.1%, and then appropriate amount of natural seawater is added to the system every three days to increase the salinity of the system by 0.1%, that is, the overall salinity of the system is: 0.1% on the third day, 0.2% on the sixth day, 0.3% on the ninth day, and so on, 1% on the thirtieth day, to obtain the acclimated daphnia.

[0054] Test example

[0055] Take the daphnia acclimated by the traditional method (the method in comparative example 1), the daphnia acclimated by the new method (the method in example 3 of the present application), and the daphnia not acclimated as examples, natural seawater is diluted with distilled water to a salinity of 30‰, 26‰, 20‰, 16‰, 10‰ and 5‰ for standby. About 200 daphnia are taken from the original cultivation water body using a 120 mesh net, dried and then put into each beaker, and the time is started to count, and all daphnia sinking to the bottom without movement is taken as the death time, which is repeated for 3 times, and the average value of the time is taken. Finally, the death time data of the unacclimated, newly acclimated and traditionally acclimated daphnia are summarized, and the specific results are shown in Table 1. 图1 .

[0056] 图1The survival time statistics chart of the daphnia in different salinity seawater shows that the death time of the daphnia is shortened with the increase of the salinity of seawater, wherein the daphnia without domestication appears sharp decline when the salinity of seawater increases to 2.5‰, the daphnia domesticated by the traditional method appears sharp decline when the salinity of seawater increases to 15‰, and the daphnia domesticated by the new method appears decline trend when the salinity of seawater increases to 24‰, which proves that the salt tolerance of the daphnia domesticated by the new method is improved.

Claims

1. A method for domesticating freshwater daphnia using marine bacteria, characterized in that: The following steps are involved: (1) Cultivate the captured freshwater daphnia; (2) Add the freeze-dried powder of Bacillus velezensis to fresh water for activation, then add it to seawater culture medium for expansion, and finally collect the bacteria by centrifugation and store them at low temperature for future use; (3) When the culture is expanded in step (1) to the point where young freshwater daphnia appear, the Bacillus Velezii cells collected in step (2) are fed to the freshwater daphnia as food until the freshwater daphnia adapt to the target salinity seawater.

2. The method for acclimating freshwater daphnia using marine bacteria according to claim 1, wherein: The freshwater daphnia in step (1) includes any one of the genus Daphnia, Daphnia and Daphnia.

3. The method for acclimating freshwater daphnia using marine bacteria according to claim 1, wherein: The expansion method in step (1) is as follows: the captured freshwater daphnia are placed in clean fresh water to prepare daphnia liquid, and then yeast powder particles and concentrated green algae are used as feed for expansion and breeding, and the feed is fed once every 10-12 hours.

4. The method for acclimating Daphnia pulex using marine bacteria according to claim 3, wherein: The concentration of freshwater daphnia in the daphnia magna is 0.3-0.7 / ml; the feeding amount of yeast powder granules is 1 granule per 200-300ml of daphnia magna; the feeding amount of concentrated green algae is 200-400w cells / ml.

5. The method for acclimating freshwater daphnia using marine bacteria according to claim 3, wherein: During the expansion and rearing process in step (1), an animal calcium source is added to the water.

6. The method for acclimating Daphnia pulex using marine bacteria according to claim 1, wherein: In step (2), the inoculation concentration of Bacillus velezensis in the seawater culture medium is 0.2-2 mg / ml.

7. The method for acclimating freshwater daphnia using marine bacteria according to claim 1, wherein: In step (2), the seawater culture medium is seawater beef extract peptone culture medium or seawater potato culture medium, and the salinity of the seawater culture medium is 2.6-4%; the expansion culture time is 6-8 days.

8. The method for acclimating Daphnia pulex using marine bacteria according to claim 1, wherein: In step (2), the centrifugation parameters are 2000-4000 rpm, the centrifugation time is 3-5 min, and after centrifugation, sterilized seawater of the same salinity is added to the collected bacteria for preservation.

9. The method for acclimating Daphnia pulex using marine bacteria according to claim 1, wherein: In step (3), when young individuals reappear in the freshwater Daphnia after consuming the Bacillus Velezii cells, natural seawater is added to the system to increase the salinity of the system by 0.2-0.4%, and the above steps are repeated until the system reaches the target salinity.

10. The method for acclimating Daphnia pulex using marine bacteria according to claim 1, wherein: In step (3), while adding Bacillus Velez cells as food, concentrated green algae is also added as food. The feeding amount of Bacillus Velez cells and concentrated green algae is 200-400w cells / ml.

Images