Method for domesticating daphnia magna by using marine bacteria

Abstract

The invention discloses a method for domesticating daphnia magna by using marine bacteria, which belongs to the technical field of daphnia magna cultivation and comprises the following steps: carrying out expanding cultivation on captured daphnia magna; the method comprises the following steps: adding bacillus velezensis freeze-dried powder into fresh water for activation, then adding into a seawater culture medium for expanding culture, finally centrifugally collecting thalli, and storing at low temperature for later use; when the daphnia magna is subjected to expanding culture until young individuals appear, the collected bacillus velezensis thalli are used as food to feed the daphnia magna until the daphnia magna adapts to the seawater with the target salinity. According to the domestication method, the salinity tolerance of the freshwater daphnia magna is improved by changing the food source of the daphnia magna, and the problem that the domestication result is poor in the prior art is effectively solved.

Description

Technical Field

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

[0002] Daphnia are an important type of zooplankton belonging to the order Cladocera of the class Crustacea of ​​the phylum Arthropoda. They have a flattened body and often make short-distance jumps in the water, hence the name "water flea" or simply "daphnia". Daphnia play an important role in the ecosystem, especially as primary consumers in the food chain and a source of live food for fish and shrimp seedlings.

[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 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 large production but cannot survive in seawater. Daphnia cultured in freshwater will only survive 30 minutes in 1% salinity seawater before all of them die. Dead daphnia have poor attractant effect on fish, and if 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 ways to gradually domesticate by changing the salinity of water bodies. This is to change the external environment, cause stress to Daphnia, and force Daphnia to adapt to the external environment, but there are problems such as slow domestication, low breeding salinity, and low total lethal salinity. Taking the method of domestication using the salinity slow 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 salinity slow change method domestication, the tolerance of freshwater Daphnia to salinity can be increased 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, but the rainy season is not the fry breeding season, and the market demand for Daphnia is extremely low. Obviously, this domestication result is not satisfactory and cannot be used in production. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for domesticating freshwater daphnia using marine bacteria. The domestication method improves the tolerance of freshwater daphnia to salinity by changing the food source of algae, effectively solving the problem of poor domestication results in the prior art.

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

[0008] A method for taming 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 Velezii to fresh water for activation, then adding it to seawater culture medium for expansion, and finally centrifuging to collect the bacteria and storing them at low temperature for future use;

[0011] (3) When young freshwater daphnia appear in the culture in step (1), 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.

[0012] Furthermore, the freshwater daphnia in step (1) includes any one of the genus Daphnia, Daphnia and Daphnia.

[0013] Furthermore, the expansion method in step (1) is: placing the captured freshwater daphnia in clean fresh water to prepare daphnia liquid, and then expanding and raising them with yeast powder particles and concentrated green algae as feed, feeding them once every 10-12 hours.

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

[0015] Furthermore, an animal calcium source is added to the water during the expansion and breeding process.

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

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

[0018] Furthermore, 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%; and the expansion culture time is 6-8 days.

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

[0020] Furthermore, 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.

[0021] Furthermore, in step (3), while adding Bacillus Velez cells as food, concentrated green algae are also added as food, and the feeding amount of Bacillus Velez cells and concentrated green algae is 200-400w 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 mainly determined by the amount of the substance in the animal's internal environment. If it is not absorbed, there will be no effect. In addition to injection, direct intake through food can quickly increase the amount of the substance taken up by the organism. Due to the characteristics of the marine bacteria themselves, the amount of salt taken up by freshwater daphnia can be greatly increased, thereby increasing the salt content in the freshwater daphnia, stimulating the expression of genes that adapt to salinity changes in daphnia, increasing the number of corresponding receptors and transport proteins on the surface of daphnia cells, and increasing the activity of corresponding enzymes, thereby gradually improving the salt tolerance of daphnia.

[0024] 2. By feeding marine bacteria to acclimate freshwater daphnia to salinity, daphnia can 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, adding an animal calcium source during the expansion of freshwater daphnia can replenish the calcium source for freshwater daphnia in time, promote the rapid calcification of the exoskeleton of freshwater daphnia, 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] Figure 1 This is a statistical chart of the survival time of Daphnia in seawater of different salinity. DETAILED DESCRIPTION

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

[0028] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making 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 such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

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

[0031] Example 1

[0032] A method for taming freshwater daphnia using marine bacteria comprises the following steps:

[0033] (1) The captured algae of the genus Bosmina were cultured. The specific method was as follows: freshwater daphnia captured from rice fields were placed in mineral water to prepare daphnia liquid. The concentration of freshwater daphnia in the daphnia liquid was 0.3 daphnia / ml, and crushed shells were added to the algae liquid. Yeast powder particles and concentrated Chlorella were then used as feed for culture. The feeding amount of yeast powder particles was 1 particle per 200ml of daphnia liquid. The feeding amount of concentrated Chlorella was 200w cells / ml, and it was fed once every 10 hours.

[0034] (2) 0.1 g of Bacillus velezensis freeze-dried powder was added to fresh water for activation, and then added to 500 ml of seawater beef extract peptone medium for expansion. 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 expansion time was 6 days. Finally, centrifugation was performed at 2000 rpm for 3 min, the supernatant was removed, the bacteria were collected, and sterile seawater of the same salinity was added to the bacteria for elution. The centrifugation was repeated, and then the bacteria were collected and sterile seawater of the same salinity (1 times the weight of the bacteria) was added to the bacteria for low-temperature storage for later use.

[0035] (3) When young freshwater daphnia appear in step (1), the Bacillus Velez subtilis collected in step (2) is used as food to feed the freshwater daphnia at a feeding amount of 200w cells / ml. At the same time, concentrated Chlorella vulgaris is also added as food at a feeding amount of 200w 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, 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.

[0036] In the above operation, adding natural seawater to the system can speed up the acclimation time of freshwater Daphnia, but if no seawater is added, the purpose of acclimation can be achieved by only feeding Bacillus Velezii bacteria, but the acclimation time is relatively long.

[0037] Example 2

[0038] A method for taming freshwater daphnia using marine bacteria comprises the following steps:

[0039] (1) The captured algae of the genus Ceriodaphnia were cultured. The specific method was as follows: freshwater daphnia captured from rice fields were placed in clean fresh water to prepare daphnia liquid. The concentration of freshwater daphnia in the daphnia liquid was 0.7 daphnia / ml, and crushed animal bones were added to the algae liquid; yeast powder particles and concentrated Nannochloropsis algae were then used as feed for culture. The feeding amount of yeast powder particles was 1 particle per 300ml of daphnia liquid; the feeding amount of concentrated Nannochloropsis algae was 400w cells / ml, and it was fed once every 12 hours;

[0040] (2) 1 g of Bacillus velezensis freeze-dried powder was added to fresh water for activation, and then added to 500 ml of seawater beef extract peptone medium for expansion. The salinity of the seawater medium was 4%, and the inoculation concentration of Bacillus velezensis in the seawater potato medium was 2 mg / ml. The expansion time was 8 days. Finally, centrifugation was performed at 4000 rpm for 5 min, the supernatant was removed, the bacteria were collected, and sterile seawater of the same salinity was added to the bacteria for elution. The centrifugation was repeated, and then the bacteria were collected and sterile seawater of the same salinity was added to the bacteria at a weight of 3 times the weight of the bacteria for low-temperature storage for later use.

[0041] (3) When young freshwater daphnia appear in step (1), the Bacillus Velez subtilis collected in step (2) is used as food to feed the freshwater daphnia at a feeding amount of 400w cells / ml. At the same time, concentrated Nannochloropsis chlororaphis is also added as food at a feeding amount of 400w 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, 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.

[0042] In the above operation, adding natural seawater to the system can speed up the acclimation time of freshwater Daphnia, but if no seawater is added, the purpose of acclimation can be achieved by only feeding Bacillus Velezii bacteria, but the acclimation time is relatively long.

[0043] Example 3

[0044] A method for taming freshwater daphnia using marine bacteria comprises the following steps:

[0045] (1) The captured algae of the genus Chydorus were cultured. The specific method was as follows: freshwater daphnia captured from rice fields were placed in mineral water to prepare daphnia liquid. The concentration of freshwater daphnia in the daphnia liquid was 0.4 daphnia / ml, and crushed shells were added to the algae liquid; yeast powder granules and concentrated Chlorella were then used as feed for culture. The feeding amount of yeast powder granules was 1 granule per 250ml of daphnia liquid; the feeding amount of concentrated Chlorella was 300w cells / ml, and it was fed once every 11 hours;

[0046] (2) 0.5 g of Bacillus velezensis lyophilized powder was added to fresh water for activation, and then added to 500 ml of seawater beef extract peptone medium for expansion. The salinity of the seawater medium was 3.5%, and the inoculation concentration of Bacillus velezensis in the seawater medium was 1 mg / ml. The expansion time was 7 days. Finally, the culture was centrifuged at 3000 rpm for 4 min, the supernatant was removed, the cells were collected, and sterile seawater of the same salinity was added to the cells for elution. The centrifugation was repeated, and then the cells were collected and sterile seawater of the same salinity, which was twice the weight of the cells, was added to the cells for low-temperature storage for later use.

[0047] (3) When young freshwater daphnia appear in step (1), the Bacillus Velez subtilis collected in step (2) is used as food to feed the freshwater daphnia at a feeding amount of 300w cells / ml. At the same time, concentrated Chlorella vulgaris is also added as food at a feeding amount 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, 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.

[0048] In the above operation, adding natural seawater to the system can speed up the acclimation time of freshwater Daphnia, but if no seawater is added, the purpose of acclimation can be achieved by only feeding Bacillus Velezii bacteria, but the acclimation time is relatively long.

[0049] Example 4

[0050] A method for taming freshwater daphnia using marine bacteria comprises the following steps:

[0051] (1) The captured algae of the genus Bosmina were cultured. The specific method was as follows: freshwater daphnia captured from rice fields were placed in mineral water to prepare daphnia liquid. The concentration of freshwater daphnia in the daphnia liquid was 0.0.6 daphnia / ml, and crushed shells were added to the algae liquid. Yeast powder particles and concentrated Chlorella were then used as feed for culture. The feeding amount of yeast powder particles was 1 particle per 250 ml of daphnia liquid. The feeding amount of concentrated Chlorella was 250w cells / ml, and it was fed once every 10 hours.

[0052] (2) 0.7 g of Bacillus velezensis lyophilized powder was added to fresh water for activation, and then added to 500 ml of seawater beef extract peptone medium for expansion. 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, centrifugation was performed at 3800 rpm for 4 min, the supernatant was removed, the bacteria were collected, and sterile seawater of the same salinity was added to the bacteria for elution. The centrifugation was repeated, and then the bacteria were collected and sterile seawater of the same salinity was added to the bacteria at a weight of 3 times the weight of the bacteria for low-temperature storage for later use.

[0053] (3) When young freshwater daphnia appear in step (1), the Bacillus Velez subtilis collected in step (2) is used as food to feed the freshwater daphnia at a feeding amount of 400w cells / ml. At the same time, concentrated Chlorella vulgaris is also added as food at a feeding amount 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, 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.

[0054] In the above operation, adding natural seawater to the system can speed up the acclimation time of freshwater Daphnia, but if no seawater is added, the purpose of acclimation can be achieved by only feeding Bacillus Velezii bacteria, but the acclimation time is relatively long.

[0055] Comparative Example 1

[0056] A method for taming freshwater daphnia comprises the following steps:

[0057] The obtained freshwater Bosmina daphnia was placed in water for 12 hours, and then concentrated Chlorella was added as food. The feeding amount was 300w cells / ml, and a slow salinity acclimation method with a high survival rate of daphnia was adopted. Natural seawater was added to the system on the third day of feeding to increase the salinity of the system by 0.1%. Then, an appropriate amount of natural seawater was 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 was: 0.1% on the third day, 0.2% on the sixth day, 0.3% on the ninth day...1% on the thirtieth day, and the acclimated freshwater daphnia were obtained.

[0058] Test example

[0059] Taking the traditional method of domestication (the method in comparative example 1), the new method of domestication (the method in Example 3 of the present application) and undomesticated freshwater daphnia as examples, natural seawater was diluted with distilled water to a salinity of 30‰, 26‰, 20‰, 16‰, 10‰, and 5‰ respectively for use. Take 800ml of the prepared seawater of each salinity in each 1000ml beaker, and use a 120-mesh scoop net to scoop out about 200 freshwater daphnia from the original breeding water body. After shaking off the water, put them into each beaker and start timing. The time of death is when all daphnia sink to the bottom without movement. Repeat 3 times and take the average time. Finally, the death time data of undomesticated, newly domesticated, and traditionally domesticated daphnia are summarized. The specific results are shown in Figure 1 .

[0060] Figure 1The figure is a statistical chart of the survival time of Daphnia in seawater of different salinities. The results show that with the increase of seawater salinity, the death time of Daphnia shortens. Among them, the death time of Daphnia that has not been domesticated drops sharply when the seawater salinity increases to 2.5‰; the death time of Daphnia that has been domesticated by the traditional method drops sharply when the seawater salinity increases to 15‰; the death time of Daphnia that has been domesticated by the new method shows a downward trend only when the seawater salinity increases to 24‰, which proves that the salt tolerance of Daphnia domesticated by the new method of the present invention 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) Adding the freeze-dried powder of Bacillus Velezii to fresh water for activation, then adding it to seawater culture medium for expansion, and finally centrifuging to collect the bacteria and storing them at low temperature for future use; (3) When young freshwater daphnia appear in the culture in step (1), 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 taming freshwater daphnia using marine bacteria as claimed in claim 1, characterized in that: The freshwater daphnia in step (1) includes any one of the genus Daphnia, Daphnia and Daphnia.

3. The method for taming freshwater daphnia using marine bacteria as claimed in claim 1, characterized in that: The expansion method in step (1) is: placing the captured freshwater daphnia in clean fresh water to prepare daphnia liquid, and then expanding and raising them with yeast powder particles and concentrated green algae as feed, feeding them once every 10-12 hours.

4. The method for taming freshwater daphnia using marine bacteria as claimed in claim 3, characterized in that: The concentration of freshwater daphnia in the daphnia grana is 0.3-0.7 daphnia / ml; the feeding amount of yeast powder granules is 1 granule per 200-300ml of daphnia grana; the feeding amount of concentrated green algae is 200-400w cells / ml.

5. The method for taming freshwater daphnia using marine bacteria as claimed in claim 3, characterized in that: During the expansion and breeding process, add animal calcium sources to the water.

6. The method for taming freshwater daphnia using marine bacteria as claimed in claim 1, characterized in that: In step (2), the inoculation concentration of Bacillus velez in the seawater culture medium is 0.2-2 mg / ml.

7. The method for taming freshwater daphnia using marine bacteria as claimed in claim 1, characterized in that: 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%; and the expansion time is 6-8 days.

8. The method for taming freshwater daphnia using marine bacteria as claimed in claim 1, characterized in that: In step (2), the centrifugation parameters are 2000-4000 rpm, the centrifugation time is 3-5 min, and after centrifugation, sterile seawater of the same salinity is added to the collected bacteria for preservation.

9. The method for taming freshwater daphnia using marine bacteria as claimed in claim 1, characterized in that: 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 taming freshwater daphnia using marine bacteria as claimed in claim 1, characterized in that: In step (3), while adding Bacillus Velez cells as food, concentrated green algae are also added as food. The feeding amount of Bacillus Velez cells and concentrated green algae is 200-400w cells / ml.

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