A method for raising seedlings for improving disease resistance of Chinese mitten crab larvae

Abstract

The present invention relates to the field of aquaculture technology, and in particular to a method for raising seedlings to improve the disease resistance of Chinese mitten crab larvae. 5 The brooding crabs of Chinese mitten crabs were twice immersed in seawater containing 100 CFU / mL of inactivated Mycobacterium bicuspidatum to stimulate the brooding crabs. The zoeae larvae produced by the stimulated brooding crabs were then hatched, thereby obtaining disease-resistant Chinese mitten crab larvae. Cultivating brooding crabs using the seedling raising method provided by the present invention can promote the development of Chinese mitten crab embryos and improve the resistance of the zoeae larvae to infection by Mycobacterium bicuspidatum.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a seedling raising method for improving the disease resistance of Chinese mitten crab larvae. Background Art

[0002] Yeast is a kind of fungi that is widely found in nature. Many species of yeast have been confirmed to be the main pathogens of aquaculture animals. Torulopsis mogii ), Portuguese Candida ( Candida lusitaniae ) and Metschnikowia bicuspidatum ( Metschnikowia bicuspidata ) can infect a variety of aquatic animals alone or together with other pathogens, causing death of aquatic animals and seriously threatening the high-quality development of my country's aquaculture industry. Daphnia magna ) and is now known to infect species including Macrobrachium rosenbergii ( Macrobrachium rosenbergii )、Portunus trituberculatus( Portunus trituberculatus ), Chinook salmon ( Oncorhynchus tshawytscha ) and Chinese mitten crab ( Eriocheir sinensis There is currently no effective treatment for a variety of aquaculture animals, including ).

[0003] The Chinese mitten crab (Eriocheir sinensis) is a large, commercial freshwater crab unique to my country, possessing significant economic value. my country is the world's largest producer of Chinese mitten crab. However, with the continuous expansion of aquaculture scale and density, various diseases have emerged, including tremor disease, water-shriveled crab disease, gill rot, enteritis, and ciliates. Metschnikoff's yeast has been reported as a pathogen causing "milk disease." Developing green control technologies for this disease is urgent. Summary of the Invention

[0004] In order to solve the problems in the background technology, the present invention provides a method for raising seedlings of Chinese mitten crabs for improving the disease resistance of juveniles.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for raising seedlings to improve the disease resistance of Chinese mitten crab larvae, characterized in that the method comprises: 5 The brooding crabs of Chinese mitten crab are stimulated by immersing them twice in seawater containing inactivated yeast of 100 CFU / mL. After the stimulation, the brooding crabs produce zoanthid larvae, thus obtaining disease-resistant Chinese mitten crab larvae.

[0007] Specifically, the egg-bearing crabs in the pre-nauplius stage of Chinese mitten crab were treated with a solution containing a concentration of 1x10 5CFU / mL of inactivated Mycobacterium bicuspidatum was immersed in seawater for stimulation. After stimulation, the crabs were cultured in normal seawater until the protozoan larvae stage. Then, the crabs cultured to this stage were treated with a solution containing 1x10 5 The crabs were immersed in seawater containing inactivated Mycobacterium bicuspidatum at 100 CFU / mL to stimulate the production of zoanthellae larvae, and the disease-resistant Chinese mitten crab larvae were obtained after two stimulations.

[0008] The concentration is 1x10 5 The seawater of inactivated yeast was prepared by inactivating yeast with formaldehyde solution and the concentration was adjusted to 1x10 CFU / mL using sterile seawater. 5 CFU / mL.

[0009] The formaldehyde solution-inactivated yeast is prepared by adding formaldehyde solution to a resuspended bacterial solution containing yeast, incubating the solution, and then centrifuging and washing to obtain the formaldehyde solution-inactivated yeast; wherein the concentration of yeast in the resuspended bacterial solution is adjusted to 2×10 7 CFU / mL, and the final concentration of formaldehyde solution in the resuspended bacterial solution was 0.5% (v / v).

[0010] The formaldehyde solution-inactivated yeast is prepared by inoculating yeast into YPD liquid culture medium for amplification and culture, removing excess culture medium, and repeatedly resuspending the yeast with sterile distilled water to obtain a resuspended bacterial solution; adding formaldehyde solution to the resuspended bacterial solution, mixing, and incubating at 4°C for 24 hours to obtain a yeast suspension, and then centrifuging and washing to obtain the formaldehyde solution-inactivated yeast.

[0011] The amplification culture temperature is 28° C., the time is 24 h, and the rotation speed is 180 r / min.

[0012] Compared with the prior art, the present invention has the following effects:

[0013] This invention is the first to utilize the principles of higher animal vaccine immunization and invertebrate immune sensitization to use a relatively low concentration (1x10 5 An innovative Chinese mitten crab (Eriocheir sinensis) seedling culture technique has been established by immersing brooding crabs in inactivated Mycobacterium bicuspidatum (M. bicuspidatum) at 100 CFU / mL. Using the method provided by this invention to cultivate brooding crabs promotes the development of zoanthid larvae and enhances their resistance to infection by Mycobacterium bicuspidatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This figure shows the changes in dry weight, wet weight, and egg diameter of Chinese mitten crab (Eriocheir sinensis) embryos at various stages after stimulation with inactivated Metschnikoff yeast in Example 1. A: Change in dry weight of embryonic eggs; B: Change in wet weight of embryonic eggs; C: Change in egg diameter. E1: Post-nauplius stage; E2: Early protozoan stage; E3: Protozoan stage; E4: Late protozoan stage; E5: Early larval stage. Significant differences between groups are indicated by "*" (*: p <0.05;**: p <0.01).

[0015] Figure 2 This figure shows the changes in carapace width, abdominal segment length, and telson length of Chinese mitten crab larvae at different stages after stimulation with inactivated Metschneider yeast in Example 1. A: Changes in carapace width; B: Changes in abdominal segment length; C: Changes in telson length. Z1: zoa stage I; Z2: zoa stage II; Z3: zoa stage III; Z4: zoa stage IV; Z5: zoa stage V; M: megalopa stage. Significant differences between groups are indicated by "*" (*: p <0.05;**: p <0.01;***: p <0.001; ****: p <0.0001).

[0016] Figure 3 The mortality and metamorphosis rates of Daphnia stage I larvae in the experimental group and the control group in Example 1, where A: metamorphosis rate of Daphnia stage I larvae; B: mortality rate of Daphnia stage I larvae. Significant differences between different groups at the same time point are marked with "**" ( p <0.01) and “***” ( p <0.001).

[0017] Figure 4 1x10 in Example 1 5 The mortality of Z. fasciatus I juveniles in the experimental and control groups after challenge with live yeast strains of CFU / mL. The significant differences between different groups at the same time point are marked with "*" ( p <0.05). DETAILED DESCRIPTION

[0018] The present invention is described in detail below through embodiments and test examples. It is necessary to point out that the embodiments and test examples are only used to further illustrate the present invention, but the content of the present invention is not limited to the content involved in the embodiments and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art in this field can make some non-essential improvements and adjustments based on the above content of the present invention.

[0019] The invention utilizes inactivated Mycobacterium bicuspidatum to soak Chinese mitten crab embryos and larvae twice, and cultivates the brooding crabs by adopting a seedling raising method, thereby promoting the development of Chinese mitten crab embryos and improving the ability of zoeae larvae to resist infection by Mycobacterium bicuspidatum.

[0020] The yeast Metschnikowia bicuspidatum used in the following is publicly available from known channels.

[0021] The embodiment of the present invention provides a method for raising seedlings for improving the disease resistance of Chinese mitten crab larvae, comprising:

[0022] The pre-nauplius stage of Chinese mitten crab was placed in a solution containing 1x10 5 CFU / mL of inactivated Mycobacterium bicuspidatum was immersed in seawater for stimulation for 12 h. After stimulation, the crabs were cultured in normal seawater until the protozoan larvae stage. Then, the crabs were placed in a water bath containing 1×10 5 The crabs were immersed in seawater containing CFU / mL of inactivated Mycobacterium bicuspidatum for stimulation for 12 hours, and then transferred to normal seawater for continued cultivation. The egg-bearing crabs after two stimulations were used to produce Zygophyllum larvae.

[0023] In the present invention, formaldehyde solution is used to inactivate yeast. The inactivation method comprises: inoculating yeast into YPD liquid culture medium for amplification culture, removing excess culture medium, and repeatedly resuspending the bacteria with sterile distilled water to obtain a resuspended bacterial solution. Formaldehyde solution is added to the resuspended bacterial solution and mixed, and then incubated at 4°C for 24 hours to obtain a suspension of yeast, which is then centrifuged and washed to obtain yeast inactivated by formaldehyde solution. Before adding formaldehyde solution to the resuspended bacterial solution, the concentration of the resuspended bacterial solution is adjusted to 2x10 7 CFU / mL, the final concentration of the added formaldehyde solution is 0.5% (v / v). The amplification culture temperature is 28°C, the time is 24 hours, and the rotation speed is 180 r / min.

[0024] As some specific embodiments, the specific method of the present invention for promoting the development of Chinese mitten crab embryos and zoanthid larvae and enhancing their disease resistance comprises the following steps:

[0025] Cultivation of brooding crabs and zoa larvae: The brooding crabs were provided by Photosynthetic Crab Company and maintained in seawater with water changes and feed every two days. After embryos hatched, the zoa larvae were separated from the mother crabs and fed rotifers daily.

[0026] (2) Preparation of inactivated yeast: The yeast obtained from public channels was inoculated into YPD liquid medium for amplification culture (28°C, 180 rpm, 24 h). Centrifuge (6800 × gAfter removing the excess culture medium, resuspend the cells with sterile distilled water and repeat 3 times. Adjust the concentration of the yeast in the resuspended cell suspension to 2x10 7 CFU / mL, and add formaldehyde solution with a final concentration of 0.5% (v / v). Incubate the yeast suspension containing formaldehyde solution at 4℃ for 24 h. g The cells were centrifuged for 5 min and washed twice with sterile distilled water to remove formaldehyde.

[0027] (3) Immersion stimulation of inactivated yeast Bicuspidatum on Chinese mitten crab embryos: The concentration of formaldehyde-inactivated yeast Bicuspidatum was adjusted to 1x10 5 CFU / mL, the pre-nauplius stage of Chinese mitten crab was placed in a solution containing 1x10 5 CFU / mL of inactivated Mycobacterium bicuspidatum was immersed in seawater for stimulation for 12 h. After stimulation, the crabs were cultured in normal seawater until the protozoan larvae stage. Then, the crabs were placed in a water bath containing 1×10 5 CFU / mL of inactivated Mycobacterium bicuspidatum was immersed in seawater for stimulation for 12 h, and then transferred to normal seawater for continued cultivation.

[0028] (4) Monitoring of the dry weight, wet weight, and egg diameter of Chinese mitten crab embryos at different stages: Each day, Chinese mitten crab embryos were taken with tweezers and observed under a microscope for their developmental stages and counted. After removing surface moisture, the wet weight of the embryos was accurately weighed using a balance and the wet weight of each egg was calculated. The embryos were then wrapped in tin foil and dried in a 70°C oven for 24 h. The dry weight was then accurately weighed and the dry weight of each egg was calculated. The egg diameter of the embryos was statistically measured daily under a microscope using an eyepiece micrometer.

[0029] (5) Monitoring of the carapace width, abdominal segment length and tail segment length of Chinese mitten crab larvae at different stages: After the embryos are hatched, the larvae are taken out with a dropper and observed under a microscope every day, and the carapace width, abdominal segment length and tail segment length of the larvae are statistically measured using an eyepiece micrometer.

[0030] (6) Monitoring of metamorphosis rate and mortality of stage I zoanthid larvae of Chinese mitten crab: After embryonic hatching, the larval development period was observed under a microscope every day and the metamorphosis rate was calculated. 150 zoanthid larvae hatched from each brooding crab were temporarily raised in a 1 L beaker for calculating the mortality rate.

[0031] (7) Detection of resistance of stage I larvae of Z. bicuspidatum to yeast: After embryonic hatching, 150 stage I larvae of Z. bicuspidatum from each brooding crab were taken out of each brooding crab and temporarily cultured in a 1 L beaker. 5The first-stage larvae of Z. bicuspidatum were challenged by immersing them in seawater containing 0.1 CFU / mL of Metschnikowia bicuspidatum. After 12 h, the larvae were transferred to normal seawater for continued culture, and the mortality rate of the first-stage larvae of Z. bicuspidatum was counted daily.

[0032] The method for promoting the development of Chinese mitten crab embryos and zoanthid larvae and enhancing their immunity provided by the present invention is described in detail below with reference to the examples.

[0033] Example 1

[0034] The yeast was inoculated into YPD liquid medium for amplification culture (28℃, 180 rpm, 24 h). g After removing the excess culture medium, resuspend the cells with sterile distilled water and repeat 3 times. Adjust the concentration of the yeast in the resuspended cell suspension to 2x10 7 CFU / mL, and add formaldehyde solution with a final concentration of 0.5% (v / v). Incubate the yeast suspension containing formaldehyde solution at 4℃ for 24 h. g The cells were centrifuged for 5 min and washed twice with sterile distilled water to remove formaldehyde.

[0035] Adjust the concentration of formaldehyde-inactivated yeast to 1x10 5 CFU / mL (i.e., seawater containing inactivated Metschnikowia bicuspidatum). When the Chinese mitten crab embryos developed to the pre-nauplius stage, the brooding crabs were placed in the above-mentioned seawater containing inactivated Metschnikowia bicuspidatum and immersed for 12 hours, and then transferred to normal seawater for continued cultivation. When the brooding crabs developed to the protozoan larvae stage, they were placed in the above-mentioned seawater containing inactivated Metschnikowia bicuspidatum and immersed for 12 hours, and then transferred to normal seawater for continued cultivation as the experimental group; the brooding crabs in the control group were not treated. After immersion stimulation, the growth and development of the embryos in the experimental and control groups were observed under a microscope every day, and the dry weight, wet weight and egg diameter of each egg at each developmental stage of the embryos were monitored (Table 1 and Figure 1 ).

[0036] Table 1 Development of Chinese mitten crab embryos and larvae after two consecutive stimulations with inactivated yeast

[0037] As shown in Table 1, on the eighth day after all the larvae were hatched, 66.67% of the larvae in the experimental group stimulated by two soakings of inactivated yeast Bicoidea entered the stage of stage II larvae, while all the larvae in the control group remained in the stage of stage I larvae. Figure 1It can be seen that after the first stimulation, the dry weight of single eggs in the experimental group embryos decreased to a lower level, and no significant difference was found in the dry weight of single eggs at different stages. In contrast, the dry weight of single eggs in the control group embryos in the early stage of emergence was significantly lower than that in the protozoan stage. The wet weight of single eggs in both the experimental and control groups showed an overall downward trend, and a significant difference appeared between the two in the late protozoan stage after the second stimulation. After the first inactivated yeast stimulation, the egg diameter of the experimental group embryos had already increased to a higher level and was significantly higher than that of the control group, while the control group embryos only began to show an upward trend after the post-nauplius stage. This indicates that the first and second stimulations with inactivated Metschnikowia bicuspidatum may have promoted embryonic development and accelerated the consumption of nutrients.

[0038] After the embryos hatched, the larvae were observed under a microscope daily to observe their developmental stages and calculate the metamorphosis rate. The carapace width, abdominal segment length, and telson length of the experimental and control groups at each stage were calculated. 150 zoanthid larvae hatched from each egg-carrying crab were temporarily housed in a 1 L beaker for mortality statistics. The carapace width, abdominal segment length, and telson length of the experimental group larvae at each stage were mostly greater or significantly greater than those of the control group ( Figure 2 The metamorphosis rate of the first stage larvae of the experimental group was significantly higher than that of the control group, and the mortality rate was lower than that of the control group ( Figure 3 ).

[0039] After the embryos were hatched, 150 zoeae I larvae from each brooding crab in the experimental group and the control group were taken out in a 1 L beaker and temporarily raised. 5 The first-stage larvae of Z. bicuspidatum were challenged by immersing them in seawater containing 100 CFU / mL of Metschnikowia bicuspidatum. After 12 hours, the larvae were transferred to normal seawater for continued culture. The mortality rate of the first-stage larvae of Z. bicuspidatum in each group after challenge was counted daily. Three replicates were set up for each group. The overall mortality rate of the first-stage larvae of Z. bicuspidatum in the control group after challenge was significantly higher than that in the experimental group ( Figure 4 ).

[0040] From the above, it can be seen that soaking and stimulating Chinese mitten crab embryos with inactivated M. bicuspidatum yeast can promote the development of Chinese mitten crab embryos and the development of the zoeae hatched therefrom, and improve the ability of the zoeae larvae to resist infection by M. bicuspidatum yeast.

[0041] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for raising seedlings for improving the disease resistance of Chinese mitten crab larvae, characterized in that: With a concentration of 1x10 5 The pre-nauplius stage of Chinese mitten crab was stimulated by immersing it in seawater containing 1×10 CFU / mL of inactivated yeast. After stimulation, the crabs were cultured in normal seawater until they reached the protozoan stage. The crabs were then cultured in normal seawater until they reached the protozoan stage. 5 The disease-resistant Chinese mitten crab larvae were obtained by immersing the crab in seawater containing inactivated Mycobacterium bicuspidatum at a concentration of 100 CFU / mL and using the crabs stimulated twice to produce zoanthid larvae.

2. The method for raising seedlings for improving the disease resistance of Chinese mitten crab larvae according to claim 1, characterized in that: The concentration is 1x10 5 The seawater of inactivated yeast was prepared by inactivating yeast with formaldehyde solution and the concentration was adjusted to 1x10 CFU / mL using sterile seawater. 5 CFU / mL.

3. The method for raising seedlings for improving the disease resistance of Chinese mitten crab larvae according to claim 2, characterized in that: The formaldehyde solution-inactivated yeast is prepared by adding formaldehyde solution to a resuspended bacterial solution containing yeast, mixing and incubating, and then centrifuging and washing to obtain the formaldehyde solution-inactivated yeast; wherein the concentration of yeast in the resuspended bacterial solution is adjusted to 2×10 7 CFU / mL, and the final concentration of formaldehyde solution in the resuspended bacterial solution was 0.5% (v / v).

4. The method for raising seedlings for improving the disease resistance of Chinese mitten crab larvae according to claim 3, characterized in that: The formaldehyde solution-inactivated yeast is prepared by inoculating yeast into YPD liquid culture medium for amplification and culture, removing excess culture medium, and repeatedly resuspending the yeast with sterile distilled water to obtain a resuspended bacterial solution; adding formaldehyde solution to the resuspended bacterial solution, mixing, and incubating at 4°C for 24 hours to obtain a yeast suspension, and then centrifuging and washing to obtain the formaldehyde solution-inactivated yeast.

5. The method for raising seedlings for improving the disease resistance of Chinese mitten crab larvae according to claim 4, characterized in that: The amplification culture temperature was 28°C, the time was 24 h, and the rotation speed was 180 r / min.

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