Seedling raising method for improving disease resistance of eriocheir sinensis larvae

Abstract

The invention relates to the technical field of aquaculture, in particular to a breeding method for improving the disease resistance of eriocheir sinensis larvae. The method comprises the following steps: soaking and stimulating egg-carrying crabs of the Eriocheir sinensis twice by using seawater containing inactivated Meischwa yeast with the concentration of 1 * 10 < 5 > CFU / mL, and hatching the stimulated egg-carrying crabs to generate daphnia larvae, namely the disease-resistant Eriocheir sinensis larvae. By adopting the breeding method provided by the invention to breed the egg-carrying crabs, the development of Eriocheir sinensis embryos can be promoted, and the resistance of zoea larvae to infection of Meischwa's yeast is improved.

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 fungus that is ubiquitous in nature. Many types of yeast have been confirmed to be the main pathogens of aquatic animals. For example, Torulopsis mogii, Candidalusitaniae and Metschnikowia bicuspidata can infect a variety of aquatic animals alone or together with other pathogens, inducing the death of aquatic animals, which seriously threatens the high-quality development of my country's aquaculture industry. Among them, Metschnikowia bicuspidata was first isolated from Daphnia magna in 1884. It is now known that it can infect a variety of aquatic animals including Macrobrachium rosenbergii, Portunus trituberculatus, Oncorhynchus tshawytscha and Eriocheir sinensis. There is currently no effective treatment.

[0003] Chinese mitten crab is a large freshwater aquaculture economic crab unique to my country, with very high economic value. my country is the world's largest producer of Chinese mitten crab, but with the continuous expansion of aquaculture scale and density, various diseases of Chinese mitten crab, such as tremor disease, "water-shrinking" disease, gill rot, enteritis, ciliates, etc., have broken out one after another. The "milk disease" first discovered in Panjin City, Liaoning Province in 2019 caused a large-scale death of Chinese mitten crabs. It is a highly harmful epidemic with a mortality rate of more than 20%, causing huge economic losses to farmers. It has been reported that the two-pointed mitten crab yeast is one of the pathogens that induce "milk disease". It is urgent to develop green prevention and control technologies for the "milk disease" of Chinese mitten crabs. Summary of the invention

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

[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 larvae are prepared by using a 1x10 5The Chinese mitten crab (Eriocheir sinensis) was stimulated to soak in seawater containing inactivated Saccharomyces cerevisiae at 100 CFU / mL twice, and the stimulated crab produced Zygophyllum larvae, thus obtaining disease-resistant Chinese mitten crab larvae.

[0007] Specifically, the pre-nauplius stage of Chinese mitten crab was treated with a solution containing 1x10 5 CFU / mL of inactivated Mycosaccharomyces cerevisiae was used for immersion stimulation. After stimulation, the crabs were cultured in normal seawater until the zoanthid stage. Then, the crabs cultured to this stage were treated with a solution containing 1x10 5 The crabs were stimulated by immersion in seawater containing inactivated Mycotoxins bicuspidatum at 10 CFU / mL. After two stimulations, the crabs produced Zygophyllum larvae, thus obtaining disease-resistant Chinese mitten crab larvae.

[0008] The concentration is 1x10 5 The seawater for inactivating the yeast Mycobacterium bicuspidatum with a concentration of 1x10 CFU / mL was the seawater for inactivating the yeast Mycobacterium bicuspidatum with a formaldehyde solution. The concentration was adjusted to 1x10 CFU / mL using sterile seawater. 5 CFU / mL.

[0009] The formaldehyde solution-inactivated yeast is prepared by adding a 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, 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 the yeast into a YPD liquid culture medium for amplification 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 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] The present 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 CFU / mL) of inactivated Mycosaccharomyces cerevisiae was used to soak and stimulate the brooding crabs, and an innovative Chinese mitten crab seedling raising technology was established. By adopting the method provided by the present invention to cultivate brooding crabs, the development of zoanthid larvae can be promoted, and the resistance of zoanthid stage I larvae to infection by Mycosaccharomyces cerevisiae can be enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The following is a graph showing the changes in the dry weight, wet weight and diameter of a single egg of Chinese mitten crab embryos at different stages after stimulation with inactivated Mycosaccharomyces cerevisiae in Example 1, wherein A: changes in the dry weight of a single egg of an embryo; B: changes in the wet weight of a single egg of an embryo; C: changes in the diameter of an embryo. E1: post-nauplius stage; E2: early protozoan stage; E3: protozoan stage; E4: late protozoan stage; E5: early stage of emergence. Significant differences between different groups are indicated by "*" (*: p<0.05; **: p<0.01).

[0015] Figure 2 The figure shows the changes in the width of the cephalothorax, the length of the abdominal segment and the length of the tail segment of the larvae of Chinese mitten crab at different stages after being stimulated by the inactivated yeast of Bicuspidae in Example 1, wherein A: the change in the width of the cephalothorax of the larvae; B: the change in the length of the abdominal segment of the larvae; C: the change in the length of the tail segment of the larvae. Z1: zoanthid stage I; Z2: zoanthid stage II; Z3: zoanthid stage III; Z4: zoanthid stage IV; Z5: zoanthid stage V; M: megalopa stage. The significant differences between different groups are indicated by "*" (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001).

[0016] Figure 3 The mortality rate and metamorphosis rate of the larvae of stage I of the zoea in the experimental group and the control group in Example 1, wherein A: metamorphosis rate statistics of larvae of stage I of the zoea; B: mortality rate statistics of larvae of stage I of the zoea. The significant differences between different groups at the same time point are indicated by "**" (p<0.01) and "***" (p<0.001).

[0017] Figure 4 1x10 in Example 1 5 The mortality rate of Z. divaricata stage I larvae in the experimental group and the control group after challenge with live Saccharomyces cerevisiae at CFU / mL. The significant differences between different groups at the same time point are indicated by “*” (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. Technical personnel skilled in the art can make some non-essential improvements and adjustments based on the above-mentioned content of the present invention.

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

[0020] The yeast used in the following is available to the public 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 Mycosaccharomyces cerevisiae was immersed in seawater for stimulation for 12 h. After stimulation, the crabs were cultured in normal seawater until the zoanthid stage. Then, the crabs were placed in a humidified water bath containing 1x10 5 The crabs were stimulated by immersion in seawater containing inactivated Saccharomyces cerevisiae at CFU / mL for 12 hours, and then transferred to normal seawater for continued cultivation. The egg-bearing crabs stimulated twice were used to produce Zygophyllum larvae.

[0023] In the present invention, formaldehyde solution is used to inactivate the yeast. The inactivation method comprises: inoculating the yeast into a YPD liquid medium for amplification culture, removing excess culture medium, and repeatedly resuspending the bacteria with sterile distilled water to obtain a resuspended bacterial solution. Adding formaldehyde solution to the resuspended bacterial solution and mixing well, incubating at 4°C for 24 hours to obtain a suspension of yeast, centrifuging and washing to obtain a yeast inactivated by formaldehyde solution. Before adding formaldehyde solution to the resuspended bacterial solution, adjust the concentration of the resuspended bacterial solution 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 24h, and the rotation speed is 180r / 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] (1) Cultivation of brooding crabs and zoa larvae: brooding crabs were provided by Photosynthetic Crab Company and were raised in seawater. The water was changed and feed was given once every two days. After the embryos were hatched, the zoa larvae were cultured separately from the mother crabs. The zoa larvae needed to be fed rotifers every day.

[0026] (2) Preparation of inactivated yeast: The yeast obtained from public channels was inoculated into YPD liquid medium for amplification culture (28°C, 180 r / min, 24 h). After centrifugation (6800×g, 25°C, 5 min) to remove excess culture medium, the cells were resuspended in sterile distilled water and repeated three times. The concentration of yeast in the resuspended culture was adjusted to 2×10 7 CFU / mL, and add a formaldehyde solution with a final concentration of 0.5% (v / v). The suspension of Saccharomyces cerevisiae containing the formaldehyde solution was incubated at 4°C for 24 hours. The bacterial suspension was centrifuged at 6800×g for 5 minutes at 4°C and washed twice with sterile distilled water to remove formaldehyde.

[0027] (3) Immersion stimulation of inactivated yeast on Chinese mitten crab embryos: The concentration of formaldehyde-inactivated yeast was adjusted to 1×10 5 CFU / mL, the pre-nauplius stage of Chinese mitten crab was placed in a solution containing 1x10 5 CFU / mL of inactivated Mycosaccharomyces cerevisiae 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 humidified water bath containing 1x10 5 CFU / mL of inactivated Metschnikowia bicuspidatum was immersed in seawater for stimulation for 12 hours, and then transferred to normal seawater for continued cultivation.

[0028] (4) Monitoring of dry weight, wet weight and egg diameter of Chinese mitten crab embryos at different stages: Chinese mitten crab embryos were taken with tweezers daily to observe the developmental stages under a microscope and the number was counted. After removing the surface water, the wet weight of the embryos was accurately weighed with a balance and the wet weight of each egg was calculated. The embryos were then wrapped with 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 zoan 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 zoan larvae hatched from each brooding crab were temporarily kept in a 1L beaker for calculating the mortality rate.

[0031] (7) Detection of resistance of stage I zoanthellae to Saccharomyces cerevisiae: After embryonic hatching, 150 stage I zoanthellae hatched from each brooding crab were temporarily raised in a 1L beaker and then incubated with a 1x10 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, 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 embodiments.

[0033] Example 1

[0034] The yeast was inoculated into YPD liquid medium for amplification culture (28°C, 180r / min, 24h). After removing the excess culture medium by centrifugation (6800×g, 25°C, 5min), the cells were resuspended with sterile distilled water and repeated 3 times. The concentration of the yeast in the resuspended solution was adjusted to 2x10 7 CFU / mL, and add a formaldehyde solution with a final concentration of 0.5% (v / v). The suspension of Saccharomyces cerevisiae containing the formaldehyde solution was incubated at 4°C for 24 hours. The bacterial suspension was centrifuged at 6800×g for 5 minutes at 4°C and washed twice with sterile distilled water to remove formaldehyde.

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

[0038] 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 entered the stage of stage II larvae, while all the larvae in the control group remained in the stage of stage I larvae. Figure 1 It 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 there was no significant difference in the dry weight of single eggs in each period. 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 of embryos in both the experimental and control groups showed an overall downward trend, and there was a significant difference between the two in the late protozoan stage after the second stimulation. After the first inactivated yeast stimulation, the egg diameter of the embryos in the experimental group had increased to a higher level and was significantly higher than that in the control group, while the embryos in the control group began to increase after the post-nauplius stage. This shows that the first and second inactivated Metschnikowia bicuspid stimulation may have promoted embryonic development and accelerated the consumption of nutrients.

[0039] After the embryos were hatched, the larval development period was observed under a microscope every day and the metamorphosis rate was calculated. The carapace width, abdominal segment length and tail segment length of the larvae in the experimental group and the control group at each stage were calculated. 150 zoanthid larvae hatched from each egg-carrying crab were temporarily kept in a 1L beaker for calculating the mortality rate. The carapace width, abdominal segment length and tail segment length of the larvae in the experimental group at each stage were mostly greater or significantly greater than those in the control group ( Figure 2 The metamorphosis rate of the first-stage larvae of the experimental group was also significantly higher than that of the control group, and the mortality rate was lower than that of the control group ( Figure 3 ).

[0040] After the embryos were hatched, 150 Zygomorpha I larvae from each egg-carrying crab in the experimental group and the control group were taken out in a 1L beaker and then kept in a 1x10 5 The first-stage larvae of Z. bicuspidatum were challenged by immersion 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 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 ).

[0041] It can be seen from the above that soaking and stimulating Chinese mitten crab embryos with inactivated Saccharomyces cerevisiae can promote the development of Chinese mitten crab embryos and the development of the zoanthid larvae hatched therefrom, and improve the ability of the zoanthid larvae to resist infection by Saccharomyces cerevisiae.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for raising seedlings for improving the disease resistance of Chinese mitten crab larvae, characterized in that: The concentration of 1x10 5 The Chinese mitten crab (Eriocheir sinensis) was stimulated to soak in seawater containing inactivated Saccharomyces cerevisiae at 100 CFU / mL twice, and the stimulated crab produced Zygophyllum larvae, thus obtaining disease-resistant Chinese mitten crab 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 was 1x10 5 The pre-nauplius stage of Chinese mitten crab was stimulated by immersion in seawater containing 1x10 CFU / mL of inactivated Metschnikowia bicuspidatum. After stimulation, the crabs were cultured in normal seawater until the protozoan stage. The crabs were then treated with 1x10 5 The crabs were immersed in seawater containing inactivated Saccharomyces cerevisiae at 100 CFU / mL for stimulation, and the brooding crabs were used to produce Zygophyllum larvae after two stimulations, thus obtaining disease-resistant Chinese mitten crab larvae.

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

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 adding a 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, the final concentration of formaldehyde solution in the resuspended bacterial solution was 0.5% (v / v).

5. The method for raising seedlings for improving the disease resistance of Chinese mitten crab larvae according to claim 4, characterized in that: The formaldehyde solution-inactivated yeast is prepared by inoculating the yeast into a YPD liquid culture medium for amplification 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 centrifuging and washing to obtain the formaldehyde solution-inactivated yeast.

6. 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 is 28° C., the time is 24 h, and the rotation speed is 180 r / min.

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