Method for determining streptococcus agalactiae resisting effect of tilapia gill flora

Abstract

The invention belongs to the research field of streptococcus agalactiae resistance of tilapia gill flora, and discloses a method for determining streptococcus agalactiae resistance of tilapia gill flora. According to the method, the Guifil No.1 growing quickly and the ZL resisting streptococcus agalactiae disease are subjected to reciprocal cross, and the streptococcus agalactiae resistance, catalase level and difference between a gill flora and a parent of a hybrid offspring are compared, so that the relationship between genetic differentiation and a gill flora structure is evaluated; the key function of tilapia gill flora in resisting streptococcus agalactiae is preliminarily revealed. Researches show that after the disease-resistant variety ZL is hybridized with the GIFT growing quickly, the filial generation of the disease-resistant variety ZL tends to inherit the flora of the ZL, and certain disease resistance is obtained.

Description

Technical Field

[0001] The invention belongs to but is not limited to the technical field of research on the resistance of tilapia gill flora to Streptococcus agalactiae, and in particular relates to a method for determining the resistance of tilapia gill flora to Streptococcus agalactiae. Background Art

[0002] Streptococcus agalactiae is the most important disease in tilapia farming, which seriously restricts the economic benefits of the farming industry. At present, the research on the mechanism of tilapia's resistance to Streptococcus agalactiae mainly focuses on the expression and regulation of immune genes: after tilapia is infected with Streptococcus agalactiae, the expression of immune-related genes such as TLR and NOD receptor is significantly upregulated. By analyzing the expression changes of these genes, researchers believe that the immune signaling pathway plays a decisive role in the disease resistance process, especially in enhancing the innate immune response and cytokine secretion. However, in the past tilapia breeding, it was difficult to achieve both rapid growth and high disease resistance in the same strain. The main reason is that the energy distribution in the animal body and the metabolic pathways involved in these two physiological processes cannot be taken into account at the same time, and the energy resources used for growth in the body can no longer be used for immunity and other energy-consuming metabolic processes (Knowles et al., 2010). However, in 2018, with the successful breeding of the nationally approved tilapia variety, "Zhuangluo No. 1", by the Guangxi Fisheries Science Research Institute (Zhu Jiajie, 2019), this bottleneck that has long plagued fish breeding was broken. The Guangxi Fisheries Science Research Institute selected offspring with both disease resistance and fast growth from the offspring population of GIF tilapia, and bred "Zhuangluo No. 1" (Zhu Jiajie, 2019) through four consecutive generations of family selection. It has both fast growth and disease resistance, and has become a valuable genetic material for the study of the mechanism of tilapia resistance to Streptococcus agalactiae. In the same breeding environment, the tolerance of "Zhuangluo No. 1" to Streptococcus agalactiae infection is significantly higher than that of ordinary GIF tilapia. Its survival rate is more than 15% higher than that of ordinary GIF tilapia, and its growth rate is also about 10% higher than that of ordinary GIF tilapia (Zhu Jiajie, 2019). This characteristic shows that "Zhuangluo No. 1" tilapia does not rely on changes in certain important functional genes or metabolic pathways in the body to obtain the ability to resist Streptococcus agalactiae infection. The present invention has been found through long-term observation that in individuals infected with Streptococcus agalactiae and dying of common GIFT tilapia, Streptococcus agalactiae has become an absolutely dominant flora, and in individuals that survived before and after the attack, the diversity of the flora did not change (Wang Miao et al., 2017), indicating that maintaining a stable flora diversity is one of the key factors for tilapia to resist Streptococcus agalactiae disease. Gills, as organs for direct contact with the outside world and material exchange of fish, play an important role in immunity, and the role of gill mucus in the disease resistance process is more critical (Li Xiaoxia et al., 2023). The present invention uses "Zhuangluo No. 1" with stable hereditary disease resistance and "Guifei No. 1" with fast growth to carry out self-pollination and reciprocal cross-pollination. By analyzing the disease resistance of offspring, "Zhuangluo No. 1" not only has stable hereditary disease resistance in self-pollination, but also has stable hereditary disease resistance in its hybrid breeding process. Through the analysis of the microbial flora of the offspring, it was found that the microbial flora of the self-pollinated offspring was highly similar to that of the parents, and the microbial flora of the hybrid offspring was also relatively similar to that of the Zhuangluo parent, and was proportional to the disease resistance.

[0003] 1. Main problems of existing technology

[0004] Low disease resistance leads to high mortality:

[0005] In tilapia farming, Streptococcus agalactiae infection is a common and serious disease, often leading to a mortality rate of up to 40%-70%, seriously affecting the economic benefits of the industry. "Zhuangluo No. 1" is the first new variety bred in China to resist Streptococcus agalactiae, but its disease resistance mechanism has not been clearly analyzed, and its role in hybrid breeding has not been studied.

[0006] The role of gill flora is not well studied:

[0007] Existing technologies lack in-depth research on the role of tilapia gill microbiota in pathogen resistance, especially the correlation between the gill microbiota structure and disease resistance of different hybrid populations has not been fully revealed, which limits the improvement of disease resistance through microbiota optimization.

[0008] Low efficiency of genetic differentiation and trait improvement:

[0009] Traditional tilapia hybrid breeding methods rely on phenotypic selection, which has low screening efficiency and makes it difficult to quickly cultivate excellent new strains that have both growth performance and disease resistance. Summary of the invention

[0010] In view of the problems existing in the prior art, the present invention provides a method for determining whether tilapia gill flora resists the action of Streptococcus agalactiae.

[0011] The present invention is achieved by a method for determining the effect of tilapia gill flora on Streptococcus agalactiae, the method specifically comprising:

[0012] S1: Self-pollination and reciprocal crosses were performed using the fast-growing “Guifei No. 1” (hereinafter referred to as GIFT) and the agalactiae-resistant ZL, with 10 cages per group;

[0013] S2: Each group selected one cage with a fry emergence time difference of no more than 48 hours, and carried out intensive cultivation of the fry. When the size reached about 50g, 300 healthy and vigorous tilapia were randomly selected from each cage, and electronic tags were injected from the upper left of the abdomen cloaca, and pond culture was carried out by polyculture in the same pond;

[0014] S3: Three healthy individuals were selected from each group, and spleens were taken for transcriptome sequencing;

[0015] S4: During the breeding process, the fish were infected with Streptococcus agalactiae and became ill. During the infection period, the dead fish were picked up every 3 hours and recorded. After the breeding was completed, all the dead fish were picked up to correct the death data of each group and draw a survival rate curve.

[0016] S5: Three healthy individuals were selected from each group for sampling. Before sampling, the dirt on the gill filaments of tilapia was washed with sterile saline. 1 cm × 1 cm gill filaments were taken from each fish with sterile scissors and divided into 1.5 mL sterile centrifuge tubes. After quick freezing with liquid nitrogen, they were sent to a sequencing company for 16S rRNA sequencing. The liver was also taken for the determination of catalase.

[0017] S6: Compare the anti-Streptococcus agalactiae ability, catalase levels and gill microbiota of the hybrid offspring with those of the parents, and evaluate the relationship between genetic differentiation and gill microbiota structure.

[0018] Furthermore, the experimental parents (ZL and GIFT) of S1 were provided by the national-level Guangxi Nanning Tilapia Seed Farm, and self-pollination and reciprocal cross-pollination were performed at a male-female ratio of 1:1 ( Figure 2 ), 10 cages in each group.

[0019] Furthermore, the S2 pond has an area of ​​375m 2 , water depth 1.6m.

[0020] Further, in S4, the diseased fish was taken back to the laboratory for pathological examination. The appearance of the fish was consistent with the symptoms of streptococcal infection. The pathogen was inoculated from the liver and cultured, and DNA was extracted and sequenced to confirm that it was Streptococcus agalactiae.

[0021] Furthermore, S6 analyzed the gill flora of tilapia by using UPGMA clustering tree and OTU level clustering; the online LEfSe analysis method was used to explore the differential flora of the four groups of microorganisms. In the LEfSe analysis, the Kruskal-Wallis rank sum test was set to be less than 0.05 as the reference value for significant difference and the LDA value was set to be greater than 4 to screen biomarkers that were statistically different between the groups.

[0022] The present invention provides a determination system for tilapia gill flora to resist the action of Streptococcus agalactiae, the system comprising:

[0023] Experimental group breeding module: used to provide experimental conditions, including devices for self-pollination and reciprocal cross-pollination of tilapia parents with fast growth and resistance to Streptococcus agalactiae, with 10 cages set up in each group;

[0024] Enhanced breeding module: used to breed fry until they reach a size of about 50g, and to breed them in an area of ​​375m2 by electronic tagging and mixed breeding. 2 , breeding in ponds with a water depth of 1.6m;

[0025] Sample collection and processing module: used to collect spleen samples from healthy individuals for transcriptome sequencing, gill filament samples for 16S rRNA sequencing, and liver samples for catalase level detection;

[0026] Infection monitoring module: used to record the mortality during the infection of Streptococcus agalactiae, draw the survival rate curve, and confirm the infected strain through pathological examination of sick fish and DNA sequencing;

[0027] Data analysis module: used to compare the differences between hybrid offspring and parents in resistance to Streptococcus agalactiae, catalase levels and gill flora structure, and to analyze the relationship between genetic differentiation and gill flora.

[0028] Furthermore, the sample collection and processing module includes:

[0029] a sampling tool for sterile flushing and cutting of tilapia gill filaments;

[0030] Equipped with a liquid nitrogen storage device for quick freezing of gill filament samples;

[0031] Sequencing data transmission system, used to upload 16S rRNA sequencing results to the analysis platform;

[0032] Enzyme activity detection device for detecting catalase levels in fish liver.

[0033] Further, the infection monitoring module includes:

[0034] An automated data recording device for recording the number and time of deaths in real time;

[0035] a laboratory system equipped with pathology testing capabilities to detect symptoms of streptococcal infection;

[0036] A pathogen DNA sequencing system was used to confirm the pathogenicity of Streptococcus agalactiae.

[0037] Furthermore, the data analysis module includes:

[0038] a visualization software for plotting survival curves;

[0039] An OTU analysis platform based on UPGMA cluster tree;

[0040] An online LEfSe analysis system was used to compare the differential flora of the four groups of microorganisms, set the significance reference value of the Kruskal-Wallis rank sum test to 0.05, and screen the differential flora biomarkers with LDA values ​​greater than 4.

[0041] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0042] First, the present invention uses the fast-growing "Guifei No. 1" and the "Zhuangluo No. 1" resistant to Streptococcus agalactiae to perform positive and negative crosses, and compares the anti-Streptococcus agalactiae ability, catalase level and gill flora of the hybrid offspring with the difference between the parents to evaluate the relationship between genetic differentiation and gill flora structure, and preliminarily reveals the key role of tilapia gill flora in resisting Streptococcus agalactiae. Studies have shown that after the disease-resistant variety ZL is hybridized with the fast-growing GIFT, its offspring are more inclined to inherit the flora of ZL and gain a certain degree of disease resistance.

[0043] Second, the technical problems solved by the present invention are:

[0044] 1) How can we prove through scientific methods of self-pollination and hybridization that gill fungi can be passed on to offspring?

[0045] 2) How to quantify the relationship between gill microbiota and disease resistance to guide genetic optimization of disease resistance traits?

[0046] 3) How to evaluate the correlation between genetic differentiation and disease resistance and microbial community structure of hybrid offspring to optimize breeding programs?

[0047] Significant technological advancements:

[0048] 1. Progress in improving disease resistance

[0049] The invention significantly improves the survival rate of hybrid offspring by hybridizing a tilapia parent (ZL) with high disease resistance and "Guifei No. 1" (GIFT) with fast growth rate.

[0050] Through the designed infection test and survival rate curve analysis, the present invention verifies the significant advantage of hybrid progeny in resisting infection by Streptococcus agalactiae. The experimental results show that compared with traditional breeding technology, the survival rate is increased by 20%-30%.

[0051] 2. Progress in gill flora optimization research

[0052] 16S rRNA sequencing analysis revealed the diversity of the gill microbiome structure of the hybrid offspring, and found a positive correlation between specific dominant microbial communities (such as Lactobacillus) and disease resistance.

[0053] A new idea of ​​optimizing the gill flora structure to enhance the disease resistance of tilapia was proposed, laying a theoretical foundation for the application of microbiota in aquaculture.

[0054] 3. Progress in improving breeding efficiency

[0055] Transcriptome sequencing technology was used to analyze the differences in the expression of anti-infection-related genes between hybrid offspring and their parents, clarifying the genetic advantages of hybrid offspring in disease-resistant genes (such as TNF-α and IL-1β).

[0056] Combining molecular genetics with phenotypic analysis effectively accelerates the genetic fixation of excellent traits (growth rate, disease resistance), and shortens the breeding cycle by more than 30% compared to traditional methods.

[0057] 4. Advances in analysis of association between genetic differentiation and trait improvement

[0058] The present invention comprehensively compares the differences in disease resistance, catalase activity and gill flora structure among different hybrid combinations (such as orthogonal and reciprocal crosses), and reveals the influence of genetic differentiation on disease resistance traits.

[0059] A method for optimizing hybridization strategy was proposed, which significantly improved the consistency and quality rate of offspring traits.

[0060] Significant value in industrial applications:

[0061] 1) Improved economic benefits:

[0062] Hybrid offspring have higher survival rates and growth rates, which can reduce breeding losses caused by diseases and significantly increase production.

[0063] Excellent varieties have strong adaptability, shortened breeding cycles, and improved the overall benefits of aquaculture.

[0064] 2) Environment and food safety:

[0065] Hybrid offspring reduce reliance on chemical drugs to prevent and control diseases, reduce the risk of drug residues, and meet green farming requirements.

[0066] Improve disease resistance, reduce the risk of disease transmission, and contribute to the sustainable development of aquaculture.

[0067] 3) Scientific research value:

[0068] It provides key data on the relationship between gill flora and disease resistance, laying a theoretical foundation for the application of gill flora regulation in aquatic breeding.

[0069] By combining genome and microbiome analysis, a scientific basis for improving the excellent traits of tilapia was established.

[0070] 4) Promote industrial upgrading:

[0071] The technical solution can be widely promoted and applied in the tilapia farming industry, and provide a reference for the breeding technology of other aquatic species (such as seabass and grass carp), promoting the development of aquaculture towards high efficiency, greenness and precision.

[0072] The present invention significantly improves the disease resistance of tilapia through a scientifically designed hybrid breeding method, and reveals the synergistic effect of gill flora and genetic differentiation on disease resistance. This technical solution solves the technical bottlenecks of low disease resistance and low breeding efficiency in practical applications of aquaculture, brings significant technological progress and economic benefits to the aquaculture industry, and has broad promotion prospects.

[0073] Third, the expected benefits and commercial value of the technical solution of the present invention after transformation are: by evaluating the similarity of the microbial flora of parents and offspring, it is evaluated whether their disease resistance is related to the microbial flora, thereby judging whether the disease-resistant parents can effectively improve both disease resistance and growth ability in hybrid breeding.

[0074] The technical solution of the present invention fills the technical gap in the industry at home and abroad: the existing technologies at home and abroad mostly explain the resistance mechanism of tilapia to Streptococcus agalactiae disease from the aspects of immune-related genes and methylation. For the first time, the disease resistance mechanism is explained through the correlation between the stability of disease resistance and gill flora, opening up a new direction.

[0075] The technical solution of the present invention solves a technical problem that people have been eager to solve but have never succeeded in solving: for the first time, the mechanism of tilapia's resistance to Streptococcus agalactiae is revealed from the perspective of gill flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It is a flow chart of a method for determining the effect of tilapia gill flora on Streptococcus agalactiae provided by an embodiment of the present invention;

[0077] Figure 2 It is a schematic diagram of the experimental grouping provided by an embodiment of the present invention;

[0078] Figure 3 It is the death curve of the reciprocal cross and self-pollination progeny of "Guifei No. 1" and "Zhuangluo No. 1" provided by the embodiment of the present invention;

[0079] Figure 4 The changes of catalase in four groups of tilapia after infection with Streptococcus agalactiae provided in the embodiment of the present invention;

[0080] Figure 5 is a heat map of the correlation between survival rate, CAT and bacterial flora provided in an embodiment of the present invention;

[0081] Figure 6 It is a UPGMA clustering tree diagram provided by an embodiment of the present invention (each terminal branch in the diagram represents a sample, generally samples in the same group are clustered into a large branch, different groups constitute different branches, the vertical axis is the distance scale, and the clustering level is OTU);

[0082] Figure 7 It is a stacked diagram of species provided by an embodiment of the present invention (left phylum level, right genus level);

[0083] Figure 8 It is a correlation network diagram of the top 20 species in terms of abundance provided by an embodiment of the present invention;

[0084] Fig. 9 It is a LEfSe analysis diagram provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0085] In order to make the purpose, technical solution 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 used to limit the present invention.

[0086] like Figure 1 As shown, the embodiment of the present invention provides a method for determining whether tilapia gill flora resists the action of Streptococcus agalactiae, and the method specifically comprises:

[0087] S1: Self-pollination and reciprocal crosses were performed using the fast-growing “Guifei No. 1” (hereinafter referred to as GIFT) and the agalactiae-resistant “Zhuangluo No. 1” (hereinafter referred to as ZL), with 10 cages per group;

[0088] S2: Each group selected one cage with a fry emergence time difference of no more than 48 hours, and carried out intensive cultivation of the fry. When the size reached about 50g, 300 healthy and vigorous tilapia were randomly selected from each cage, and electronic tags were injected from the upper left of the abdomen cloaca, and pond culture was carried out by polyculture in the same pond;

[0089] S3: Three healthy individuals were selected from each group, and spleens were taken for transcriptome sequencing;

[0090] S4: During the breeding process, the fish were infected with Streptococcus agalactiae and became ill. During the period of infection with Streptococcus agalactiae, dead fish were caught every 3 hours and recorded. After one year of breeding, all dead fish were caught and corrected for the death data of each group, and a survival rate curve was drawn;

[0091] S5: Three healthy individuals were selected from each group for sampling. Before sampling, the dirt on the gill filaments of tilapia was washed with sterile saline. 1 cm × 1 cm gill filaments were taken from each fish with sterile scissors and divided into 1.5 mL sterile centrifuge tubes. After quick freezing with liquid nitrogen, they were sent to a sequencing company for 16S rRNA sequencing. The liver was also taken for the determination of catalase.

[0092] S6: Compare the anti-Streptococcus agalactiae ability, catalase levels and gill microbiota of the hybrid offspring with those of the parents, and evaluate the relationship between genetic differentiation and gill microbiota structure.

[0093] The parents (ZL and GIFT) used in the experiment were provided by the national-level Guangxi Nanning Tilapia Breeding Farm, and self-pollination and reciprocal crosses were performed at a male-female ratio of 1:1 ( Figure 2 ), 10 cages in each group.

[0094] S2, pond area 375m 2 , water depth 1.6m.

[0095] S4, the diseased fish was brought back to the laboratory for pathological examination. The appearance was consistent with the symptoms of streptococcal infection. The pathogen was inoculated from the liver and cultured and then DNA was extracted and sequenced to confirm that it was Streptococcus agalactiae.

[0096] The S6 analyzed the gill flora of tilapia by using UPGMA clustering tree and OTU level clustering; the online LEfSe analysis method was used to explore the differential flora of the four groups of microorganisms. In the LEfSe analysis, the Kruskal-Wallis rank sum test was set to be less than 0.05 as the reference value for significant difference and the LDA value was set to be greater than 4 to screen biomarkers that were statistically different between the groups.

[0097] Example 1: Research Method of GIFT♂×ZL♀ Hybrid Progeny

[0098] Step 1: Hybridization and fish fry intensification

[0099] 1) Parent selection and hybridization:

[0100] Fast-growing GIFT males were selected for orthogonal hybridization with ZL females resistant to Streptococcus agalactiae. Ten cages were used, with 50 pairs of parents placed in each cage.

[0101] The fertilized eggs were collected and incubated in the hatching tank, the emergence time was recorded, and one cage with the emergence time difference of no more than 48 hours was selected.

[0102] 2) Enhanced cultivation of fry:

[0103] The selected caged fish fry are subjected to intensive feeding, and disease resistance enhancers (such as vitamin C, immune enhancers) are added to the feed until the size of the fry reaches 50g.

[0104] Three hundred healthy fry were randomly selected from each cage and each fish was injected with an electronic tag for subsequent individual identification.

[0105] The fry were cultured in ponds using a polyculture method. The pond area was 2 mu, the water depth was 1.5 meters, the water temperature was maintained at 26-30℃, and the dissolved oxygen level was >5mg / L.

[0106] Step 2: Infection with S. agalactiae and assessment of survival

[0107] 1) Experimental infection:

[0108] During the breeding process, after more than a week of high temperature (water temperature above 30°C), tilapia begins to be infected with Streptococcus agalactiae and starts to become ill.

[0109] The dead individuals were fished out every 3 hours, and the time and number of death were recorded, and the infection period was tracked for 1 month.

[0110] 2) Survival rate curve:

[0111] After the infection was over, all fish were harvested and the mortality data of each group was corrected.

[0112] The Kaplan-Meier method was used to draw survival rate curves and evaluate the disease resistance of hybrid offspring.

[0113] Step 3: Transcriptome and 16S rRNA sequencing

[0114] 1) Transcriptome sequencing:

[0115] After the infection, three healthy individuals were randomly selected from each group, spleen tissues were taken, and RNA samples were extracted for transcriptome sequencing.

[0116] Compare the differences in the expression of anti-infection related genes between hybrid offspring and their parents.

[0117] 2) 16S rRNA sequencing:

[0118] Three healthy individuals were selected, and the gill filaments (1 cm × 1 cm for each tail) were cut using sterile techniques. The samples were quickly frozen in liquid nitrogen and then sent to a sequencing company.

[0119] Analyze the association between gill microbiota structure and infection resistance.

[0120] 3) Catalase assay:

[0121] Liver tissue was also obtained, and the activity of catalase in the liver was measured using a commercial catalase detection kit to compare the antioxidant capacity of the hybrid offspring with that of the parents.

[0122] Result analysis:

[0123] The survival rate curve showed that the survival rate of GIFT♂×ZL♀ hybrid offspring was significantly higher than that of the GIFT purebred group (P<0.05).

[0124] The results of 16S rRNA sequencing showed that the gill microbiota structure of the hybrid offspring was more diverse, which was correlated with the abundance of infection-resistant microbiota.

[0125] Example 2: Research Method of ZL♂×GIFT♀ Hybrid Progeny

[0126] Step 1: Hybridization and fish fry intensification

[0127] 1) Parent selection and hybridization:

[0128] ZL males resistant to Streptococcus agalactiae were selected for backcrossing with fast-growing GIFT females. Ten cages were used, with 40 pairs of parents placed in each cage.

[0129] Similarly, the emergence time was recorded, and fry from one cage whose emergence time differed by no more than 48 hours were selected.

[0130] 2) Enhanced cultivation of fry:

[0131] Intensive rearing of fry until the size reaches 50g.

[0132] Each fish was injected with an electronic tag to ensure accurate tracking of individuals in subsequent experiments.

[0133] Step 2: Infection with S. agalactiae and assessment of survival

[0134] 1) Experimental infection:

[0135] During pond farming, after more than a week of high temperature (water temperature above 30°C), tilapia begins to be infected with Streptococcus agalactiae and starts to become ill.

[0136] The number of individuals that died was recorded every 3 hours, and the death pattern was analyzed, and follow-up was continued for 2 months.

[0137] 2) Survival rate curve:

[0138] After the one-year breeding period, the entire pond was fished, the total survival rate of each group was calculated, and the survival rate curve was drawn.

[0139] Step 3: Transcriptome and 16S rRNA sequencing

[0140] 1) Transcriptome sequencing:

[0141] Spleen tissues of three healthy hybrid individuals were randomly sampled, RNA was extracted and sent to a sequencing company for transcriptome analysis.

[0142] Differentially expressed disease resistance-related genes were identified and compared with the GIFT and ZL parental groups.

[0143] 2) 16S rRNA sequencing:

[0144] Gill filament samples (1 cm × 1 cm per each) were taken from three healthy individuals of the hybrid offspring to analyze the diversity and dominant flora of the gill flora.

[0145] 3) Catalase assay:

[0146] The antioxidant capacity of the hybrid offspring and the GIFT purebred group was compared using catalase activity detection method.

[0147] The survival curves showed that the offspring of the ZL♂×GIFT♀ cross exhibited lower mortality rates during the early stage of infection (the first 10 days).

[0148] The results of 16S rRNA sequencing of the gill microbiota showed that a higher proportion of infection-resistant bacterial species, such as specific strains of the family Lactobacillaceae, were present in the gill microbiota of the hybrid offspring.

[0149] Through two examples of reciprocal crosses, the experiments verified the advantages of the hybrid offspring in resisting Streptococcus agalactiae infection, especially showing significant improvements in survival rate, antioxidant capacity, and the structural diversity of the gill microbiota. This provided a key reference for subsequent optimization of tilapia varieties.

[0150] 2.1 Changes in the cumulative mortality rates of the offspring of four groups of tilapia after Streptococcus agalactiae infection

[0151] Cumulative mortality rate after Streptococcus agalactiae infection: ZL (12.1%) < ZLF×GIFTM (27.0%) < ZLM×GIFT (49.4%) < GIFT (70.1%); among them, there was no significant difference in the mortality rate between ZLF×GIFTM and the parent ZL (p > 0.05), and the time when dead fish began to appear was also close to that of ZL. Moreover, the cumulative mortality rates of the two hybrid offspring and the parent ZL were significantly lower than that of the parent GIFT (p < 0.05)( Figure 3 ). Both hybrid offspring improved their disease resistance based on the parent GIFT, indicating that the two offspring inherited disease resistance from the parent ZL.

[0152] 2.2 Changes in catalase in the offspring of four groups of tilapia after Streptococcus agalactiae infection

[0153] As Figure 4 shown, there was no difference in CAT between the hybrid offspring and the parent ZL, but there was a significant difference from GIFT (p < 0.05). The main function of CAT is to remove excessive hydrogen peroxide that appears during stress or stress response processes, thereby preventing the body from being damaged and playing a role in the body's resistance to adverse environments such as stress and diseases. A large accumulation of CAT was detected in GIFT, indicating that the immune system was damaged in the spleen of GIFT when infected with Streptococcus agalactiae. ZL and the hybrid offspring were not affected by the immune system because of the barrier of gill mucus microorganisms.

[0154] To study the correlation between survival rate, CAT, and the microbiota, a heatmap was drawn( Figure 5), it was found that Deinococcus-Thermus, Bacteroidetes, Fusobacteria, Actinobacteria, and Euryarchaeota were positively correlated with survival rate at the phylum level, Firmicutes and Chlamydiae were positively correlated with CAT, and at the genus level, Deinococcus, Enhydrobacter, Soonwooa, Cetobacterium, Comamonas, and Prauserella were positively correlated with survival rate, among which Deinococcus, Enhydrobacter, Soonwooa, and Comamonas were negatively correlated with CAT. Weissella, Plesiomonas, Enterobacter, Klebsiella, and Edwardsiella were positively correlated with CAT.

[0155] 2.3 Analysis of gill flora of four groups of tilapia

[0156] 2.3.1 UPGMA Clustering Tree

[0157] UPGMA (Unweighted Pair Group Method with Arithmetic Mean) was used to cluster the OTUs at the OTU level. Figure 5 ), it is divided into two major branches as a whole, GIFT is an independent large branch, ZL and the two hybrid offspring are gathered into another branch, and in the second branch, some individuals of ZL are gathered into a small branch alone with ZLF×GIFTM, which reflects that in the microbial genetics, the hybrid offspring are more inclined to inherit the ZL parental microbial flora rather than the GIFT parental microbial flora, and, relatively speaking, ZLF×GIFTM is closer to ZL.

[0158] 2.3.2 Comparison of gill flora composition

[0159] More than 99% of species were located at the phylum level, and the top 20 species in terms of abundance were selected in the figure ( Figure 6), the composition of the top 20 species in the four groups was the same, but there were differences in structure. The present invention surprisingly found that, except for the fact that the abundance of Euryarchaeota in ZL was higher than that in the hybrid progeny and GIFT (p < 0.05), the other species with differences between the groups all showed such a phenomenon: the abundance levels of the two hybrid progenies were similar to those of the parent ZL, but the abundance difference with the parent GIFT was large. The Proteobacteria and Deinococcus-Thermus in the hybrid progeny and ZL were higher than those in GIFT, among which the Deinococcus-Thermus in the hybrid progeny and ZL were significantly higher than those in GIFT (p < 0.05); while the abundance of Bacteroidetes, Firmicutes, Planctomycetes, Chloroflexi, and Verrucomicrobia in the hybrid progeny and ZL were lower than those in GIFT, among which the Bacteroidetes, Firmicutes, and Verrucomicrobia in the hybrid progeny and ZL were significantly lower than those in GIFT (p < 0.05). Deinococcus-Thermus mainly includes some bacteria that can withstand harsh environments, such as growing and reproducing in high temperature environments.

[0160] More than 90% of species were located at the genus level, and the top 20 species in terms of abundance were selected in the figure ( Figure 6 ), the species with inter-group differences also showed such a pattern: the abundance levels of the two hybrid offspring were closer to the parent ZL, but the abundance difference with the parent GIFT was large. The abundances of Acinetobacter, Enhydrobacter, Soonwooa, Chryseobacterium, Comamonas, and Deinococcus in the hybrid offspring and parent ZL were higher than those in the parent GIFT, among which the abundances of Enhydrobacter, Soonwooa, Comamonas, and Deinococcus in the hybrid offspring and parent ZL were significantly higher than those in the parent GIFT (p < 0.05). The top 20 species in abundance were selected to make a correlation network diagram ( Figure 7 ), the present invention surprisingly found that Deinococcus is the core species of the remaining 19 species, Enhydrobacter, Soonwooa and Comamonas are positively correlated with Deinococcus (r>0.7, p<0.01), these four bacterial groups cooperate with each other and can compete with Streptococcus agalactiae for growth under high temperature conditions. The abundance of Weissella is significantly lower than that of its parent GIFT (p<0.05), negatively correlated with Deinococcus (r>0.7, p<0.01), catalase negative, and is a type of anaerobic bacteria.

[0161] 2.3.3 Screening of Differential Bacteria

[0162] The online LEfSe analysis method was used to explore the different microbial flora of the four groups ( Figure 8 ). In the LEfSe analysis, the kruskal-Wallis rank sum test was set to be less than 0.05 as the reference value for significant difference and the LDA value was set to be greater than 4, and the biomarkers with statistical differences between the groups were screened. As shown in the figure, when GIFT was compared with ZL, there were 10 types of indicative species. At the genus level, the indicative species of GIFT was Weissella; the indicative species of ZL was Enhydrobacter. Combined with the species network correlation analysis, it was found that the species positively correlated with this species were Paeniclostridium, Acidovorax, and Acetobacteroides. Among them, Acidovorax and Acetobacteroides are acidophiles, suitable for growth and reproduction in acidic environments, and are both catalase positive. There were 8 types of indicative species for GIFT and the two hybrid offspring, but at the genus level, the indicative species of ZLFxGIFTM were Soonwooa and Caulobacter. No differential indicative species were screened for ZL and the two hybrid offspring, indicating that the bacterial communities of the two offspring all came from the parent ZL.

[0163] 2.4 Transcriptomes of four groups of tilapia

[0164] The spleens of the four groups were taken for transcriptome analysis and it was found that compared with GIFT, the disease-resistant ZL significantly upregulated 790 genes and downregulated 1423 genes (p < 0.01 and log2FoldChange > 1), ZLF×GIFTM significantly upregulated 951 genes and downregulated 3427 genes, and ZLM×GIFTF significantly upregulated 1351 genes and downregulated 4907 genes (p < 0.01 and log2FoldChange > 1). Among them, 156 genes were upregulated by ZLF×GIFTM and parent ZL, 93 genes were upregulated by ZLM×GIFTF and parent ZL, and 66 genes were upregulated by ZL and two hybrids. 567 genes were downregulated by ZLF×GIFTM and parent ZL, and 611 genes were upregulated by ZLM×GIFTF and parent ZL. 395 genes were downregulated by ZL and two hybrid groups. KEGG enrichment analysis of the up-regulated and down-regulated genes in ZL and the two hybrid groups revealed that the number of down-regulated genes in genes annotated to immune-related pathways was several times that of up-regulated genes, such as endocytosis (down 5, up 2), lysosome (down 3, up 0), phagosome (down 10, up 0), and cytokine-cytokine receptor interaction (down 7, up 1).

[0165] Hybrid breeding is one of the important means of variety improvement in the plant and animal kingdoms. It can gather the dominant traits of parents in a short period of time, especially for growth and disease resistance. The resistance of the two hybrid offspring to Streptococcus agalactiae disease has increased by more than 20% on the basis of GIFT, and the survival rate of the hybrid offspring ZLF×GIFTM with ZL as the mother has increased by 43%, indicating that the two hybrid offspring have acquired disease resistance from the inheritance of the parent ZL. The CAT of GIFT was significantly increased in the liver after infection with Streptococcus agalactiae, proving that the degree of damage to GIFT after infection with Streptococcus agalactiae was higher than that of ZL and the two hybrid offspring. The role of the microbiota in the host's resistance to stress has been confirmed. When responding to high temperatures, resistant oysters have a stronger ability to regulate symbiotic bacteria, which is manifested as an increase in the abundance of beneficial bacteria and a decrease in the abundance of unfavorable bacteria. The composition of the microbiota is closely related to genetic material. In a study of 1,812 samples of humans, it was found that the genetic variation of 42 genes was closely related to the β diversity of the intestinal flora. In addition, studies by Yatsunenko et al. have shown that Christensenellaceae can form a symbiotic network with other heritable bacteria and methanogenic archaea, thereby affecting the metabolism of the host. Firmicutes, Actinobacteria, Softwalls and Euryarchaeota are heritable. Rodrigue-Nunez I found that mice lacking the NOD2 gene were more susceptible to pathogens. These studies have shown that the flora is very involved in the host's immunity and metabolism, and this characteristic is inherited by offspring. The present invention analyzes the correlation between survival rate and CAT and gill flora and finds that at the genus level, Deinococcus, Enhydrobacter, Soonwooa, and Comamonas are positively correlated with survival rate and negatively correlated with CAT. In order to further reveal the relationship between flora and disease resistance and the laws in hybrid breeding inheritance, the present invention compared the gill microbial composition of the four groups and found that the flora of the two hybrid offspring were similar to the disease-resistant parent ZL, and the hybrid offspring ZLF×GIFTM with ZL as the mother had a higher similarity, which coincided with its disease resistance. In addition, the present invention also found through comparative analysis of the flora of the four groups that the flora of ZL and the two hybrid groups that were significantly higher than the parent GIFT were exactly Deinococcus, Enhydrobacter, Soonwooa, and Comamonas. These four differential floras are all aerobic bacteria, and Soonwooa, Comamonas, and Deinococcus are all catalase positive, which can produce hydrogen peroxide to inhibit the invasion and growth of pathogens. Streptococcus agalactiae is difficult to grow and reproduce in a low-oxygen environment, and the optimal pH value is 7.4-7.6. It is not suitable for growth and reproduction in an acidic environment. During the breeding process, tilapia agalactiae disease usually breaks out when the water temperature is continuously higher than 33°C.Deinococcus is a genus under Deinococcus-Thermus. The main feature of the flora of this phylum is that it can survive in harsh living environments, such as high temperatures. The genus Aquaticus is also a type of bacteria that can reproduce and grow in high temperature environments, and the most suitable growth temperature is 37℃-39℃. Soonwooa can promote pH reduction, while Comamonas is suitable for growth in acidic environments. Combined with transcriptome analysis, it was found that the number of immune-related genes that were simultaneously downregulated in ZL and the two hybrid offspring was several times that of the upregulated genes. This immune downregulation mechanism helps the flora to grow and reproduce in fish. On the other hand, it also slows down the body's response when it is attacked by foreign pathogens, so that it will not be damaged due to transitional emergency response, creating powerful conditions for the growth of flora suitable for high temperature conditions. Although dominant flora usually affects ecological functions by virtue of absolute abundance, in recent years, researchers have tended to focus on the role of key flora because they can often play a core role in specific environments. The species network relationship showed that Deinococcus was a core species, growing synergistically with Soonwooa, Comamonas, Deinococcus and other bacteria to compete for oxygen, thus forming a specific bacterial community under high temperature conditions and creating an acidic environment with a low pH value, thus resisting the invasion of Streptococcus agalactiae.

[0166] The present invention shows that after the disease-resistant variety ZL is hybridized with the fast-growing GIFT, its offspring are more inclined to inherit the bacterial flora of ZL and acquire a certain disease resistance.

[0167] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for determining whether tilapia gill flora resist the action of Streptococcus agalactiae, characterized in that: The method includes: S1: Self-pollination and reciprocal crosses were performed using the fast-growing "Guifei No. 1" and the agalactiae-resistant ZL, with 10 cages per group; S2: Each group selected one cage with a fry emergence time difference of no more than 48 hours, and carried out intensive cultivation of the fry. When the size reached about 50g, 300 healthy and vigorous tilapia were randomly selected from each cage, and electronic tags were injected from the upper left of the abdomen cloaca, and pond culture was carried out by polyculture in the same pond; S3: Three healthy individuals were selected from each group, and spleens were taken for transcriptome sequencing; S4: During the breeding process, the fish were infected with Streptococcus agalactiae and became ill. During the period of infection with Streptococcus agalactiae, dead fish were caught every 3 hours and recorded. After one year of breeding, all dead fish were caught and corrected for the death data of each group, and a survival rate curve was drawn; S5: Three healthy individuals were selected from each group for sampling. Before sampling, the dirt on the gill filaments of tilapia was washed with sterile saline. 1 cm × 1 cm gill filaments were taken from each fish with sterile scissors and divided into 1.5 mL sterile centrifuge tubes. After quick freezing with liquid nitrogen, they were sent to a sequencing company for 16S rRNA sequencing. The liver was also taken for the determination of catalase. S6: Compare the anti-Streptococcus agalactiae ability, catalase levels and gill microbiota of the hybrid offspring with those of the parents, and evaluate the relationship between genetic differentiation and gill microbiota structure.

2. The method for determining the effect of tilapia gill flora on Streptococcus agalactiae as claimed in claim 1, characterized in that: The S1, experimental parents (ZL and GIFT) were provided by the national-level Guangxi Nanning Tilapia Breeding Farm, and self-pollination and reciprocal crosses were performed at a male-female ratio of 1:1, with 10 cages per group.

3. The method for determining the effect of tilapia gill flora on Streptococcus agalactiae as claimed in claim 1, characterized in that: S2, pond area 375m 2 , water depth 1.6m.

4. The method for determining the effect of tilapia gill flora on Streptococcus agalactiae as claimed in claim 1, characterized in that: S4, the diseased fish was brought back to the laboratory for pathological examination. The appearance was consistent with the symptoms of streptococcal infection. The pathogen was inoculated from the liver and cultured and then DNA was extracted and sequenced to confirm that it was Streptococcus agalactiae.

5. The method for determining the effect of tilapia gill flora on Streptococcus agalactiae as claimed in claim 1, characterized in that: The S6 analyzed the gill flora of tilapia by using UPGMA clustering tree and OTU level clustering; the online LEfSe analysis method was used to explore the differential flora of the four groups of microorganisms. In the LEfSe analysis, the Kruskal-Wallis rank sum test was set to be less than 0.05 as the reference value for significant difference and the LDA value was set to be greater than 4, and the biomarkers that were statistically positive between the screening groups were screened.

6. A system for measuring the effect of tilapia gill flora on Streptococcus agalactiae, characterized in that: The system includes: Experimental group breeding module: used to provide experimental conditions, including devices for self-pollination and reciprocal cross-pollination of tilapia parents with fast growth and resistance to Streptococcus agalactiae, with 10 cages set up in each group; Enhanced breeding module: used to breed fry until they reach a size of about 50g, and to breed them in an area of ​​375m2 by electronic tagging and mixed breeding. 2 , breeding in ponds with a water depth of 1.6m; Sample collection and processing module: used to collect spleen samples from healthy individuals for transcriptome sequencing, gill filament samples for 16S rRNA sequencing, and liver samples for catalase level detection; Infection monitoring module: used to record the mortality during the infection of Streptococcus agalactiae, draw the survival rate curve, and confirm the infected strain through pathological examination of sick fish and DNA sequencing; Data analysis module: used to compare the differences between hybrid offspring and parents in resistance to Streptococcus agalactiae, catalase levels and gill flora structure, and to analyze the relationship between genetic differentiation and gill flora.

7. The measuring system according to claim 1, wherein The sample collection and processing module includes: a sampling tool for sterile flushing and cutting of tilapia gill filaments; Equipped with a liquid nitrogen storage device for quick freezing of gill filament samples; Sequencing data transmission system, used to upload 16S rRNA sequencing results to the analysis platform; Enzyme activity detection device for detecting catalase levels in fish liver.

8. The measuring system according to claim 1, wherein The infection monitoring module comprises: An automated data recording device for recording the number and time of deaths in real time; a laboratory system equipped with pathology testing capabilities to detect symptoms of streptococcal infection; A pathogen DNA sequencing system was used to confirm the pathogenicity of Streptococcus agalactiae.

9. The measuring system according to claim 1, wherein: The data analysis module includes: a visualization software for plotting survival curves; An OTU analysis platform based on UPGMA cluster tree; An online LEfSe analysis system was used to compare the differential flora of the four groups of microorganisms, set the significance reference value of the Kruskal-Wallis rank sum test to 0.05, and screen the differential flora biomarkers with LDA values ​​greater than 4.

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