Screening method of microbial indicator species for water quality grade of river crab earth pond breeding water body and application of screening method

Abstract

The invention belongs to the technical field of aquaculture, and particularly relates to a screening method of microbial indicator species for river crab earth pond seedling water quality grades and application of the screening method. Physical and chemical index data of water bodies of different river crab breeding earth ponds in different development periods of river crabs are obtained, physical and chemical indexes with violent data changes serve as water quality indexes, and water quality grades are divided; high-throughput sequencing is carried out, and microorganism species and composition of different river crab breeding earth ponds in different water quality grade groups in water bodies of the river crabs in different development periods are obtained and are relatively rich; calculating an indicator value by adopting an indicator value analysis method, and carrying out statistical test by utilizing multiple replacement test to obtain primarily screened microbial indicator species; and carrying out correlation analysis on the primarily screened microbial indicator species and the obtained physicochemical index data, and screening out the final microbial indicator species for evaluating the water quality grade of the river crab earth pond breeding water body. The method has the advantages of being strict in logic, scientific in system and reliable in result, and valuable reference is provided for dividing the water quality grade of the river crab earth pond breeding water body.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture, and particularly relates to a method for screening microbial indicator species for the water quality grade of Eriocheir sinensis larvae cultured in soil ponds and its application. Background Art

[0002] Chinese mitten crab (Eriocheir sinensis) is one of the most economically valuable crustaceans in China. Due to its rich nutrition and profound cultural connotations, Chinese mitten crab is very popular in China, and its aquaculture output reached more than 800,000 tons in 2023. The aquaculture of Chinese mitten crab usually includes three stages: the larval (zoea larvae, megalopa larvae) cultivation stage, the juvenile crab cultivation stage, and the adult crab cultivation stage. Among them, the larval cultivation stage is crucial for the Chinese mitten crab aquaculture industry and is the most technically demanding and risky link in the Chinese mitten crab aquaculture process. The cultivation of Chinese mitten crab seedlings mainly has two methods: indoor cement pond factory seedling cultivation and outdoor pond ecological seedling cultivation. Due to the advantages of less infrastructure investment, high seedling quality, and low seedling cultivation cost in outdoor soil pond ecological seedling cultivation, at present, Eriocheir sinensis larvae cultured in soil ponds have become the main source of Eriocheir sinensis seedlings (megalopa larvae) in China. However, since the outdoor soil pond seedling cultivation is an open system and has disadvantages such as poor controllability of environmental conditions and unstable seedling cultivation yield, it is very important to screen indicator species and monitor the aquaculture water environment.

[0003] The water environment of pond seedling cultivation usually includes phytoplankton, zooplankton, microorganisms, Eriocheir sinensis larvae, benthic organisms, etc. The composition and quantity of the microbial community play a crucial role in the pond water quality and Eriocheir sinensis larvae, etc. On the one hand, the water body microorganisms are very sensitive to the water quality changes in the aquaculture pond, and the abundance of some microorganisms can be used to evaluate the pond water quality. For example, the number of Escherichia coli can be used to evaluate the quality of drinking water. On the other hand, some bacteria themselves are pathogens or interact with pathogens, which can directly or indirectly affect the health status of Eriocheir sinensis seedlings and cause disease outbreaks. Therefore, water body microorganisms may be one of the ideal indicator organisms for the water quality of Eriocheir sinensis seedling ponds. Screening microbial indicators that can be used to indicate the health status of aquaculture water bodies has important practical significance for monitoring the water quality of seedling ponds.

[0004] In the current national environmental assessment standard (HJ 100I-2018), microorganisms are also one of the important assessment indicators. However, only the total number of Escherichia coli is involved due to the consideration of drinking risk. And the aquaculture water body pays more attention to physical and chemical indicators such as dissolved oxygen and ammonia nitrogen that directly affect the survival rate of aquaculture animals rather than the potability of water. Therefore, investigating the number of Escherichia coli cannot achieve the purpose of monitoring the aquaculture water body. There is an urgent need to establish a method that can simply, quickly, and accurately screen microbial indicator species to monitor the aquaculture water environment status to overcome the limitations of the existing technology.

[0005] In indicator species screening, phytoplankton and zooplankton are the most commonly used species, while microorganisms are rarely mentioned. The main reason is the difficulty in quantifying and identifying microorganisms. High-throughput DNA sequencing technology can identify microbial species and obtain their abundance by determining the barcode gene sequences of aquatic bacteria and comparing them with reference databases.

[0006] In indicator species screening methods, existing studies only rely on calculating the correlation between species and environmental factors (e.g., "Exploring the Indicative Role of Phytoplankton in Water Quality in the Ganjiang River Using Environmental DNA and Ecological Network Analysis") or critical species indicator analysis (e.g., "Characteristics of Attached Diatom Communities in Shanghai Urban River Networks and Their Indicative Role in Dissolved Total Nitrogen and Total Phosphorus"). The former essentially calculates whether a species has a linear response to continuously changing environmental factors, while the latter calculates the response threshold of a species to various environmental factors. Neither of these methods can screen for species that represent different levels of aquatic environments.

[0007] In conclusion, identifying microbial indicator species that can represent the water quality levels of different aquaculture water bodies has always been a challenging problem. Summary of the Invention

[0008] This invention provides a method for screening microbial indicator species for differentiating water quality grades in crab breeding ponds. This new analytical method offers a rigorous and scientifically sound approach for identifying microbial indicator species in different aquaculture water bodies. The microbial indicator species screened using this method are reliable and traceable, providing valuable indicators for the quality assessment and classification of aquaculture water bodies.

[0009] The technical solution of this invention is a method for screening microbial indicator species for water quality classification in earthen ponds for raising crab seedlings, comprising the following steps: (1) Obtain the physicochemical index data of the water in different crab seedling ponds at different development stages of crabs, and use the physicochemical indexes with drastic changes as water quality indicators, and classify the water quality grades according to the national local water environment quality standards. (2) High-throughput sequencing, clustering and species annotation were performed to obtain the composition and relative abundance of microbial species in different crab breeding ponds located in different water quality grade groups at different developmental stages of crabs; (3) The indicator value analysis method is used to calculate the indicator value and perform statistical tests using multiple permutation tests. The species that meet the statistical significance level (p<0.05) are the microbial indicator species for initial screening. The formula for the indicator value analysis method is as follows: IndVal ij = X ij × Y ij In the formula X ij = nInd ij / nInd j Y represents the proportion of the average abundance of species i in aquaculture water samples with water quality grade j to the total abundance of all samples; ij =nSt ij / nSt j , represents the proportion of the number of aquaculture water samples with species i present in water quality grade j to the total number of aquaculture environment samples of all grade j. (4) Perform a correlation analysis between the microbial indicator species initially screened in step (3) and the physicochemical index data obtained in step (1) to screen out the final microbial indicator species if there is a significant correlation (p<0.05).

[0010] In step (1), the physicochemical index data include, but are not limited to, nitrite, ammonia nitrogen, phosphate, nitrate, total nitrogen, total phosphorus, chlorophyll a, pH, dissolved oxygen, water temperature, etc.

[0011] In step (1), the physicochemical indicator with drastic data changes is ammonia nitrogen. The drastic data changes refer to the largest difference between the maximum and minimum values.

[0012] In step (1), the water quality is classified as follows: Class I water quality, ammonia nitrogen less than or equal to 0.15 mg / L; Class II water quality, ammonia nitrogen greater than 0.15 mg / L.

[0013] In step (2), clustering is used to obtain operational classification units, and species annotation is performed based on the operational classification units. Specifically, genomic DNA was extracted from the water bodies of different crab breeding ponds located in different water quality groups at different developmental stages to obtain total microbial genomic DNA. Primer pairs 338F (5'-ACTCCTACGGGAGGCAGAG-3', SEQ ID NO.1) and 806R (5'-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO.2, where H represents A, C, or T, V represents A, C, or G, and W represents A or T) were used to amplify the V3-V4 region of the microbial 16S rRNA gene. After PCR purification, PCR amplification and library construction were performed, followed by high-throughput sequencing. The high-throughput sequencing results were filtered using FastP and merged using FLASH. After quality control, high-quality sequences were obtained. The filtered sequences were clustered using the Deblur plugin in Qiime2 to obtain operational taxonomic units (OTUs). The Naive Bayes species annotation classifier in Qiime2 was used, based on SILVA 16S... The rRNA database (v138) provides species annotation for OTUs.

[0014] In step (2), the microbial species in the water bodies of different crab seedling ponds located in different water quality grade groups at different developmental stages of crabs are mainly composed of phyla such as Actinobacteriota, Cyanobacteria, Bacteroidota, Firmicutes, Patescibacteria, Proteobacteria and Verrucomicrobiota.

[0015] In step (3), the multiple permutation test is performed 1000 times.

[0016] In step (3), the microbial indicator species screened initially include Microbacteriaceae and Rhodobacteraceae.

[0017] In step (4), the final screened microbial indicator species include Microbacteriaceae and Rhodobacteraceae. Rhodobacteraceae showed a significant correlation with Class I water quality, which uses ammonia nitrogen concentration as an indicator, while Microbacteriaceae showed a significant correlation with Class II water quality, which uses ammonia nitrogen concentration as an indicator. In step (4), the relative abundance values ​​of the finally screened microbial indicator species in the water quality at different developmental stages of the crab are obtained, as well as the relative abundance trends in the timeline of the crab development cycle (Daphnia stage I, Daphnia stage II, Daphnia stage III, Daphnia stage IV, Daphnia stage V, and Megaptera stage). In the timeline of the crab development cycle, the relative abundance of Rhodobacteraceae shows a decreasing trend, while the relative abundance of Microbacteriaceae shows an increasing trend. The intersection point of the relative abundance of Rhodobacteraceae and Microbacteriaceae is obtained. Before the intersection point, the relative abundance of Rhodobacteraceae is higher than that of Microbacteriaceae, which belongs to Class I water quality with ammonia nitrogen concentration as the indicator. After the intersection point, the relative abundance of Rhodobacteraceae is lower than that of Microbacteriaceae, which belongs to Class II water quality with ammonia nitrogen concentration as the indicator. Furthermore, for Class I water quality using ammonia nitrogen concentration as an indicator, the relative abundance of Rhodobacteraceae should be no less than 30%-35%, and the relative abundance of Microbacteriaceae should be less than 30%-35%. More specifically, the relative abundance of Rhodobacteraceae should be no less than 35%, and the relative abundance of Microbacteriaceae should be less than 35%. For Class II water quality using ammonia nitrogen concentration as an indicator, the relative abundance of Rhodobacteraceae should be less than 30%-35%, and the relative abundance of Microbacteriaceae should be no less than 30%-35%. More specifically, the relative abundance of Rhodobacteraceae should be less than 35%, and the relative abundance of Microbacteriaceae should be no less than 35%.

[0018] Microbacteriaceae and Rhodobacteraceae can be used to evaluate the water quality grade of crab seedling ponds using ammonia nitrogen concentration as an indicator. Specifically, the relative abundance values ​​of Microbacteriaceae and Rhodobacteraceae in the water at different developmental stages of crabs are detected. The relative abundance trends of Microbacteriaceae and Rhodobacteraceae along the timeline of the crab development cycle are obtained, as well as the intersection point of the relative abundance of Rhodobacteraceae and Microbacteriaceae. Before the intersection point, the relative abundance of Rhodobacteriaceae is higher than that of Microbacteriaceae, which belongs to Class I water quality using ammonia nitrogen concentration as an indicator. After the intersection point, the relative abundance of Rhodobacteriaceae is lower than that of Microbacteriaceae, which belongs to Class II water quality using ammonia nitrogen concentration as an indicator.

[0019] A method for evaluating the water quality grade of crab seedling ponds, particularly a method using ammonia nitrogen concentration as an indicator, includes the following steps: detecting the relative abundance values ​​of Microbacteriaceae and Rhodobacteraceae in the water at different developmental stages of crabs; obtaining the relative abundance change trends of Microbacteriaceae and Rhodobacteraceae along the timeline of the crab development cycle; and identifying the intersection point of the relative abundance of Rhodobacteraceae and Microbacteriaceae. Before the intersection point, if the relative abundance of Rhodobacteriaceae is higher than that of Microbacteriaceae, the water quality is classified as Class I (using ammonia nitrogen concentration as an indicator); after the intersection point, if the relative abundance of Rhodobacteriaceae is lower than that of Microbacteriaceae, the water quality is classified as Class II (using ammonia nitrogen concentration as an indicator). Furthermore, if the relative abundance of Rhodobacteraceae is not less than 30%-35% and the relative abundance of Microbacteriaceae is less than 30%-35%, and even further, if the relative abundance of Rhodobacteraceae is not less than 35% and the relative abundance of Microbacteriaceae is less than 35%, the water quality is classified as Class I. If the relative abundance of Rhodobacteraceae is less than 30%-35% and the relative abundance of Microbacteriaceae is not less than 30%-35%, and even further, if the relative abundance of Rhodobacteraceae is less than 35% and the relative abundance of Microbacteriaceae is not less than 35%, the water quality is classified as Class II. Attached Figure Description

[0020] Figure 1 The relative abundance of Microbacteriaceae is shown as follows: from the zoea larvae I stage to the megalopa larvae stage, representing the timeline of the crab's developmental cycle.

[0021] Figure 2 The relative abundance of Rhodobacteraceae is shown as follows: from the zoea larvae I stage to the megalopa larvae stage, representing the timeline of the crab's developmental cycle. Detailed Implementation

[0022] Example 1 1.1 Experimental Design and Sampling A survey was conducted on three crab larvae rearing ponds (each approximately 1666 square meters in size) in Rudong County, Nantong City, Jiangsu Province. Water samples were collected at different developmental stages, including Daphnia I (Z1), Daphnia II (Z2), Daphnia III (Z3), Daphnia IV (Z4), Daphnia V (Z5), and megalopa (DY) larvae, and environmental physicochemical factors were measured simultaneously. 1 L of each sample was filtered onto a 0.2 μm pore size polycarbonate membrane, rapidly frozen in liquid nitrogen, and stored at -80°C for further analysis. A total of 18 water samples were collected.

[0023] 1.2 DNA extraction, PCR amplification, and sequencing analysis Total microbial genomic DNA was extracted from 18 samples. The V3-V4 region of the microbial 16S rRNA gene was amplified using primer pairs 338F (5'-ACTCCTACGGGAGGCAGAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). After purification of the PCR products, libraries were constructed using the NEXTFLEX Rapid DNA-Seq Kit. Sequencing was performed using an Illumina PE250 platform.

[0024] After obtaining high-throughput sequencing results, sequences were filtered using FastP (v0.19.6) and paired-end merging was performed using FLASH (v1.2.11). After quality control, a total of 753,823 high-quality sequences were obtained, with 30,347-68,429 reads per sample. The filtered sequences were clustered using the Deblur plugin in Qiime2 (v2020.2) to obtain operational taxonomic units (OTUs). At a 97% similarity level, these sequences were clustered into 1,431 OTUs. To reduce the impact of sequencing depth on α and β diversity, the sequences per sample were flattened to 30,347, with an average coverage of 97.90%. The Naive Bayes species annotation classifier in Qiime2 was used to annotate the OTUs based on the SILVA 16S rRNA database (v138).

[0025] 1.3 Data Analysis Physicochemical analysis showed that the highest ammonia nitrogen concentration during aquaculture was more than 10 times the lowest, making it the most volatile water quality indicator (Table 1). According to the National Surface Water Environmental Quality Standard (GB3838-2002), samples were divided into two groups based on ammonia nitrogen concentration: Class I water bodies (≤0.15 mg / L) and Class II water bodies (≥0.15 mg / L). IndVal values ​​for each group were calculated, and the reliability of the results was verified using a multiple permutation test (1000 permutations). Pearson correlation analysis was performed using SPSS software to verify the indicative relationship between the selected groups and each eutrophication indicator and environmental factors.

[0026] 2. Research Results The microbial community mainly consists of phyla such as Actinobacteriota, Cyanobacteria, Bacteroidota, Firmicutes, Patescibacteria, Proteobacteria, and Verrucomicrobiota. Statistical tests after IndVal calculations showed that Microbacteriaceae (Actinobacteriota) and Rhodobacteraceae (Proteobacteria) are two ideal indicator microorganisms (Table 2). These represent Class II aquatic environments with high ammonia nitrogen concentration and Class I environments with low ammonia nitrogen concentration, respectively. The relative abundance of the former increases during seedling cultivation (…). Figure 1 The relative abundance of the latter decreases during the seedling stage. Figure 2 Meanwhile, the relative abundance of Microbacteriaceae suddenly and sharply increased to 35% during Zoe IV, while the abundance of Rhodobacteraceae decreased sharply, accounting for less than 35%, and further decreased to below 30% during Zoe V. Combined with water quality index analysis, significant correlations were found between Microbacteriaceae and Rhodobacteraceae and nitrite, ammonia nitrogen, and phosphate (Table 3). Furthermore, although the relative abundances of Microbacteriaceae and Rhodobacteraceae fluctuated after Zoe IV, they remained above 35% and below 40%, respectively. Therefore, the relative abundances of Microbacteriaceae and Rhodobacteraceae can effectively indicate the water quality status of crab seedling culture water bodies.

[0027] Table 1 Physicochemical factors of water samples from various crab breeding bodies Table 2. Screened microbial indicator species and their indicator values Table 3. Pearson correlation analysis between abundance of Microbacteriaceae and Rhodobulariaceae and nutrient indices. Example 2 Verification experiments were conducted in three crab breeding ponds in Sheyang, Jiangsu Province. High-throughput sequencing revealed that the microbial community mainly consisted of phyla such as Actinobacteriota, Cyanobacteria, Bacteroidota, Firmicutes, Patescibacteria, Proteobacteria, and Verrucomicrobiota. Among the various physicochemical indicators, ammonia nitrogen showed the greatest variation across the samples, with the lowest and highest concentrations differing by more than 10 times, indicating drastic changes. Therefore, ammonia nitrogen was used as an indicator for water quality classification. Statistical tests after IndVal calculations showed that the Microbacteriaceae (Actinobacteriota) and Rhodobulariaceae (Proteobacteria) were indicator microorganisms that indicated ammonia nitrogen concentration levels (Table 2). Microbacteriaceae represented Class II water environments with high ammonia nitrogen concentrations, while Rhodobulariaceae represented Class I environments with low ammonia nitrogen concentrations. Microbacteria and Rhodobulariaceae showed significant correlations with nitrite, ammonia nitrogen, and phosphate, and were influenced by more environmental factors. In Class I water bodies, except for DY-1, the relative abundance of Rhodobulbaceae was not less than 35% and the relative abundance of Microbacteriae was less than 35%, with an accuracy rate of 88.9%; except for Z2-3 and Z4-1, in Class II water bodies, the relative abundance of Microbacteriae was not less than 35% and the relative abundance of Rhodobulbaceae was less than 35%, with an accuracy rate of 77.8%.

[0028] Table 4. Changes in physicochemical factors in the seedling ponds of Sheyang River crabs Table 5. Microbial indicator species and their indicator values ​​selected for the Sheyang River crab nursery pond. Table 6. Pearson correlation analysis between the abundance of the two microorganisms and nutrient indicators. Table 7. Relative abundance of Rhodobulariaceae and Microbacteriaceae in various water samples from the Sheyang River crab breeding area. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for screening microbial indicator species for water quality classification in earthen ponds for raising crab seedlings, characterized by the following steps: include: (1) Obtain the physicochemical index data of the water in different crab seedling ponds at different development stages of crabs, and use the physicochemical indexes with drastic changes as water quality indicators, and classify the water quality level according to the national local water environment quality standards. (2) High-throughput sequencing, clustering and species annotation were performed to obtain the composition and relative abundance of microbial species in different crab breeding ponds located in different water quality grade groups at different developmental stages of crabs; (3) The indicator value analysis method was used to calculate the indicator value and perform statistical tests using multiple permutation tests to screen microbial indicator species. The formula for the indicator value analysis method is as follows: IndVal ij = X ij × Y ij In the formula X ij = nInd ij / nInd j Y represents the proportion of the average abundance of species i in aquaculture water samples with water quality grade j to the total abundance of all samples; ij =nSt ij / nSt j , represents the proportion of the number of aquaculture water samples with species i present in water quality grade j to the total number of aquaculture environment samples of all grade j. (4) Perform correlation analysis between the microbial indicator species initially screened in step (3) and the physicochemical index data obtained in step (1) to screen out the final microbial indicator species, and obtain the relative abundance values ​​of the final screened microbial indicator species in the water quality of different development stages of crabs, as well as the relative abundance change trend in the time axis of crab development cycle.

2. The screening method according to claim 1, characterized in that, In step (1), the physicochemical index data include nitrite, ammonia nitrogen, phosphate, nitrate, total nitrogen, total phosphorus, chlorophyll a, pH, dissolved oxygen, and water temperature.

3. The screening method according to claim 1, characterized in that, In step (1), the physicochemical index with drastic data changes is ammonia nitrogen.

4. The screening method according to claim 1, characterized in that, In step (1), the water quality is classified as follows: Class I water quality, ammonia nitrogen less than or equal to 0.15 mg / L; Class II water quality, ammonia nitrogen greater than 0.15 mg / L.

5. The screening method according to claim 1, characterized in that, In step (2), the primer pairs used for amplification in high-throughput sequencing are as follows: ACTCCTACGGGAGGCAGAG (SEQ ID NO.1) and GGACTACHVGGGTWTCTAAT (SEQ ID NO.2).

6. The screening method according to claim 1, characterized in that, In step (3), the microbial indicator species screened initially include Microbacteriaceae and Rhodobacteraceae.

7. The screening method according to claim 1, characterized in that, In step (4), the final screened microbial indicator species include Microbacteriaceae and Rhodobacteraceae. In the timeline of the crab development cycle, the relative abundance of Rhodobacteraceae shows a decreasing trend, while the relative abundance of Microbacteriaceae shows an increasing trend. The intersection point of the relative abundance of Rhodobacteriaceae and Microbacteriaceae is obtained. Before the intersection point, the relative abundance of Rhodobacteriaceae is higher than that of Microbacteriaceae, which belongs to Class I water quality with ammonia nitrogen concentration as the indicator. After the intersection point, the relative abundance of Rhodobacteriaceae is lower than that of Microbacteriaceae, which belongs to Class II water quality with ammonia nitrogen concentration as the indicator.

8. Microbacteriaceae and Rhodobacteraceae are used to evaluate the water quality grade of crab seedling ponds using ammonia nitrogen concentration as an indicator. The characteristic of these components is that... The relative abundance values ​​of Microbacteriaceae and Rhodobacteraceae in water bodies at different developmental stages of Chinese prawns were detected. The relative abundance trends of Microbacteriaceae and Rhodobacteraceae along the timeline of the Chinese prawn development cycle were obtained, as well as the intersection point of the relative abundance of Rhodobacteraceae and Microbacteriaceae. Before the intersection point, the relative abundance of Rhodobacteriaceae was higher than that of Microbacteriaceae, which belonged to Class I water quality with ammonia nitrogen concentration as the indicator. After the intersection point, the relative abundance of Rhodobacteriaceae was lower than that of Microbacteriaceae, which belonged to Class II water quality with ammonia nitrogen concentration as the indicator.

9. A method for evaluating the water quality grade of a crab seedling pond, characterized by the following steps: include: The relative abundance values ​​of Microbacteriaceae and Rhodobacteraceae in water bodies at different developmental stages of Chinese prawns were detected. The relative abundance trends of Microbacteriaceae and Rhodobacteraceae along the timeline of the Chinese prawn development cycle were obtained, as well as the intersection point of the relative abundance of Rhodobacteraceae and Microbacteriaceae. Before the intersection point, the relative abundance of Rhodobacteriaceae was higher than that of Microbacteriaceae, which belonged to Class I water quality with ammonia nitrogen concentration as the indicator. After the intersection point, the relative abundance of Rhodobacteriaceae was lower than that of Microbacteriaceae, which belonged to Class II water quality with ammonia nitrogen concentration as the indicator.

10. The evaluation method according to claim 9, characterized in that, If the relative abundance of Rhodobacteraceae is not less than 30%-35% and the relative abundance of Microbacteriaceae is less than 30%-35%, the water quality is classified as Class I; if the relative abundance of Rhodobacteraceae is less than 30%-35% and the relative abundance of Microbacteriaceae is not less than 30%-35%, the water quality is classified as Class II.

Images