A detection method for evaluating the food diversity of Procambarus clarkii
By determining the positive correlation between the number of intestinal microorganisms of the protozoa carp and the number of feeding plants, the problem of time-consuming and labor-intensive identification of the protozoa carp in traditional methods is solved, and a rapid and low-cost dietary diversity evaluation is achieved, which reduces the establishment The cost of the gene bank provides an efficient evaluation method.
Patent Information
- Application Number
- CN202311457224.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-03
- Publication Date
- 2024-12-06
- Estimated Expiration
- 2043-11-03
AI Technical Summary
Traditional methods are time-consuming and labor-intensive for identification of the diet of Crayfish, low resolution, and DNA macro-barcoding technology requires the establishment of a local biological gene bank, which is costly and difficult to quickly and accurately evaluate its dietary diversity.
By determining the positive correlation between the number of intestinal microorganisms of Protozoa Crayfish and the number of plants it feeds, high-throughput technology and DNA metagenomic technology were used to combine ethanol cleaning and cryopreservation of samples to quickly evaluate the diversity of Protozoa Crayfish's diet .
It has achieved rapid, low-cost and reliable evaluation of the dietary diversity of Crayfish, reduced labor intensity and cost, and did not require a large-scale establishment of environmental plant DNA database. It is simple to operate and has broad application prospects.
Smart Images
Figure CN117385069B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of animal feeding habit evaluation, and in particular to a detection method for evaluating the feeding habit diversity of Procambarus clarkii. Background Art
[0002] The study of animal diet generally includes two parts: field surveys and indoor experiments. Field surveys can provide a large amount of information about animal diets and are the basis for laboratory analysis. Laboratory analysis can provide correction information for the diet structure observed in the field. Traditional laboratory analysis usually uses feeding experiments and microscopic analysis of gastrointestinal contents or feces. The transfer effect of chemical elements and macromolecules between trophic levels can also determine the food source information of animals, including stable isotope technology and biomarker methods. At the same time, with the rapid development of biochemical and molecular technologies, especially the maturity of sequencing technology, DNA macrobarcoding technology has become an important method for studying animal diets. This method is fast, accurate, and has high resolution. It has been applied in the identification, differentiation, and coexistence of species in the same area of animal diets.
[0003] Procambarus clarkii is an important omnivorous animal that plays the role of a key species in the ecosystem. Omnivorous animals have complex roles in aquatic communities, so their ecological benefits are huge. Identifying the composition of Procambarus clarkii's food can provide key information for evaluating its trophic status and how it affects Procambarus clarkii in aquatic ecosystems. Food selection depends on several key factors, such as resource availability, preference, and size. Resource division among individuals of the same species can lead to differentiation in nutritional selection. Previous studies on the nutritional ecology of Procambarus clarkii have shown that although Procambarus clarkii has an opportunistic feeding habit, this species prefers large aquatic plants and large benthic invertebrates.
[0004] The diet of Procambarus clarkii is usually observed using traditional microscopic observation, which is usually time-consuming, laborious, and has low resolution. DNA metabarcoding technology can obtain more information and higher resolution, and can better study the diet of Procambarus clarkii. However, DNA metabarcoding technology requires the establishment of a local biological gene bank, which is relatively expensive. Intestinal microorganisms, as the most important "microbial organs" in the animal body, participate in individual nutrient absorption, metabolic regulation and immune function, and can well reflect the diet of omnivorous animals. Summary of the invention
[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a detection method for evaluating the diversity of the diet of Procambarus clarkii. The number of intestinal microorganisms of Procambarus clarkii is determined by high-throughput technology, and the number of plants consumed by Procambarus clarkii is determined by DNA metagenomic technology, thereby obtaining the correlation between the number of intestinal microorganisms of Procambarus clarkii and the number of plants consumed by Procambarus clarkii. Based on the correlation between the two, we can effectively characterize the diversity of Procambarus clarkii's diet. The detection method provided by the present invention is fast, convenient and inexpensive.
[0006] Technical solution: The present invention provides a detection method for evaluating the diversity of Procambarus clarkii's diet. The method utilizes the positive linear correlation between the number of intestinal microorganisms of Procambarus clarkii and the number of plants it feeds on, and evaluates the diversity of Procambarus clarkii's diet by detecting the number of OTUs of Procambarus clarkii's intestinal microorganisms.
[0007] Furthermore, the method specifically includes the following steps:
[0008] S1. Take an adult Procambarus clarkii and clean the body surface with ethanol; then dissect the Procambarus clarkii, take the intestinal contents, and freeze them;
[0009] S2. Determining the number of microorganisms in the intestinal contents using high throughput technology;
[0010] S3. Evaluation of the diversity of Procambarus clarkii's diet based on the number of microorganisms measured.
[0011] Furthermore, in S3, the number of measured microorganisms was positively correlated with the diversity of the diet of Procambarus clarkii. The more the number of measured microorganisms, the more diverse the diet of Procambarus clarkii was.
[0012] Furthermore, in S1, the ethanol is specifically ethanol with a concentration of 99%.
[0013] Furthermore, in S1, the temperature of the frozen storage is -80°C.
[0014] Beneficial effects: Compared with the prior art, the present invention is a new detection method, which uses the number of intestinal microorganisms of Procambarus clarkii as an indicator of the feeding habits of Procambarus clarkii, and further evaluates the number of plant species ingested by Procambarus clarkii. The present invention does not require the establishment of a large-scale environmental plant DNA library, greatly reducing labor intensity and saving costs. The present invention has a simple operation method, low cost, and high reliability, and has broad application prospects in evaluating the feeding habits of Procambarus clarkii. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 A diagram showing the relationship between the intestinal microorganisms of Procambarus clarkii and its feeding habits in Implementation Example 1;
[0016] Figure 2 The number of OTUs of intestinal microorganisms of Procambarus clarkii after different artificial cultivation methods in Implementation Method 2 (C1 and C2 represent the treatment of Alternanthera philoxeroides-Elodea, and C3 and C4 represent the treatment of Alternanthera philoxeroides-Elodea-willow leaf). DETAILED DESCRIPTION
[0017] The present invention is described in detail below in conjunction with the embodiments.
[0018] Implementation 1:
[0019] Establish the quantitative relationship between the number of intestinal microorganisms of Procambarus clarkii and the number of plants it ingests:
[0020] From July to September 2023, three adult Procambarus clarkii were captured using ground traps in different habitats in different watersheds in Lianshui County. Each collected individual was immediately placed in a 20 ml centrifuge tube filled with 99% ethanol, brought back to the laboratory, and refrigerated at –80°C until dissection. The samples to be dissected were removed from the refrigerator and immediately rinsed with 75% ethanol to wash away all possible non-host plant debris on the outside of the Procambarus clarkii body. The intestines (including the foregut, midgut, and hindgut) of the Procambarus clarkii samples were dissected and the contents were flushed into 5 ml cryovials. The intestinal content samples were numbered and stored in a –80°C refrigerator until processing.
[0021] Plant ingestion in Crayfish: DNA was extracted from each homogenized gut content (20–30 mg wet weight) sample using the DNAsecure Plant Kit (Cat # DP320-03, Tiangen Biotech 210 Corporation, Beijing, China). DNA concentration was monitored using a Qubit 3.0 fluorometer (Invitrogen Corporation, Carlsbad, CA, USA). Next-generation sequencing (NGS) library preparation and Illumina MiSeq sequencing were performed at Shanghai Meiji Biotech Co., Ltd. (Shanghai, China). The library was constructed from a 10 ng DNA aliquot isolated from the total DNA of each gut content sample using the MetaVxTM kit (GENEWIZ, Inc., South Plainfield, NJ, USA). The ITS2 molecular fragment was also amplified by PCR using primers S2F and S3R. PCR used a 25 μl system containing 2.5 μl TransStart Buffer, 2 μl dNTPs, 1 μl of each primer, and 20 ng template DNA. PCR conditions were: 94°C for 5 min, 40 cycles (94°C for 30 s, 56°C for 30 s, and 72°C for 45 s), and a final extension at 72°C for 10 min and held at 4°C. At the same time, index adapters were added to the ends of the ITS2 amplicons to generate index libraries in preparation for downstream next-generation sequencing on the MiSeq platform. DNA libraries were verified by Qubit3.0. DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) according to the manufacturer's instructions and sequenced using 2×300 paired-ends and image analysis.
[0022] Determination of the number of intestinal microorganisms in Procambarus clarkii: Three groups of samples were taken out from the -80℃ ultra-low temperature refrigerator, fully ground with an electric tissue grinder under sterile conditions, and total DNA was extracted according to the instructions of the fecal genomic DNA extraction kit. 2μL of the extracted DNA was detected by electrophoresis (120V, 30min), and the DNA with a single band was selected as a template to amplify its 16S rRNA V4-V5 region. The amplification primers used were 515F and 907R. PCR used a 20μL amplification system; 4μL 5×Fast Pfu Buffer, 2μL 2.5mmol / L dNTPs, 0.4μL upstream primer, 0.4μL downstream primer, 0.4μL Fast Pfu Polymerase, 10ng Template DNA, and sterilized water was added to make up to 20μL. Amplification conditions: 95°C pre-denaturation for 2 min; 25 cycles, 95°C for 30 s, 55°C for 30 s, 72°C for 45 s; 72°C extension for 10 min. PCR products were verified by 2% agarose gel electrophoresis, and the target band gel was recovered and purified using the QIA quick GelExtraction Kit (Qiagen, Germany). The purified product was sent to Shanghai Meiji Biotechnology Co., Ltd., and the sequencing platform was Illumina MiSeq. DNA samples were sent to Shanghai Biotech for Illumina Hiseq high-throughput sequencing analysis.
[0023] The number of plants consumed by Procambarus clarkii was analyzed with the number of intestinal microorganisms. There was a significant positive correlation between the number of plants consumed by Procambarus clarkii and the number of intestinal microorganisms ( Figure 1 ).
[0024] The above experimental results show that there is a significant positive correlation between the number of plants consumed by Procambarus clarkii and the number of its intestinal microorganisms. Therefore, the method of using the number of intestinal microorganisms of Procambarus clarkii as an indicator of the feeding habits of Procambarus clarkii is feasible and accurate.
[0025] Implementation 2:
[0026] From July to September 2023, Procambarus clarkii was artificially cultured in the Aquaculture Laboratory of the College of Modern Fisheries Industry of Huaiyin Normal University. The experiment was divided into two groups, one group added with Alternanthera philoxeroides and Elodea, and the other group added with Alternanthera philoxeroides, Elodea and willow leaves. In the late stage of cultivation, two adult Procambarus clarkii were harvested from each treatment group. Each collected individual was immediately placed in a 20 ml centrifuge tube filled with 99% ethanol, brought back to the laboratory, and refrigerated at –80°C until dissection. The samples to be dissected were removed from the refrigerator and immediately rinsed with 75% ethanol to wash away all possible non-host plant fragments on the outside of the Procambarus clarkii body. The intestines (including the foregut, midgut, and hindgut) of the Procambarus clarkii samples were dissected and the contents were flushed into 5 ml cryovials. The intestinal content samples were numbered and stored in a –80°C refrigerator until processing.
[0027] Determination of the number of intestinal microorganisms of Procambarus clarkii is the same as in implementation method 1
[0028] The results showed that the number of OTUs of intestinal microorganisms of Procambarus clarkii treated with Alternanthera philoxeroides-Elodea-willow leaves was 105 and 241, while the number of OTUs of intestinal microorganisms of Procambarus clarkii treated with Alternanthera philoxeroides-Elodea was 46 and 52. The latter was much smaller than the former, so intestinal microorganisms can be used as an indicator of the food types of Procambarus clarkii.
[0029] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for evaluating the food diversity of Procambarus clarkii, characterized in that: Taking advantage of the positive linear correlation between the number of intestinal microorganisms of Procambarus clarkii and the number of plants it feeds on, the diversity of Procambarus clarkii's diet was evaluated by detecting the number of OTUs in the intestinal microorganisms of Procambarus clarkii. Among them, when detecting the number of intestinal microorganisms of Procambarus clarkii, the amplification primers used were 515F and 907R; the PCR used a 20μL amplification system: 4μL 5×Fast Pfu Buffer, 2μL 2.5mmol / L dNTPs, 0.4μL upstream primer, 0.4μL downstream primer, 0.4μL Fast Pfu Polymerase, 10ng Template DNA, and sterilized water was added to 20μL; amplification conditions: pre-denaturation at 95℃ for 2min; 25 cycles of 95℃ for 30s, 55℃ for 30s, and 72℃ for 45s; extension at 72℃ for 10min.
2. The method for evaluating the food habit diversity of Procambarus clarkii according to claim 1, characterized in that: The specific steps include: S1. Take an adult Procambarus clarkii and clean the body surface with ethanol; then dissect the Procambarus clarkii, take the intestinal contents, and freeze them; S2. Determining the number of microorganisms in the intestinal contents using high throughput technology; S3. Evaluation of the diversity of Procambarus clarkii's diet based on the number of microorganisms measured.
3. The detection method for evaluating the feeding diversity of Procambarus clarkii according to claim 2, characterized in that: In S3, the number of measured microorganisms was positively correlated with the diversity of the diet of Procambarus clarkii. The more the number of measured microorganisms, the more diverse the diet of Procambarus clarkii was.
4. The method for evaluating the feeding diversity of Procambarus clarkii according to claim 2, characterized in that: In S1, the ethanol is specifically ethanol with a concentration of 99%.
5. The method for evaluating the feeding diversity of Procambarus clarkii according to claim 2, characterized in that: In S1, the freezing storage temperature is -80°C.