Method for detecting lake and reservoir living eukaryotic plankton based on environmental DNA technology and application thereof

Abstract

The invention discloses a method for detecting lake and reservoir living eukaryotic plankton based on an environmental DNA technology and application of the method, and relates to the technical field of biology, ecology and environmental science. The method comprises the following steps: S1, determining a sampling site; s2, collecting a water sample; s3, filtering the sample; s4, PMA treatment is carried out; and S5, storing the sample. The PMA dyeing treatment is used in the eukaryotic plankton sample collection process, living eukaryotic plankton information in the environment can be obtained, the species and number of overall and living eukaryotic planktons can be detected and distinguished, the detection efficiency is improved, and the detection time is shortened. Therefore, the diversity of plankton communities with different particle sizes in different water layers of a water body can be quickly and accurately quantified.

Description

Technical Field

[0001] The invention relates to the technical fields of biology, ecology and environmental science, and specifically to a method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology and application thereof. Background Art

[0002] Eukaryotic plankton is a type of eukaryotic organism that lives in the water. It has extremely high biodiversity and is an important component of freshwater and marine ecosystems, playing a key role in material circulation and energy flow. When the water body is deep, the water body forms thermal stratification in the vertical profile due to differences in water temperature, resulting in significant differences in water properties between different water layers. Therefore, there are significant differences in the types and numbers of eukaryotic plankton living in different water layers. However, in the past, the collection of eukaryotic plankton samples in lakes and reservoirs was mainly concentrated on surface water bodies, while less attention was paid to stratified water bodies. At the same time, eukaryotic plankton can be divided into small plankton (microplankton, 20-200μm), microplankton (nanoplankton, 3-20μm), and ultra-microplankton (picoplankton, 0.2-3μm) according to size.

[0003] Traditional sampling methods generally target specific types of eukaryotic plankton, such as phytoplankton, protozoa and rotifers, cladocerans and copepods, etc., and require a large amount of water (e.g., cladocerans and copepods are large and low in abundance, and usually more than 20L of water samples need to be collected from reservoirs). The water samples then need to be filtered with a plankton net, and the samples need to be further concentrated and counted under a microscope. The entire process is time-consuming, has low coverage of biological groups, does not consider or distinguish between eukaryotic plankton of different sizes, and is difficult to quickly and accurately quantify the diversity of plankton communities of different particle sizes in water bodies.

[0004] Environmental DNA (eDNA) technology is a revolutionary biodiversity monitoring method. It can directly extract the DNA of organisms from environmental samples and obtain species information through high-throughput sequencing of specific genes. It is non-invasive, efficient, highly sensitive and low-cost. The use of eDNA technology in the analysis of eukaryotic plankton samples can easily obtain eukaryotic plankton of different particle sizes and enhance the understanding of the diversity of eukaryotic plankton communities. However, the DNA directly extracted from environmental samples includes DNA from living cells with intact biofilms, extracellular DNA, and DNA from non-living cells with damaged membranes. Therefore, conventional eDNA detection cannot distinguish between the dead and alive states of plankton. In order to distinguish between total DNA and living organism DNA in the environment, rRNA sequencing can be used to quantify living biodiversity, but RNA is easily degraded and difficult to purify. In recent years, propidium monoazide (PMA) staining has been used to eliminate residual DNA in the environment, but its application in eDNA technology is still very limited.

[0005] Therefore, we proposed a method and its application for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology in order to solve the above-mentioned problems.

[0006] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to ordinary technicians in this field. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology and its application, so as to solve the problems raised by the above-mentioned background technology.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology, comprising the following steps:

[0009] S1. Determine the sampling site:

[0010] S1-1: Based on the rate of change of water temperature in the vertical section of the lake water body, the water body is divided into the upper lake layer, the thermocline layer and the lower lake layer;

[0011] S1-2: If the water depth is less than 3m or there is no thermal stratification, collect water samples at 0.5m from the surface;

[0012] S1-3: If the water depth is 3-10m, collect water samples from the surface 0.5m and 1-2m above the bottom sediment;

[0013] S1-4: If the water depth is greater than 10m, collect samples from 3-5 water layers, including water samples 0.5m above the upper lake layer, the thermocline water layer, 1-2m above the sediments of the lower lake layer, and the middle position between the upper and lower lake layers;

[0014] S2. Water sample collection: After the sampling site is identified, water samples are collected from each water layer, and at least 10L of water samples are collected from each sampling point;

[0015] S3. Sample filtering:

[0016] S3-1: Use 200 μm nylon mesh to pre-filter to remove large particles and large plankton in the water, and use a polycarbonate filter membrane with a pore size of 3 μm to filter. After the filtration is completed, remove the filter membrane and wrap it with aluminum foil, and store it in a sterilized 2 mL centrifuge tube. Collect the filtered water sample produced in this step;

[0017] S3-2: Filter using a polycarbonate filter membrane with a pore size of 0.22 μm. After filtration, remove the filter membrane and wrap it in aluminum foil, then store it in a sterilized 2 mL centrifuge tube;

[0018] S3-3: Filter and collect 3-6 replicates of each sample;

[0019] S4. PMA treatment: The collected filter membranes were divided into two parts. One part of the filter membranes was treated with PMA. The filter membranes were placed in a culture dish with a diameter of 5 cm and evenly immersed in 500 μL of PMA dye with a final concentration of 50 μg / mL for 20 minutes in the dark. Then, they were exposed to a 200 W LED light at a distance of 20 cm from the culture dish for 15 minutes. The other part of the filter membranes that were not treated with PMA were also illuminated together with the filter membranes that were treated with PMA, and finally non-PMA (total DNA) and PMA (live biological DNA) samples were obtained.

[0020] S5. Sample storage: After the illumination, all filters were stored in a -80°C ultra-low temperature freezer for subsequent DNA extraction, PCR amplification and DNA sequencing.

[0021] Preferably, in S3, each independent sample ensures that the total filtration time of the water sample is greater than 30 minutes.

[0022] Preferably, in the S3-1, small and micro plankton are obtained in the range of 3-200 μm, and in S3-2, ultramicro plankton are obtained in the range of 0.22-3 μm.

[0023] The present invention also provides the application of the above-mentioned method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology in environmental detection and water body detection.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention uses PMA staining treatment in the process of collecting eukaryotic plankton samples, which can obtain information on living eukaryotic plankton in the environment, detect and distinguish the types and quantities of overall and living eukaryotic plankton, improve detection efficiency, and thus quickly and accurately quantify the diversity of plankton communities of different particle sizes in different water layers of the water body.

[0026] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the process of the present invention;

[0028] Figure 2 Schematic diagram of the differences in eukaryotic plankton communities in the Nuozhadu Reservoir on the Lancang River. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] 1. Determine the sampling site

[0032] The number of sampling points is determined based on the area, morphology, ecological distribution characteristics of plankton, and survey objectives of the water body. The sampling points should be representative and reflect the basic situation of plankton in the entire water body. For lakes, sampling points should be set both near the shore and in the middle. They can be dispersed in the center of the lake, the center of a large bay, near the inlet and outlet, and in the shallow water area along the coast (with and without aquatic plants) according to the shape of the lake; reservoirs should be set in the center of the reservoir (river-type reservoirs should be set in the center of the upper, middle, and lower reaches respectively) and the center of the large bay, near the main inlet and outlet, the main sewage outlet, and the confluence of rivers entering the reservoir. Taking the Nuozadu Reservoir on the Lancang River as an example, sampling points were set up in the upper, middle, and lower reaches of the reservoir.

[0033] Use a depth sounder to measure the water depth, and use a multi-parameter water quality analyzer to determine the vertical profile temperature of the water body at the sampling station. According to the rate of change of water temperature in the vertical profile, the water body is divided into thermocline (water layer with a sharp change in temperature), and its upper and lower layers are respectively the upper lake layer and the lower lake layer. If the water body is shallow (water depth is less than 3m) or there is no thermal stratification, surface (0.5m) water samples can be collected. When the water depth is 3-10m, collect surface (0.5m) and bottom water (1-2m above the surface of the sediment) samples respectively. If the water depth is greater than 10m, generally collect samples from at least 3-5 water layers, including the upper lake layer (0.5m) water body, thermocline water layer, lower lake layer (1-2m above the surface of the sediment) water body samples, and the middle position between the upper lake layer and the lower lake layer.

[0034] The depth of the upstream station of Nuozadu Reservoir is about 50m. The five sampling depths set according to the thermocline are: surface layer (0.5m), middle layer between surface layer and thermocline (3.5m), thermocline (7m), middle layer between thermocline and bottom layer (27m), bottom layer (48m). The depth of the midstream station is about 112m. The five sampling depths set according to the thermocline are: surface layer (0.5m), middle layer between surface layer and thermocline (10m), thermocline (40m), middle layer between thermocline and bottom layer (80m), bottom layer (110m). The depth of the downstream station is about 182m. The five sampling depths set according to the thermocline are: surface layer (0.5m), middle layer between surface layer and thermocline (10m), thermocline (40m), middle layer between thermocline and bottom layer (100m), bottom layer (180m).

[0035] 2. Water sample collection

[0036] After the sampling water layer is determined, use a 5L water sampler or water pump to collect water samples from different water layers. Collect at least 10L of water samples at each sampling point and fill them into polyethylene plastic barrels with sample numbers pre-written. Before filling the water samples, rinse them three times with in-situ water samples of the corresponding depth to prevent sample contamination. The collected water samples should be transported back to the laboratory within 1 hour for immediate processing, or stored in a car-mounted low-temperature refrigerator at 4°C and transported back to the laboratory as soon as possible.

[0037] 3. Sample Filtering

[0038] Clean the filtration equipment, including the filter cup, filter element, measuring cup, measuring cylinder and suction bottle, with ultrapure water, and assemble the filtration device. Mix the raw water in the polyethylene plastic bucket up and down and shake it evenly. Use 200μm nylon mesh to pre-filter to remove large particles and large plankton in the water. Then use a polycarbonate filter membrane with a pore size of 3μm (diameter 47mm, Millipore, USA) for filtration, start the vacuum pump, adjust the air pressure in the suction filtration system to 0.02MPa, rinse the measuring cup and measuring cylinder with raw water, and add water samples in small amounts and multiple times. Generally, 50–400mL is added each time. For each independent sample, ensure that the total filtration time of the water sample is greater than 30min to ensure that enough plankton is collected. After the filtration is completed, cut the aluminum foil to a suitable size, clamp the white edge of the filter membrane with tweezers, transfer the filter membrane to the center of the aluminum foil, fold the aluminum foil into a long strip and put it into a sterilized 2mL centrifuge tube for storage. During this process, the tip of the tweezers must not touch the sample enrichment area in the center of the filter membrane. When folding the aluminum foil, ensure that the sample area in the center of the filter membrane is not contaminated by the aluminum foil. If the sample is contaminated or the filter membrane is damaged, the sample will be discarded. The particle size range of 3-200μm is small and micro plankton. The volume of filtered water samples from severely eutrophic water bodies is about 100-300mL, the volume of filtered water samples from mesotrophic water bodies is about 800-1000mL, and the volume of filtered water samples from oligotrophic water bodies can exceed 2000mL. The volume of filtered water samples from the Nuozadu Reservoir of the Lancang River is about 1200-2400mL.

[0039] The water samples were collected after filtering through a 3 μm filter membrane and then filtered through a polycarbonate filter membrane with a pore size of 0.22 μm (diameter 47 mm, Millipore, USA). The total filtration time for each independent sample was at least 30 min. The filtration volume of the water sample from the Nuozadu Reservoir of the Lancang River was approximately 500-1000 mL. After the filtration was completed, the filter membrane was removed and wrapped with aluminum foil and stored in a sterilized 2 mL centrifuge tube. The samples obtained in the particle size range of 0.22-3 μm were ultra-microplankton.

[0040] The outer wall of the centrifuge tube should indicate the sample number, the name of the filter person, the filtering time, the filtering volume, the pore size of the filter membrane, and the number of filter membranes in the tube. In order to ensure the accuracy of the results, 3-6 replicate samples of different particle sizes are collected for each sample. Before filtering each water sample of a different sample, the filter cup, filter element, measuring cup and measuring cylinder must be cleaned with ultrapure water, and then rinsed with the original water of the sample to be filtered to avoid cross contamination between samples. After filtering, use ultrapure water to clean the glass sand core filter device, suction bottle, etc. and let them air dry naturally to prevent bacterial growth and contamination.

[0041] 4. PMA treatment

[0042] The collected filter membranes were divided into two parts. One part of the filter membrane was further treated with PMA staining to inhibit the PCR amplification of dead cells and free DNA, thereby separating the living organism DNA from the total DNA. The other part of the filter membrane was not treated with PMA. During the PMA staining treatment, the filter membrane carrying plankton was placed in a culture dish with a diameter of 5 cm, evenly immersed in 500 μL of PMA dye (final concentration of 50 μg / mL), and treated in the dark for 20 minutes. It was then exposed to an LED light (200W) 20 cm away from the culture dish for 15 minutes. The filter membrane that was not treated with PMA was also illuminated together with the filter membrane treated with PMA to eliminate the effect of light on DNA. Finally, samples of non-PMA (total DNA) and PMA (living organism DNA) were obtained.

[0043] 5. Sample storage

[0044] After the illumination, all filters were stored in a -80°C ultra-low temperature freezer for subsequent DNA extraction, PCR amplification, and DNA sequencing.

[0045] 6. Differences in sample composition

[0046] A total of 60 samples were collected from the upstream, midstream and downstream of the Nuozadu Reservoir on the Lancang River, including 3 sampling stations, 5 water layers, 2 different particle sizes, and 2 types of PMA treatment samples. Figure 2 As shown in the figure, there are significant differences in the composition of the eukaryotic plankton community, among which the eukaryotic plankton is significantly different among the three sampling stations and the five water layers. There are also significant differences between different particle sizes, and between the total plankton community and the living plankton community.

[0047] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0048] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology, characterized in that: The steps include: S1. Determine the sampling site: S1-1: Based on the rate of change of water temperature in the vertical section of the lake water body, the water body is divided into the upper lake layer, the thermocline layer and the lower lake layer; S1-2: If the water depth is less than 3m or there is no thermal stratification, collect water samples at 0.5m from the surface; S1-3: If the water depth is 3-10m, collect water samples from the surface 0.5m and 1-2m above the bottom sediment; S1-4: If the water depth is greater than 10m, collect samples from 3-5 water layers, including water samples 0.5m above the upper lake layer, the thermocline water layer, 1-2m above the sediments of the lower lake layer, and the middle position between the upper and lower lake layers; S2. Water sample collection: After the sampling site is identified, water samples are collected from each water layer, and at least 10L of water samples are collected from each sampling point; S3. Sample filtering: S3-1: Use 200 μm nylon mesh to pre-filter to remove large particles and large plankton in the water, and use a polycarbonate filter membrane with a pore size of 3 μm to filter. After the filtration is completed, remove the filter membrane and wrap it with aluminum foil, and store it in a sterilized 2 mL centrifuge tube. Collect the filtered water sample produced in this step; S3-2: Filter using a polycarbonate filter membrane with a pore size of 0.22 μm. After filtration, remove the filter membrane and wrap it in aluminum foil, then store it in a sterilized 2 mL centrifuge tube; S3-3: Filter and collect 3-6 replicate samples for each sample; S4. PMA treatment: The collected filter membranes were divided into two parts. One part of the filter membranes was treated with PMA staining. The filter membranes were placed in a culture dish with a diameter of 5 cm and evenly immersed in 500 μL of PMA dye with a final concentration of 50 μg / mL for 20 minutes in the dark. Then, they were exposed to a 200 W LED light at a distance of 20 cm from the culture dish for 15 minutes. The other part of the filter membranes that were not treated with PMA were also illuminated together with the filter membranes that were treated with PMA, and finally non-PMA (total DNA) and PMA (live biological DNA) samples were obtained. S5. Sample storage: After the illumination, all filters were stored in a -80°C ultra-low temperature freezer for subsequent DNA extraction, PCR amplification and DNA sequencing.

2. The method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology and its application according to claim 1, characterized in that: In S3, each independent sample ensures that the total filtration time of the water sample is greater than 30 minutes.

3. The method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology and its application according to claim 1, characterized in that: In the S3-1, the micro- and micro-plankton were obtained in the range of 3-200 μm, and the ultra-micro-plankton were obtained in the range of 0.22-3 μm in S3-2.

4. Application of the method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology as described in any one of claims 1 to 3 in environmental testing and water body testing.

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