A method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology and its application

Abstract

The present invention discloses a method for detecting living eukaryotic plankton in lakes and reservoirs based on environmental DNA technology and its application, relating to the fields of biology, ecology, and environmental science and technology. The method comprises the following steps: S1, determining a sampling site; S2, collecting water samples; S3, filtering the samples; S4, PMA treatment; and S5, storing the samples. The present invention utilizes PMA staining during the eukaryotic plankton sample collection process to obtain information about living eukaryotic plankton in the environment. It is capable of detecting and distinguishing the species and number of both total and living eukaryotic plankton, improving detection efficiency, and thereby rapidly and accurately quantifying the diversity of plankton communities of different particle sizes in different water layers of a water body.

Description

Technical Field

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

[0002] Eukaryotic plankton are eukaryotic organisms that live in the water body. They have extremely high biodiversity and are an important component of freshwater and marine ecosystems. They play 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 the difference in water temperature, which leads to significant differences in water properties between different water layers. Therefore, there are significant differences in the species and number 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 in surface water bodies, and less involved in 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 typically target specific types of eukaryotic plankton, such as phytoplankton, protozoa, rotifers, cladocerans, and copepods. These methods require large volumes of water (cladocerans and copepods are large and low in abundance, typically requiring more than 20 liters of water to be collected from reservoirs). The water samples must then be filtered through a plankton net and further concentrated and counted using a microscope. This entire process is time-consuming, provides low coverage of biological groups, and fails to consider or differentiate between eukaryotic plankton of varying sizes, making it difficult to quickly and accurately quantify the diversity of plankton communities of varying particle sizes in water.

[0004] Environmental DNA (eDNA) technology is a revolutionary biodiversity monitoring method. It allows direct extraction of biological DNA from environmental samples and provides species information through high-throughput sequencing of specific genes. It is non-invasive, efficient, highly sensitive, and low-cost. Applying eDNA technology to the analysis of eukaryotic plankton samples allows convenient access to eukaryotic plankton of varying sizes, enhancing our understanding of the diversity of eukaryotic plankton communities. However, DNA directly extracted from environmental samples includes DNA from living cells with intact biofilms, extracellular DNA, and DNA from non-viable cells with damaged membranes. Therefore, conventional eDNA testing cannot distinguish between living and dead plankton. To distinguish between total DNA in the environment and DNA from living organisms, rRNA sequencing can be used to quantify living biodiversity, but RNA is easily degraded and difficult to purify. Propidium monoazide (PMA) staining has recently been used to eliminate residual DNA in the environment, but its application to eDNA technology is still 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 problems raised above.

[0006] The above information disclosed in this background technology is only for enhancing understanding of the background technology of the present invention and therefore it may contain information that does not constitute the prior art that is already known to a person of ordinary skill in the art. 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 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 from the surface 0.5m;

[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 10 m, collect samples from 3-5 water layers, including the water 0.5 m above the upper lake, the thermocline water layer, the water sample 1-2 m above the sediment in the lower lake, and the position between the upper and lower lake layers;

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

[0015] S3. Sample filtering:

[0016] S3-1: Pre-filter the water using a 200 μm nylon mesh to remove large particles and large plankton. Filter the water using a 3 μm pore size polycarbonate membrane. After filtration, remove the membrane and wrap it in aluminum foil. Store the membrane in a sterilized 2 mL centrifuge tube. Collect the filtered water sample produced in this step.

[0017] S3-2: Filter through a polycarbonate membrane with a pore size of 0.22 μm. After filtration, remove the membrane, wrap it in aluminum foil, and 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 was treated with PMA. The filter membranes were placed in a 5 cm diameter culture dish 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 membrane that was not treated with PMA was also illuminated together with the PMA-treated filter membrane to finally obtain non-PMA (total DNA) and PMA (live organism DNA) samples.

[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, the micro- and micro-plankton are obtained in the range of 3-200 μm, and the nano-plankton are obtained in the range of 0.22-3 μm in S3-2.

[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 about 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 provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] Example 1

[0031] 1. Determine the sampling site

[0032] The number of sampling points should be determined based on the area, morphology, ecological distribution of plankton, and survey objectives. These points should be representative and reflect the basic plankton profile of the entire water body. For lakes, sampling points should be located both nearshore and in the central region. Depending on the lake's shape, sampling points can be dispersed across the lake's center, the center of large bays, near inlets and outlets, and in shallow coastal areas (both with and without aquatic plants). For reservoirs, sampling points should be located in the center (for river-type reservoirs, in the center of the upper, midstream, and lower reaches, respectively), the center of large bays, near major inlets and outlets, major sewage outlets, and at confluences of rivers entering the reservoir. For the Nuozhadu Reservoir on the Lancang River, for example, sampling points were established upstream, midstream, and downstream of the reservoir.

[0033] Use a depth sounder to measure the water depth, and use a multi-parameter water quality analyzer to measure 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 (a water layer with a sharp change in temperature), the upper and lower layers of which are the upper lake layer and the lower lake layer, respectively. If the water body is shallow (less than 3m deep) 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 sediment surface) samples separately. 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, the thermocline water layer, the lower lake layer (1-2m above the sediment surface) water samples, and the middle position between the upper lake layer and the lower lake layer.

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

[0035] 2. Water sample collection

[0036] After identifying the sampling layer, use a 5L water sampler or pump to collect water samples from different layers. Collect at least 10L of water sample at each sampling point and fill it into a polyethylene plastic bucket pre-marked with the sample number. Before filling, rinse the water sample three times with in-situ water sample of the corresponding depth to prevent sample contamination. Collected water samples should be transported to the laboratory within 1 hour for immediate processing, or stored in a vehicle-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, graduated cylinder, and filtration flask, with ultrapure water and assemble the filtration apparatus. Mix the raw water in the polyethylene plastic bucket by shaking it up and down. Pre-filter it through a 200 μm nylon mesh to remove large particles and large plankton. Then, filter the water using a 3 μm pore size polycarbonate membrane (47 mm diameter, Millipore, USA). Start the vacuum pump and adjust the pressure in the filtration system to 0.02 MPa. Rinse the measuring cup and graduated cylinder with raw water and add the water sample in small, repeated additions (generally 50–400 mL at a time). Ensure that the total filtration time for each individual sample is greater than 30 minutes to ensure sufficient plankton biomass is collected. After filtration, cut aluminum foil to the appropriate size. Grasp the white edge of the filter membrane with tweezers and transfer the membrane to the center of the foil. Fold the foil in half into a long strip and place it in a sterile 2 mL centrifuge tube for storage. During this process, the tips of the tweezers must not touch the sample-enriched 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 foil. If the sample is contaminated or the filter membrane is damaged, the sample will be discarded. Particles in the 3-200μm size range represent small and microplankton. The filtered water sample volume for severely eutrophic water bodies is approximately 100-300mL, the filtered water sample volume for mesotrophic water bodies is approximately 800-1000mL, and the filtered water sample volume for oligotrophic water bodies can exceed 2000mL. The filtered water sample volume for the Nuozhadu Reservoir on the Lancang River is approximately 1200-2400mL.

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

[0040] Indicate the sample number, name of the filter operator, filtration time, filtration volume, membrane pore size, and number of membranes in the centrifuge tube on the outside of the tube. To ensure accurate results, collect 3-6 replicates of varying particle sizes for each sample. Before filtering each different sample, the filter cup, filter element, measuring cup, and graduated cylinder must be re-rinsed with ultrapure water and then rinsed with the original water from the sample to be filtered to prevent cross-contamination between samples. After filtration, rinse the glass frit filter, suction flask, and other equipment with ultrapure water and air dry 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 PMA staining, 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 that was 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 Nuozhadu 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, there are significant differences in the composition of the eukaryotic plankton community, with significant differences in eukaryotic plankton between the three sampling stations and the five water layers. Significant differences also exist between different particle sizes, and between the total plankton community and the living plankton community.

[0047] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0048] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify 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 from the surface 0.5m; 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 10 m, collect samples from 3-5 water layers, including the water 0.5 m above the upper lake, the thermocline water layer, the water sample 1-2 m above the sediment in the lower lake, and the position between the upper and lower lake layers; S2. Water sampling: After the sampling sites are identified, water samples are collected from each water layer, with at least 10L of water sample collected at each sampling point; S3. Sample filtering: S3-1: Pre-filter the water using a 200 μm nylon mesh to remove large particles and large plankton. Filter the water using a 3 μm pore size polycarbonate membrane. After filtration, remove the membrane and wrap it in aluminum foil. Store the membrane in a sterilized 2 mL centrifuge tube. Collect the filtered water sample produced in this step. S3-2: Filter through a polycarbonate membrane with a pore size of 0.22 μm. After filtration, remove the membrane, wrap it in aluminum foil, and store it in a sterilized 2 mL centrifuge tube. S3-3: Filter and collect 3-6 replicates of each sample; S4. PMA treatment: The collected filter membranes were divided into two parts. One part 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 membrane that was not treated with PMA was also illuminated together with the PMA-treated filter membrane to finally obtain non-PMA and PMA samples. Non-PMA refers to total DNA, and PMA refers to living organism DNA. 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 according to claim 1 and its application, 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 according to claim 1 and its application, characterized in that: In the S3-1, the micro- and micro-plankton were obtained in the range of 3-200 μm, and the nano-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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