CN116536433B - Turbid lake fish monitoring method based on eDNA - Google Patents

Turbid lake fish monitoring method based on eDNA Download PDF

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CN116536433B
CN116536433B CN202310722449.8A CN202310722449A CN116536433B CN 116536433 B CN116536433 B CN 116536433B CN 202310722449 A CN202310722449 A CN 202310722449A CN 116536433 B CN116536433 B CN 116536433B
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龙云川
蒋娟
郭金
蒋玄空
代亮亮
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Guizhou Institute of Biology
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Abstract

The application discloses an eDNA-based turbid water lake fish monitoring method, which comprises the following steps: (1) collecting a water environment sample; (2) pretreatment of a water environment sample; (3) eDNA enrichment; (4) eDNA extraction and metagene sequencing. According to the application, the water sample is continuously collected along the sample line in the turbid water lake, so that the problem of insufficient representativeness caused by positioning sample point collection is solved; the water sample is pre-filtered by a large-aperture mesh screen to separate out the particles, then the eDNA in the particles is separated and released by using reagents such as proteinase K and the like, and finally the total eDNA in the water environment is collected by using a small-aperture filter membrane to collect the eDNA in the water environment to the maximum extent, and the problem of difficult eDNA enrichment caused by the particles in turbid lakes is solved. The steps are cooperatively matched, so that the monitoring method can realize higher sensitivity and has great application potential in monitoring fish in turbid lakes.

Description

Turbid lake fish monitoring method based on eDNA
Technical Field
The application relates to the technical field of aquatic organism diversity monitoring, in particular to an eDNA-based turbid lake fish monitoring method.
Background
The aquatic organism diversity is scientifically monitored, and the perfect basic information is the precondition of protecting the water ecology and the organism diversity. Traditional methods of biodiversity monitoring require physical capture, which can cause interference or damage to habitats; and species diversity can be limited by professionals in the systematic taxonomic identification; in addition, small or rare populations of organisms in aqueous environments are difficult to find.
Environmental DNA (eDNA) technology, which has received widespread attention in recent years, is an effective tool for monitoring the diversity of aquatic organisms. eDNA refers to DNA left in the environment by an organism, which may originate from shed tissue, mucus, secretions, excretions, blood, cadavers, and the like. The eDNA technology is used as a non-invasive method, has small interference to a biological community and high species detectability, overcomes some defects and shortcomings of the traditional technology, is an emerging ecological system monitoring technology, and has wide application prospect.
In practical applications, the eDNA technology still presents many challenges. Microporous filter membrane is the main mode of enrichment water body eDNA, compares with the high eDNA transmissivity of large aperture filter membrane, and the small bore filter membrane can intercept most of eDNA, but the filtration pore is often blockked up by aquatic particulate matter, has reduced water sample filtration volume, leads to the sample collection volume few, and is representative poor.
Secondly, the acquired eDNA has the problems of degradation or pollution in the processes of transportation, preservation, pretreatment and the like. Thirdly, aiming at special water bodies such as rivers and lakes in remote areas (long transportation time and poor guarantee conditions, which lead to eDNA degradation risk) and turbid water bodies (a large amount of solid particles easily block the aperture of a filter membrane and influence the enrichment of eDNA), a proper eDNA technology monitoring method is lacked. In addition, unlike the complete phytoplankton and the miniature benthonic animal existing in the water environment, the water environment has no complete organism of fish, the content of fish secretion and shedding tissues in the water environment is small, the sampling area is small, the sample amount is small, and the sample amount cannot represent the species diversity condition of the whole lake. Therefore, development of a suitable monitoring method for monitoring fish in aquatic organisms is desired.
Disclosure of Invention
The application aims to provide an eDNA-based turbid water lake fish monitoring method for solving the problems in the prior art.
In order to achieve the above object, the present application provides the following solutions:
one of the technical schemes of the application is as follows: an eDNA-based turbid water lake fish monitoring method comprises the following steps:
(1) And (3) collecting a water environment sample: setting sampling lines in a lake at intervals of 500-1500m, continuously collecting surface water samples on each sampling line at a flow rate of 400-2000mL/min, defining a sampling point in a sampling interval of 500-1000m, and independently storing water environment samples collected by each sampling point;
(2) Pretreatment of water environment samples: filtering the water environment sample acquired in the step (1) by using a mesh screen on site, collecting solids by using a sterile centrifuge tube, and adding a sodium chloride solution and a benzalkonium chloride solution according to the dosage ratio of 1g to 1mL to obtain a solid sample; the filtered water sample is collected by a sterile self-sealing bag, and the dosage ratio is 1ml: (0.2-0.8) mg of solid benzalkonium chloride is added to obtain a liquid sample;
(3) enrichment of eDNA: adding proteinase K solution, sodium Dodecyl Sulfate (SDS) solution and ethylenediamine tetraacetic acid (EDTA) solution into the solid sample in the step (2), uniformly mixing, oscillating for 6-12h at 45-70 ℃, centrifuging, and reserving supernatant; uniformly mixing the supernatant with the liquid sample obtained in the step (2), filtering with a filter membrane, and intercepting eDNA in the water sample onto the filter membrane;
(4) eDNA extraction and metagene sequencing: extracting the eDNA on the filter membrane by adopting an eDNA extraction kit according to steps, and detecting the concentration and quality of the extracted genome eDNA; constructing a PE gene library, and performing bridge PCR; performing metagenome sequencing by using an Illumina HiSeq high-throughput sequencing platform; and finally, carrying out biological information analysis on the gene data to obtain fish species classification and diversity information in the turbid water lake.
Further, the water sample in the step (1) is collected by a water quality sampler, the water quality sampler is placed on a sampling ship, and the sampling ship runs at a speed of 150-300m/min on each sampling line in the sampling process.
Further, the water quality sampler is a handheld electric water quality sampler, namely, the specific operation of the step (1) is as follows: setting sampling lines every 500-1500m in a lake, driving a sampling ship on each sampling line at a speed of 150-300m/min, continuously collecting surface water samples at a flow of 400-2000mL/min by using a handheld electric water quality sampler while driving the sampling ship, and defining a sampling point as a sampling interval every 500-1000m in the whole interval range from the starting point to the ending point of the sampling lines, wherein water environment samples collected by each sampling point are independently stored.
Further, in the step (2), the mesh screen is a 500 mesh screen, the concentration of the sodium chloride solution is 0.9wt.%, and the concentration of the benzalkonium chloride solution is 0.04-0.15wt.%.
Further, the solid benzalkonium chloride added to the filtered water sample is chemically pure solid powder.
Further, in step (2), both the solid sample and the liquid sample are stored in an environment of 4 ℃.
Further, the concentration of the proteinase K solution in the step (3) is 10mg/mL, the concentration of the Sodium Dodecyl Sulfate (SDS) solution is 10wt.%, and the concentration of the ethylenediamine tetraacetic acid (EDTA) solution is 1wt.%; the dosage ratio of solids in the solid sample to the added proteinase K solution, the sodium dodecyl sulfate solution and the ethylenediamine tetraacetic acid solution is 1g to 0.1mL to 0.4mL to 2mL.
Further, the pore size of the filter membrane in the step (3) is 0.22. Mu.m.
Further, the pretreatment process of the step (2) is carried out on the water environment samples collected by each sampling point in the step (1), the number of the sampling points is set in the step (1), and the step (2) is used for obtaining solid samples and liquid samples corresponding to the number of the sampling points. And (3) independently carrying out the process of adding protease solution, sodium dodecyl sulfate solution and ethylenediamine tetraacetic acid solution into the solid samples obtained from the water environment samples collected from each sampling point, carrying out shaking centrifugation to obtain supernatant, uniformly mixing all the supernatant obtained from each solid sample with all the liquid samples in the step (2), filtering by using a filter membrane, and intercepting eDNA in the water sample onto the filter membrane.
The application discloses the following technical effects:
(1) According to the application, the water sample is continuously collected along the sample line in the turbid water lake, so that the problem of insufficient representativeness caused by positioning sample point collection is solved; the water sample is pre-filtered by a large-aperture mesh screen to separate out the particles, then the eDNA in the particles is separated and released by using reagents such as proteinase K and the like, and finally the total eDNA in the water environment is collected by using a small-aperture filter membrane to collect the eDNA in the water environment to the maximum extent, and the problem of difficult eDNA enrichment caused by the particles in turbid lakes is solved. In addition, protective agents such as benzalkonium chloride and the like are added in the process of preserving the eDNA so as to solve the problem of degradation or pollution of the eDNA in the processes of transportation, preservation, pretreatment and the like. The steps are cooperatively matched, so that the monitoring method can realize higher sensitivity and has great application potential in monitoring fish in turbid lakes.
(2) Compared with the traditional investigation method, the monitoring method of the application has the following advantages: 1) The method has the advantages of high sensitivity and high flux, and can systematically and comprehensively monitor the diversity of fishes, in particular rare fishes or tiny fishes which are difficult to capture; 2) The method does not need to collect target organisms, has little interference to habitat, has no damage to fish communities and is not limited by the forbidden fishing and the forbidden fishing of water bodies in natural protection areas; 3) The operation is simple and efficient, and the time required by investigation operation and fish species identification is saved.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
A turbid-water lake fish monitoring method based on eDNA uses a Weining county, guizhou, grass sea lake (national natural protection area, east-west maximum length about 7.0km, north-south maximum width about 4.0km, water area about 25 km) 2 ) For example, the method specifically comprises the following steps:
(1) And (3) collecting a water environment sample: sample lines are arranged in a lake every 1000m, a sampling ship runs on each sample line at the speed of 200m/min, a hand-held electric water quality sampler is used for continuously collecting surface water samples at the flow rate of 800mL/min while the sampling ship runs, each 600m sampling interval (from the starting point to the end point of the sample line) is defined as a sampling point, and water environment samples collected by each sampling point are independently stored, so that 40 water samples are collected.
(2) Pretreatment of water environment samples: the 40 water environment samples collected in the step (1) are respectively subjected to gravity filtration on site by a 500-mesh (30 mu m) mesh screen, solids are collected by a sterile centrifuge tube, and 0.9wt.% sodium chloride solution and 0.1wt.% benzalkonium chloride solution are added according to the dosage ratio of 1g to 1mL to obtain 40 solid samples; the filtered water sample is collected by a sterile self-sealing bag, and the dosage ratio is 1mL:0.5mg of solid benzalkonium chloride (chemically pure solid powder) was added to obtain 40 liquid samples; all samples were stored at 4 ℃.
(3) enrichment of eDNA: respectively adding 10mg/mL proteinase K solution, 10wt.% SDS solution and 1wt.% EDTA solution into 40 solid samples (sterile centrifuge tubes) in the step (2) (the dosage ratio of solids in the solid samples to the added proteinase K solution, sodium dodecyl sulfate solution and ethylenediamine tetraacetic acid solution is 1g:0.1mL:0.4mL:2 mL), uniformly mixing, oscillating at 55 ℃ for 10 hours, centrifuging, and reserving supernatant, wherein 40 groups of supernatant are obtained in total for the 40 solid samples; mixing the 40 groups of supernatant with the 40 liquid samples in the step (2), filtering with a filter membrane with the aperture of 0.22 mu m, and intercepting eDNA in the mixed water sample onto the filter membrane.
(4) eDNA extraction and metagene sequencing: dividing the eDNA on the filter membrane into 4 parallel samples, extracting the eDNA on the filter membrane by adopting an eDNA extraction kit (Biomarker) according to the steps, wherein the concentration of the extracted genome eDNA is 235-372 ng/mu l, the OD260/280 ratio is 1.82-1.92, and the OD260/230 ratio is 1.73-1.89; constructing a PE gene library, and performing bridge PCR; performing metagenome sequencing by using an Illumina HiSeq high-throughput sequencing platform; and finally, carrying out biological information analysis on the gene data to obtain fish species classification and diversity information in the turbid water lake.
Example 2
The only difference from example 1 is that the specific operation of the water environment sample collection in step (1) is as follows: sample lines are arranged in a lake every 500m, a sampling ship runs on each sample line at the speed of 150m/min, a hand-held electric water quality sampler is used for continuously collecting surface water samples at the flow rate of 400mL/min while the sampling ship runs, each 500m sampling interval (from the starting point to the end point of the sample line) is defined as a sampling point, and water environment samples collected by each sampling point are independently stored, so that 90 water samples are collected.
Example 3
The only difference from example 1 is that the specific operation of the water environment sample collection in step (1) is as follows: sample lines are arranged in a lake every 1500m, a sampling ship runs on each sample line at the speed of 300m/min, a hand-held electric water quality sampler is used for continuously collecting surface water samples at the flow rate of 2000mL/min while the sampling ship runs, each 1000m of sampling interval (from the starting point to the end point of the sample line) is defined as a sampling point, and water environment samples collected by each sampling point are independently stored, and 14 water samples are collected.
Comparative example 1
The only difference from example 1 is that the specific operation of the water environment sample collection in step (1) is as follows: sampling points are set at 1000m multiplied by 600m, and water environment samples are collected at a certain fixed position of each sampling point for 2.4L.
Comparative example 2
The difference from example 1 is only that benzalkonium chloride was not added to both the solid sample and the liquid sample in step (2).
Comparative example 3
The difference with example 1 is that the water environment sample in the step (2) is not filtered by a 500 mesh (30 μm) sieve by gravity, the water environment sample is directly filtered by a filter membrane with the aperture of 0.22 μm in the step (3), the filtration is stopped when the filter membrane is difficult to pass water, and the eDNA on the filter membrane is collected.
Comparative example 4
The condition of the fish communities in the grass sea was explored using traditional investigation methods (fishing + interview). The method comprises the following steps: towing according to 800m meshes at a towing speed of 0.5-1.0km/h by using a trawl (mesh less than 30 mm), individually picking out larger and different fishes in each net capturing object, cleaning the fishes, fixing the fishes with 75% alcohol, soaking and preserving the fishes, and carrying out biological characteristic analysis to identify the fishes.
The composition of the fish communities obtained by monitoring in examples 1 to 3 and comparative examples 1 to 4 is shown in Table 1, and the abundance of the fish genes is shown in Table 2.
TABLE 1 composition of fish communities
Numbering device Species of type Implementation of the embodimentsExample 1 Example 2 Example 3 Comparative example 1 Comparative example 2 Comparative example 3 Comparative example 4
1 An Shui Jinxian cheilus + + + + + +
2 Zebra fish + + + + +
3 Red and sawn capelin + + + + + +
4 Large yellow croaker + + + + +
5 Short fin medaka + + + + +
6 Wenchang fish + + + + + +
7 Turtle shell weever + + + + + +
8 Finless eel + + + + + + +
9 Huang Youyu + + + +
10 Crucian carp + + + + + + +
11 Fugu flavidus (Fugu flavidus) + + + + +
12 Carp + + + + + + +
13 Wheat head fish + + + + + +
14 Nile tilapia + + + + + +
15 Loach + + + + + + +
16 Goby of goby + + +
17 Submarine fish + + + + + +
18 Medaka + + + + + + +
19 Xiamen Wenchang fish + + + + +
20 Tibet Gao Yuan + + + + + +
21 Sturgeon with small body + + + +
TABLE 2 fish gene abundance status
As can be seen from the data in table 1: in comparative example 1, 17 kinds of fishes were detected in total by sampling at a fixed point of the sample point without using the continuous sampling method of the present application; comparative example 2 in which a protective agent such as benzalkonium chloride was not added to the solid sample and the liquid sample, the problem of degradation or contamination of eDNA during transportation, preservation, pretreatment, and the like was found, 16 species of fish were detected in total; the collection of the eDNA in comparative example 3 adopts conventional filter membrane filtration, and has the problems of small water sample amount and difficult eDNA enrichment caused by the blockage of filter holes by particles. In comparative example 4 (conventional method), 8 kinds of fish were detected, and the method of the present application detected 21 kinds of fish, and the fish type detectable by the method of the present application was more than 2 times as high as that by the conventional method. As can be seen from Table 2, the gene abundance of total eDNA detected in example 1 is 5.66 times that of the conventional eDNA technique (comparative example 3); the gene abundance of fish eDNA detected was 7.50 times that of the conventional eDNA technique (comparative example 3). The result shows that the monitoring method has high sensitivity and great application potential.
The above embodiments are only illustrative of the preferred embodiments of the present application, and not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the bridge PCR solution of the present application should fall within the scope of protection defined by the claims of the present application without departing from the spirit of the present application.

Claims (4)

1. The method for monitoring the fish in the turbid water lake based on the eDNA is characterized by comprising the following steps of:
(1) And (3) collecting a water environment sample: setting sampling lines in a lake at intervals of 500-1500m, continuously collecting surface water samples on each sampling line at a flow rate of 400-2000mL/min, defining a sampling interval of 500-1000m as a sampling point, and independently storing water environment samples collected by each sampling point;
(2) Pretreatment of water environment samples: filtering the water environment sample acquired in the step (1) by using a mesh screen on site, collecting solids by using a sterile centrifuge tube, and adding a sodium chloride solution and a benzalkonium chloride solution according to the dosage ratio of 1g to 1mL to obtain a solid sample; the filtered water sample is collected by a sterile self-sealing bag, and the dosage ratio is 1ml: (0.2-0.8) mg of solid benzalkonium chloride is added to obtain a liquid sample;
(3) enrichment of eDNA: adding proteinase K solution, sodium dodecyl sulfate solution and ethylenediamine tetraacetic acid solution into the solid sample in the step (2), uniformly mixing, oscillating at 45-70 ℃ for 6-12h, centrifuging, and reserving supernatant; uniformly mixing the supernatant with the liquid sample obtained in the step (2), filtering with a filter membrane, and intercepting eDNA in the water sample onto the filter membrane;
(4) eDNA extraction and metagene sequencing: extracting the eDNA on the filter membrane by adopting an eDNA extraction kit according to steps, and detecting the concentration and quality of the extracted genome eDNA; constructing a PE gene library, and performing bridge PCR; performing metagenome sequencing by using an Illumina HiSeq high-throughput sequencing platform; finally, biological information analysis of the gene data is carried out to obtain species classification and diversity information of fish in turbid lakes;
the mesh screen in the step (2) is a 500 mesh screen; the concentration of the sodium chloride solution is 0.9wt.%, and the concentration of the benzalkonium chloride solution is 0.04-0.15wt wt.%;
the pore size of the filter membrane in the step (3) is 0.22 μm.
2. The monitoring method of claim 1, wherein the water sample in step (1) is collected with a water quality sampler, the water quality sampler being placed on a sampling vessel, the sampling vessel traveling at a speed of 150-300m/min on each sample line during sampling.
3. The monitoring method of claim 1, wherein in step (2) both the solid sample and the liquid sample are stored in an environment of 4 ℃.
4. The method of monitoring of claim 1, wherein the concentration of proteinase K solution in step (3) is 10mg/mL, the concentration of sodium dodecyl sulfate solution is 10wt.% and the concentration of ethylenediamine tetraacetic acid solution is 1wt.%; the dosage ratio of solids in the solid sample to the added proteinase K solution, the sodium dodecyl sulfate solution and the ethylenediamine tetraacetic acid solution is 1g to 0.1mL to 0.4mL to 2mL.
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