1. A benthonic animal biological integrity evaluation method based on an eDNA macro-barcode technology is characterized by comprising the following steps:

(1) determining a reference point and a damaged point;

(2) collecting reference point and damaged point samples and extracting environmental DNA;

(3) amplifying a bar code fragment by taking the extracted environmental DNA as a template, sequencing and carrying out species annotation based on operation classification unit information corresponding to the sample;

(4) calculating a plurality of defined biological integrity index candidate indexes according to the result of the step (3);

(5) screening a final biological integrity index according to the calculation result of the step (4);

(6) calculating a zoobenthos biological integrity score according to the final index determined in step (5);

(7) the benthonic animal health rating is divided according to the benthonic animal biological integrity score.

2. The method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring as claimed in claim 2, wherein: the determination conditions of the reference points in the step (1) are as follows:

a) lake reservoir reference point: (i) the indexes of total nitrogen, total phosphorus and other nutritive salts reach IV-class water standards of surface water; (ii) the comprehensive nutritional state index is less than the 25% quantile of the monitored sample; (iii) submerged vegetation distribution; (iv) the sampling point water area has no functions of navigation channel, cultivation, entertainment and the like, and is slightly influenced by the water conservancy project;

and/or b) a river reference point: (i) the total phosphorus is less than or equal to 0.3mg/L, and the ammonia nitrogen is less than or equal to 2.0 mg/L; (ii) and (3) monitoring that the upstream of the section has a lake, a swinging and other preposed buffer areas or aquatic plant distribution along the bank.

3. The method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring according to claim 1 or 2, wherein the method comprises the following steps: the step (2) of collecting the sample comprises the following steps: collecting sediments at the bottom of the water body, and freezing and storing the sediments; the environmental DNA extraction comprises thawing, ethanol extraction, centrifugation and DNA extraction.

4. The method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring as claimed in claim 3, wherein: in the step (3), the primer pair for amplifying the environmental DNA comprises an upstream primer mlCOIintF: GGWACWGGWTGAACWGTWTAYCCYCC and the downstream primer is dgHCO 2198: TAAACYTCAGGRTGACCRAARAAYCA, respectively; the sequencing comprises high throughput sequencing of the amplification products; the species annotation comprises the step of obtaining operation classification unit information corresponding to each sample through analysis of software platforms such as qiime and usearch, and the step of performing species annotation on the OTU by adopting an NCBI database.

5. The method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring as claimed in claim 4, wherein: the biological integrity index candidate indexes defined in the step (4) need to comprise indexes for evaluating community abundance, species dominance, species composition, species pollution resistance value and genetic diversity.

6. The method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring as claimed in claim 5, wherein: the defined biological integrity index candidate indexes are not less than 27.

7. The method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring according to any one of claims 3 to 6, wherein: the defined biological integrity index candidate index and the calculation formula comprise the following steps:

8. the method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring as claimed in claim 7, wherein: the screening of the final biological integrity index in the step (5) comprises the following steps:

a) and (3) screening distribution ranges: calculating the point location ratio of the minimum value, Q25, median, Q75 and the maximum value and value >0 of each exponential distribution, keeping the indexes that the value >0 of the point location is more than 80% and the maximum value > Q75> median > Q25> minimum value, and discarding indexes with relatively narrow value range;

b) and (3) distinguishing and analyzing the box body: analyzing the distribution condition of each parameter entering the discrimination capability analysis between the reference state and the damaged state by adopting a box line diagram method, comparing the quantile ranges of 25 th-75 th of the reference state and the damaged state, and keeping the index that the overlapping condition of box bodies of the box line diagram is more than or equal to 2;

c) and (3) correlation analysis: performing Pearson correlation analysis on the indexes reserved in the step b), and checking the independence of the reflection information of each index; selecting a common index with strong representativeness as a priority retention index, and removing an index highly correlated with the priority retention index, wherein the high correlation means that a correlation coefficient | r | between the two indexes is greater than 0.750; the preferential retention index includes M01 total taxon number, M12 tubificidae cyclicaceae ratio, M27 average genetic diversity, M02 mollusc taxon or M18 mollusc ratio; m24_ BMWP index or M25_ ASPT index or M26_ BI index;

d) determining a final index: and determining the final candidate index according to the steps a), b) and c).

9. The method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring according to claim 8, wherein: the calculation of the score of the zoonosis of the benthic animals in the step (6) comprises the following steps:

a) exponential score M_iAnd (3) calculating: the stronger the interference, the lower the value of the index, taking the 95% quantile of the monitored sample value as an expected value, and the index score is equal to the monitored value divided by the expected value; for the index with stronger interference and higher value, taking the 5% quantile of the monitored sample value as an expected value, the calculation method is as follows: (maximum-monitor)/(maximum-expected), if the calculation result is larger than1, by 1, less than 0, by 0.

b) B-IBI calculation: B-IBI is calculated as shown in formula (1),

formula (1) B-IBI ═ Sigma M_i；

c) B-IBI normalization calculation: the normalization is shown as formula (2), wherein B-IBI_q9595% quantile for all monitoring sites B-IBI:

formula (2)

10. The method for evaluating the biological integrity of benthonic animals based on eDNA macro-barcode monitoring according to claim 9, wherein: in the step (7), the health grades of the benthonic animals are classified by adopting a 4-point method according to a health classification standard that 95% quantile of the B-IBI value of the monitoring sample is 'excellent', and the standard is as follows:

Background

Environmental DNA (edna) refers to the mixture of genomic DNA of all the different organisms found in an environmental sample, including water sample DNA, sediment DNA, biogenic DNA; the biogenic DNA refers to DNA extracted from a biological tissue, for example, skin, hair, etc. DNA barcodes (DNA barcodes), which refer to standard, sufficiently variable, easily amplifiable, relatively short DNA fragments that can represent the species in an organism, have become an important tool for ecological research.

The DNA macro-barcode (Metabarcoding) technology is a research method derived by combining the traditional DNA barcode and a high-throughput sequencing technology, and can non-invasively investigate the abundance of species from a plurality of ecosystems. The detection of eDNA in the environmental sample by utilizing the Metabarcoding technology can obtain taxonomy information and gene function information of species to which the DNA in the environmental sample belongs, so as to research the diversity of the species in different ecological environments and trace the source, development and change of the species, and the detection method is widely applied to environmental biological research at present.

The benthonic animals have various types and large quantity, occupy various different ecological niches, play different ecological roles and directly or indirectly influence the quantity and the distribution of different nutrition level biological groups. A number of studies have been carried out to evaluate the ecological health of water using the Benthic Index of biological Integrity (B-IBI). The B-IBI index is also incorporated into the health evaluation system of the fresh water ecosystem in the river water ecological environment quality monitoring and evaluation technical guidelines (survey papers) issued by the ecological environment department and the lake-reservoir water ecological environment quality monitoring and evaluation technical guidelines (survey papers). However, the existing benthonic animal integrity index is constructed based on morphological species identification data under a microscope, is low in monitoring efficiency, time-consuming and labor-consuming, depends heavily on professional knowledge and experience of identified people, is limited in the number of identified species, cannot reflect the change of the genetic composition of benthonic animals under the change of environmental gradient, has few candidate parameters for constructing the B-IBI index, is lack of representativeness, and cannot meet the increasing requirements for monitoring and evaluating the ecological environment.

Disclosure of Invention

1. Problems to be solved

Aiming at the problems that the existing benthonic animal integrity index construction method is low in monitoring efficiency, time-consuming and labor-consuming, seriously depends on professional knowledge and experience of identified people, is limited in the number of identified species, cannot reflect the change of the genetic composition of benthonic animals under the change of Environmental gradient, is few in candidate parameters for constructing the B-IBI index and is lack of representativeness, the invention provides the benthonic animal biological integrity evaluation method constructed based on the Environmental DNA (eDNA) macro-barcode (Metabarcoding) monitoring method, which is used for evaluating the aquatic ecological health condition more efficiently, economically and with high resolution.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention provides a primer pair for amplification and sequencing of a universal macro-barcode in a zoobenthos cytochrome C Oxidase I (COI) region, which comprises an upstream primer and a downstream primer, wherein the primer pair comprises:

the upstream primer is mlCOIintF: GGWACWGGWTGAACWGTWTAYCCYCC, respectively;

the downstream primer is dgHCO 2198: TAAACYTCAGGRTGACCRAARAAYCA are provided.

The invention also provides a benthonic animal biological integrity evaluation method based on the eDNA macro-barcode technology, which comprises the following steps:

(1) determination of Reference sites (R) and damaged sites (I);

(2) collecting samples of a reference point and a damaged point and extracting environmental DNA;

(3) amplifying a bar code fragment, sequencing and analyzing the species by taking the extracted environmental DNA as a template;

(4) calculating a plurality of defined biological integrity index candidate indexes according to the analysis result;

(5) screening a final biological integrity index according to the calculation result of the step (4);

(6) calculating a zoobenthos biological integrity score according to the final index determined in step (5);

(7) the benthonic animal health rating is divided according to the benthonic animal biological integrity score.

Preferably, in the step (1), the selection of the reference points can represent points which are not subjected to human interference or are subjected to less human interference and have optimal biological and habitat states in a monitored and evaluated water area, the points are determined according to the water quality condition, the nutrition degree, the aquatic plant coverage and the human interference strength, and the rest monitoring points are damaged points; the reference point needs to meet the conditions of relatively good water quality, relatively light eutrophication degree, relatively high aquatic vegetation coverage, relatively low man-made interference strength and the like.

Preferably, in step (1), the reference point needs to satisfy the following conditions:

the specific screening indexes of the lakes and reservoirs are as follows: (i) the indexes of total nitrogen, total phosphorus and other nutritive salts reach IV-class water standards of surface water; (ii) the comprehensive nutritional state index is less than the 25% quantile of the monitored sample; (iii) submerged vegetation distribution; (iv) the sampling point water area has no functions of navigation channel, cultivation, entertainment and the like, and is slightly influenced by the water conservancy project;

the specific screening indexes of the river are as follows: (i) the total phosphorus is less than or equal to 0.3mg/L, and the ammonia nitrogen is less than or equal to 2.0 mg/L; (ii) monitoring the upstream water incoming direction of the section to form a lake, a swinging pre-buffer zone or aquatic plant distribution along the bank; (iii) the river reach of the monitoring section has no channel, breeding, entertainment and other functions, and is less influenced by water conservancy projects.

Preferably, in step (2), the sample collection comprises: collecting sediments at the bottom of a water body, cleaning, freezing and storing; the environmental DNA extraction comprises thawing, ethanol extraction, centrifugation and DNA extraction.

Preferably, the sample collection comprises: collecting sediment at the bottom of water body with tool such as Petersen mud collector and triangular trawl, sieving, filtering, washing, draining water of mixture, and freezing at-40 deg.C to-80 deg.C for 1-3 days.

Preferably, the environmental DNA extraction is performed using means conventional in the art. Further, the environmental DNA extraction comprises: weighing after thawing, converting 1g of mixture into 1mL, adding 3-5 times of volume of absolute ethyl alcohol, extracting for 3-30 days by using ethanol, shaking up, standing, extracting 2mL of upper layer liquid, performing vacuum centrifugation to obtain dried tissue residues, and extracting environmental DNA by using a kit, wherein the centrifugation condition is 8000 rpm, and the centrifugation time is 5 minutes.

Preferably, in step (3), the amplifying and sequencing comprises: and (3) amplifying the environmental DNA in the step (2) by adopting the COI primer pair, carrying out high-throughput sequencing on the PCR product, processing sequencing data through software platforms such as qiime, usearch and the like, obtaining operation classification unit (OTU) information corresponding to each sample, and carrying out species annotation on the OTU by adopting an NCBI database.

Preferably, in step (4), the defined biological integrity index candidate indicators need to include community abundance, species dominance, species composition, species contamination resistance value, genetic diversity.

Preferably, in step (4), the defined biological integrity index candidate indexes are not less than 27.

Preferably, in step (4), the defined biological integrity index candidate index and the calculation formula include the following:

preferably, in step (5), the screening of the final index of biological integrity index comprises:

a) and (3) screening distribution ranges: calculating the minimum value, quartile (Q25), median, quartile (Q75) and point position ratio of the maximum value and the value >0 of each exponential distribution, keeping the indexes of which the value >0 of the point positions is more than 80% and the maximum value > Q75> median > Q25> minimum value, and discarding indexes with relatively narrow value range intervals;

b) and (3) distinguishing and analyzing the box body: analyzing the distribution condition of each parameter entering the discrimination capability analysis between a reference state and a damaged state by adopting a box line graph method, namely comparing the quantiles of 25 th-75 th of the reference state and the damaged state, namely the box line graph box body overlapping condition (IQ), and keeping the index that the IQ is more than or equal to 2;

c) and (3) correlation analysis: performing Pearson correlation analysis on the indexes retained in the step b), checking the independence of the information reflected by each index, selecting the commonly used and high-representativeness index as a priority retention index, and screening out indexes highly correlated with the priority retention index, wherein the high correlation means that a correlation coefficient | r | between the two indexes is greater than 0.750; preferably, the preferential retention index comprises M01 total taxon number, M12 jutrelliferae cyclicaceae ratio, M27 average genetic diversity, M02 mollusc taxon or M18 mollusc ratio; the M24_ BMWP index or the M25_ ASPT index or the M26_ BI index is ecologically generally considered that the higher the abundance of species is, the higher the biological integrity is and the more stable the ecosystem is, so that the total number of M01_ classification units is preferentially reserved, the maximum information content is also the biological index which is commonly used at present; the relative abundance of the tubificidae and the cyclamen in the eDNA biological monitoring data is higher, the representativeness is stronger, and the tubificidae and the cyclamen are stain resistant species and have good indication significance for the environment, so the M12_ tubificidae _ cyclamen proportion can be further reserved; the M27_ average genetic diversity reflects the genetic diversity of each site through sequence difference, so that the genetic diversity is higher, the biological integrity of the molecular level is higher, the resistance of the community to the external pressure is stronger, and the community can be reserved; mollusks are very sensitive to external stress and therefore the number of M02_ mollusk taxa and M18_ mollusk ratios can be selectively retained; the M24_ BMWP index, the M25_ ASPT index and the M26_ BI index are combined indexes which are more commonly used at present and consider the pollution resistance value of the species group, and one index can be selectively reserved.

d) Final candidate indices: and determining the final candidate index according to the steps a), b) and c).

Preferably, in step (6), the calculation of the score of the biological integrity of the benthic animals comprises:

a) index pointValue M_iAnd (3) calculating: the stronger the interference, the lower the value of the index, taking the 95% quantile of the monitored sample value as an expected value (namely, a ecological target value), and the index score is equal to the monitored value divided by the expected value; for the index with stronger interference and higher value, taking 5% quantile as an expected value, the calculation method is as follows: (max-monitor)/(max-desired). And if the calculation result is greater than 1, counted as 1, less than 0, counted as 0.

b) B-IBI calculation:

B-IBI＝∑M_i

c) B-IBI normalization

To facilitate the same scale for overall evaluation, the zoobenthos integrity index was normalized, where B-IBI_q9595% quantile for all monitoring sites B-IBI:

preferably, in step (7), the evaluation of the health grade of the zoobenthos integrity index is performed to monitor the sample B-IBI_{Normalization}The 95% quantile of the value is the healthy grading standard of "excellent", and then the 4-point method is adopted for grading, the standard is as follows:

3. advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) compared with the traditional method, the benthonic animal biological integrity evaluation method based on the eDNA macro-barcode technology provided by the invention generally requires less manpower, material resources and time; the method is easy to learn in operation, and does not need to have high classification and identification capacity; many individuals in benthonic animals are small and the classification characteristics are not obvious, and a large number of hidden species can be identified by eDNA macro-barcode monitoring, so that the sensitivity is higher; the confused species can be easily ignored by identifying morphology through sequence difference, and the accuracy of species identification is improved.

(2) The benthonic animal biological integrity evaluation method based on the eDNA macro-barcode technology provided by the invention can evaluate the aquatic ecological health conditions with high resolution and different human activity interference strengths. The molecular classification unit change (such as OTU) at the level below the species can be directly monitored based on the eDNA macro-barcode, the integrity of the benthonic animals can be evaluated by comparing the genetic diversity of different point positions, and the evaluation result has higher resolution.

(3) The benthonic animal biological integrity evaluation method based on the eDNA macro-barcode technology provided by the invention can be operated in a standardized manner, has strong comparability of monitoring and evaluation results, and is beneficial to comparison on large-scale time and space scales. The method adopts standardized molecular biology and bioinformatics processes, and experimental results of different samples and different personnel are comparable, so that the method is beneficial to evaluation of ecological recovery conditions of key watershed in China and comparison of water ecological health conditions among large watersheds.

Drawings

FIG. 1 is the distribution of candidate index bins after screening in example 1;

FIG. 2 is the Pearson correlation between candidate indexes in example 1;

FIG. 3 is the integrity index test of benthonic animals in lake reservoirs of the Taihu lake basin of example 1, in which: (a) discrimination of reference points and damaged points; (b) the consistency of the method with the evaluation of the integrity of the benthonic animals based on morphological monitoring;

FIG. 4 is the distribution of the candidate index bins after screening in example 2;

FIG. 5 is the Pearson correlation between candidate indices of example 2;

FIG. 6 is the integrity index test for benthonic animals of rivers in the Taihu lake basin of example 2, wherein: (a) discrimination of reference points from damaged points (b) consistency of the method with benthonic integrity assessment based on morphological monitoring.

Detailed Description

The invention is further described with reference to specific examples.

Example 1

In the embodiment, samples are collected from 30 lakes and reservoir sites in the river and lake Taihu basin, and the implementation method is specifically described in the following steps.

(1) Reference point and damaged point determination

Combining regional water ecological characteristics and an environment management target, finally selecting a pond-horse reservoir, dun, pu zhuang, pepper mountain, south of great stream reservoir, ramble mountain and east-west large leaf nodes as reference points for constructing lake and reservoir (lake reservoir) benthonic animal integrity indexes according to determination conditions of relatively good water quality, relatively light eutrophication degree, relatively high aquatic vegetation coverage and relatively low artificial interference strength and selection conditions of reference states, wherein the rest points are regarded as damaged points.

(2) Sample collection and environmental DNA extraction

Collecting sediment at the bottom of water body with tool such as Petersen mud collector and triangular trawl, filtering with screen mesh, washing, draining water, and storing in plastic bag at-20 deg.C. Thawing, weighing, converting 1g mixture into 1mL, adding 3 times volume of anhydrous ethanol, extracting with ethanol for 7 days, shaking, slightly standing, collecting 2mL supernatant, vacuum centrifuging to obtain dried tissue residue, and extracting environmental DNA with OMEGA Water kit.

(3) DNA barcode fragment amplification and high throughput sequencing and analysis

And (3) amplifying the environmental DNA in the step (2) by using a COI primer pair (the upstream primer is mlCOIintF: GGWACWGGWTGAACWGTWTAYCCYCC; the downstream primer is dgHCO 2198: TAAACYTCAGGRTGACCRAARAAYCA), carrying out high-throughput sequencing on the PCR product, processing sequencing data by a software platform such as qiime and usearch, obtaining OTU information corresponding to each sample, and carrying out species annotation on the OTU by using an NCBI database.

(4) Candidate index and calculation of benthonic animal biological integrity index

Candidate indexes of the zoobenthos biological integrity index screened based on the eDNA macro-barcode monitoring data are shown in the following table 1, and comprise community abundance parameters of 6, species dominance parameters of 2, species composition parameters of 15, species pollution resistance parameters of 3 and genetic diversity parameters of 1.

TABLE 1 candidate indices and calculation of zoobenthos biological integrity index

(5) Candidate index screening of benthonic animal biological integrity index

a) And (3) screening distribution ranges: calculating the point location ratio of the minimum value, Q25, median, Q75, maximum value and value >0 of each exponential distribution, keeping indexes of the value >0 of the point location above 80% and the value > Q75 of the maximum value > median > Q25> minimum value, preliminarily analyzing to show that the point locations of the M03_ EPT classification unit number and the M22_ EPT ratio above 20% are all 0, discarding, and entering the next analysis by 25 items of the rest parameters;

b) and (3) distinguishing and analyzing the box body: and analyzing the distribution of each parameter entering the discriminability analysis between the reference state and the damaged state by adopting a box curve graph method. Namely, the quantile range of 25 th-75 th of the reference state and the damaged state is compared, namely the box diagram box body overlapping condition (IQ), and only the parameter of IQ is more than or equal to 2 to be further analyzed. The results show that 15 candidate indexes IQ ≧ 2 in total, and the distribution of the boxes of these parameters is shown in FIG. 1.

c) And (3) correlation analysis: pearson correlation analysis is carried out on 15 biological indexes, independence of information reflected by each index is checked, a correlation coefficient | r | between two indexes is larger than 0.750 to represent high correlation between the two indexes, one of the indexes with high correlation is selected, and the result is shown in figure 2. Preferentially reserving M01_ total sorting unit number, thus eliminating the M24_ BMWP index (r is 0.87) which is highly related to the M01_ total sorting unit number; preferentially preserving M27_ average genetic diversity, thus eliminating M08_ top 3-position dominance (r ═ -0.81), M09_ shannon diversity index (r ═ 0.81), M10_ Pielou uniformity index (r ═ 0.78), M11_ Simpson index (r ═ 0.76) that are highly correlated therewith; preferentially reserving M12_ Virginiaceae _ Cervidae ratio; preferentially retaining the M25_ ASPT index, thus rejecting the M02_ mollusk taxon number (r ═ 0.95) highly correlated therewith; the M18-mollusc ratio is preferentially retained, so the M13-oligochaeta ratio (r-0.9), M16-oligochaeta-chironomidae ratio (r-0.99) are rejected, and the M19-gastropoda ratio is repeated with the molluscs, so the rejection is done.

d) Final candidate indices: the 6 parameters of reserved M01-total taxon number, M07-first dominance, M12-Tremellolidae-Cervidae proportion, M18-mollusk proportion, M25-ASPT index and M27-average genetic diversity form B-IBI together.

(6) Calculating the biological integrity of the benthonic animals, and grading the health

a) B-IBI calculation: the stronger the interference, the lower the value of the index, taking the 95% quantile of the monitored sample value as an expected value (namely, a ecological target value), and the index score is equal to the monitored value divided by the expected value; for the index with stronger interference and higher value, taking 5% quantile as an expected value, the calculation method is as follows: (max-monitor)/(max-desired). If the calculation result is more than 1, calculated according to 1, less than 0, calculated according to 0, the specific parameter calculation formula is shown in table 2, and the calculation value is more than 1 and is 1.

TABLE 2 calculation formula of integrity index of zoobenthos in lake and reservoir of Taihu lake basin

b) Normalization

To facilitate the same scale for overall evaluation, the zoobenthos integrity index was normalized, where B-IBI_q9595% quantile for all monitoring sites B-IBI:

c) health grade

To monitor the sample B-IBI_{Normalization}The 95% quantile of the value is the health grading standard of "excellent", then the 4-point method is adopted for grading, and the integrity index system of the benthonic animals in the lake and the reservoir of the Taihu lake basinThe grading criteria are shown in Table 3.

TABLE 3 rating standards for benthonic animal biological integrity index system in Taihu lake basin

(7) Reliability of benthonic animal biological integrity evaluation result

a) Discrimination between reference and damaged points

According to the constructed integrity index system of the benthonic animals in the lake and reservoir of the Taihu lake basin, B-IBI values of all the points are calculated, and box line graphs are drawn, as shown in figure 3a, the reference points are not overlapped with the box bodies of the damaged points, namely the B-IBI values constructed by the process can distinguish the integrity states of the benthonic animals of the reference points and the damaged points.

b) And consistency of results of biological integrity assessment obtained by morphological monitoring

The method has obvious positive correlation between the benthonic animal integrity evaluation result obtained based on the environmental DNA macro-barcode monitoring and the B-IBI obtained based on the morphological monitoring (figure 3B).

Example 2

Samples were taken from 37 river and stream sites in the lake Taihu basin. The results of the benthic animal integrity analysis of 37 river and stream sites obtained by the same procedure as in example 1 were as follows:

(1) according to the selection conditions of the reference states, the fishing big bridge, the river entering water channel _ building manyan, the old county, the navigation management station, the Taipu gate, the Lasuan bay and the Zhao tun are finally selected as reference points constructed by the integrity indexes of the river benthonic animals in the Taihu lake basin, and the rest non-reference points are damaged points.

(2) Through distribution range screening, all indexes have wider variable ranges and larger change space for stress response, and can participate in constructing an integrity index system, so that 27 indexes in all indexes enter a box body discrimination analysis.

(3) The results of box body discrimination analysis show that 13 candidate indexes IQ are more than or equal to 2, and the box body distribution conditions of partial parameters are shown in figure 4.

(4) After correlation analysis of 13 biomarkers, as shown in fig. 5, M09 — shannon diversity index as one of diversity indexes should be included in the evaluation system and retained, so as to eliminate the M08 — first 3-bit dominance (r ═ 0.96) highly correlated with it; indicating species aspect, M12_ tubificidae _ curculionidae proportion and M18_ mollusc proportion (r ═ -0.78) were retained, thus knockout M13_ oligopolisis proportion (r ═ 0.83), M16_ oligochaeta _ chironomidae proportion (r ═ 0.81); the M20-plectranthus ratio and M21-corbicula ratio are slightly repeated with the M18 index, so that the two are rejected.

(5) Finally, 4 parameters of M01-total taxon number, M09-Shannon diversity index, M12-Tremellidae-Cervidae ratio and M18-mollusk ratio (r is-0.78) are reserved to form B-IBI. The calculation formula of each parameter of B-IBI is shown in the following table 4, and the calculated value is more than 1 and is 1.

TABLE 4 formula for calculating integrity index of benthonic animals in Taihu lake basin river

(6) To facilitate the same scale for overall evaluation, the zoobenthos integrity index was normalized, where B-IBI_q9595% quantile for all monitoring sites B-IBI:

(7) the classification standard of the integrity index system of the benthonic animals in the rivers of the Taihu lake basin is the same as that in the table 3 after normalization.

(8) According to the constructed complete index system of the lake Tai river benthonic animals in the river basin, point positions B-IBI are calculated, box type graphs are drawn as shown in figure 6a, and correlation results of the point positions B-IBI and the B-IBI obtained based on morphological monitoring are shown in figure 6B.