Method, device for assessing the impact of human disturbance on the reproductive health of wildlife

Abstract

The application discloses a method for evaluating the influence of human interference on the reproductive health of wild animals. A steroid hormone species and an intestinal microorganism macro-genome assembly genome MAG of a sentinel animal fecal sample are obtained; based on the steroid hormone species, a first gene set is determined, the first gene set comprising a first gene, the first gene being a metabolic enzyme gene, the metabolic enzyme participating in steroid hormone metabolism; based on the first gene set and the MAGs, a first coding sequence CDS set is determined, the first coding sequence CDS set comprising a first CDS, the first CDS being from the MAGs, and the coding region of the first gene comprising the first CDS; based on the first coding sequence CDS set, a first microorganism set HCBs is determined, the HCBs being a collection of host bacteria HCBs of the first CDS, the HCBs being selected from the intestinal flora of the sentinel animal; based on the total relative abundance of the HCBs, it is determined whether human interference affects the reproductive health of wild animals. The method is scientific and reliable, and provides a scientific decision basis for wild animal protection.

Description

Technical Field

[0001] This invention relates to the field of environmental protection, and more specifically to methods and apparatus for assessing the impact of human disturbance on the reproductive health of wild animals. Background Technology

[0002] Emerging environmental pollutants refer to a class of chemical substances that are detectable in the environment and natural ecosystems and can pose significant risks and hazards to human health and environmental safety even at low doses. Extensive experimental evidence and epidemiological surveys indicate that these pollutants have a significant impact on animal reproductive hormones, and are a major contributing factor to the increased incidence of reproductive disorders, birth defects, developmental abnormalities, and metabolic disorders. Therefore, against the backdrop of increasingly severe human disturbance, detecting the impact of emerging environmental pollutants on the reproductive health of wild animals is of great significance for species conservation and population recovery.

[0003] However, emerging environmental pollutants are characterized by long-term concealment, environmental persistence, bioaccumulation, and low-dose characteristics, making them difficult to detect accurately in real-world environments. Furthermore, due to the small population size and wide range of endangered wildlife species, there is uncertainty regarding whether they are exposed to emerging environmental pollutants in their habitats, whether these pollutants transfer within their bodies, and ultimately affect their reproductive health. These factors collectively increase the difficulty of assessing the impact of emerging environmental pollutants on the reproductive health of wildlife. Currently, there is a lack of effective research methods for scientific evaluation. my country has invested significant human and material resources in the construction of nature reserves for wildlife habitats, achieving remarkable results. However, to achieve coordinated development of social, economic, and ecological benefits, and to promote the improvement of living standards for residents around protected areas and regional economic revitalization, it is necessary to moderately conduct economic activities, but this may introduce emerging environmental pollutants into wildlife habitats.

[0004] Therefore, it is urgent to establish methods that can scientifically assess the impact of human disturbance on the reproductive health of wild animals, provide technical support for coordinating human activities and wildlife conservation, and ultimately promote harmonious coexistence between humans and animals. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, the invention provides a method and apparatus for assessing the impact of human disturbance on the reproductive health of wild animals. Starting from the specific gut microbiota of wild animals, the method utilizes modern bioinformatics technology to successfully construct a complete causal chain of "human disturbance - gut microbiota - reproductive health." This method bypasses traditional habitat characteristic analysis, and through qualitative or quantitative assessment of specific gut microbiota characteristics, determines the impact of human disturbance on the reproductive health of wild animals. It is scientifically reliable and provides a scientific basis for decision-making in wildlife conservation.

[0006] This invention is based on the inventor's discoveries and understanding of the following problems:

[0007] The gut microbiota is considered the "second genome" of wild animals, playing a crucial role in their physiological and biochemical processes. Studies have shown that the bacterial gene sets encoding enzymes such as β-glucuronidase in the gut microbiome have metabolites or components closely related to estrogen metabolism. Simultaneously, the gut microbiota also participates in androgen metabolism pathways, promoting increased levels of free testosterone and dihydrotestosterone by regulating β-glucuronidase activity. Furthermore, gut microbiota possess the ability to metabolize environmental pollutants and alter their toxicity; however, their functions in endocrine regulation and reproductive-related metabolism may be disrupted during the processing of exogenous pollutants.

[0008] Using golden monkey feces, we analyzed the genes of detectable steroid hormone metabolic enzymes and the types of microorganisms involved in hormone metabolism. We found that steroid hormone metabolism-related microorganisms (HCBs) in golden monkey feces could be classified into five phyla: Spirochaetota, Bacteroidota, Bacillota, Actinomycetota, and Unclassified. Differential analysis showed that human interference significantly reduced the total relative abundance of these microorganisms, with a significant decrease in the relative abundance of Bacteroidota, Bacillota, and Actinomycetota. We also analyzed steroid hormone metabolism-resistant microorganisms (PHCBs), which, at the phylum level, could be classified into three phyla: Bacteroidota, Bacillota, and Actinomycetota. Differential analysis showed that human interference significantly increased the total relative abundance of these microorganisms, with a significant increase in the relative abundance of Bacillota and Actinomycetota, but their ability to metabolize reproductive hormones was significantly reduced.

[0009] Further validation results showed a significant positive correlation between the levels of reproductive-related hormones (10 types of steroid hormones in Table 4) and the abundance of HCBs (human steroid bacteria) involved in hormone catalysis in the golden snub-nosed monkey gut (P < 0.01). This result validates the catalytic role of gut microbiota in the reproductive hormone catalysis of wild animals. Therefore, the function and capabilities of gut microbiota can be used to predict the levels of reproductive-related hormones in wild animals.

[0010] The ability of microorganisms in wild animal feces to catalyze reproductive hormone-related reactions influenced by the metabolic function of novel environmental pollutants is significantly negatively correlated with the levels of reproductive hormones (such as estradiol, estrone, testosterone, 17-Hydroxyprogesterone, 11-Deoxycorticosterone, and Androstenedione, and Dihydrotestosterone). This validation analysis indicates that in wild animal gut microbiota, the stronger the conjugation between genes involved in the metabolism of novel environmental pollutants and genes involved in the catalytic reaction of reproductive hormones, the lower the level of reproductive hormones in feces, and the greater the negative impact of novel environmental pollutants on steroid hormones in wild animals.

[0011] Therefore, in one aspect of the invention, a method for assessing the impact of human disturbance on the reproductive health of wild animals is proposed. According to embodiments of the invention, fecal samples from sentinel animals are collected for steroid hormone detection and microbial metagenomic sequencing to obtain the types of steroid hormones in the fecal samples and the assembled genomes (MAGs) of the gut microbiota.

[0012] Based on the types of steroid hormones, a first gene set is determined, the first gene set containing a first gene, the first gene being a metabolic enzyme gene, the metabolic enzyme participating in the metabolism of the steroid hormones; based on the MAGs, the sentinel animal gut microbiota is determined.

[0013] Based on the first gene set and the MAGs, a first coding sequence (CDS) set is determined, the first coding sequence CDS set contains a first CDS, the first CDS comes from the MAGs, and the coding region of the first gene contains the first CDS;

[0014] Based on the first coding sequence CDS set, a first microbial set (HCBs) is determined, wherein the HCBs are a collection of host bacteria (HCBs) of the first CDS, and the HCBs are selected from the intestinal microbiota of the sentinel animal;

[0015] Based on the total relative abundance of HCBs, it is determined whether human disturbance affects the reproductive health of wild animals. The method according to embodiments of the present invention bypasses traditional habitat characteristic analysis, assessing the impact of human disturbance on the reproductive health of wild animals through qualitative or quantitative evaluation of specific gut microbiota characteristics. This method is scientifically reliable and provides a scientific basis for decision-making in wildlife conservation.

[0016] According to embodiments of the present invention, compared to sentinel animals accustomed to human interference, the total relative abundance of HCB in the intestines of sentinel animals habitually subjected to human interference is reduced, which is considered an impact of human interference on the reproductive health of wild animals. This further improves the accuracy of the assessment.

[0017] It should be noted that in this article, the terms "decrease" or "increase" refer to statistically significant positive or negative correlation (P < 0.05) or highly significant positive or negative correlation (P < 0.01) of the parameter value under investigation.

[0018] In this article, the term "human disturbance" refers to any event or condition, directly or indirectly caused by human activities, that can alter the structure, function, or dynamic processes of an ecosystem.

[0019] According to an embodiment of the present invention, the sentinel animal is a golden snub-nosed monkey, and the HCBs include the phyla Bacteroidota, Bacillota, and Actinomycetota. According to an embodiment of the present invention, the abundance of these three bacteria is significantly different in the intestines of Sichuan golden snub-nosed monkeys accustomed to human disturbance compared to those without human interference.

[0020] According to embodiments of the present invention, the HCBs consist of the following phyla: Spirochaetota, Bacteroidota, Bacillota, Actinomycetota, and Unclassified. According to embodiments of the present invention, the total relative abundance of these five bacteria in the intestines of Sichuan golden monkeys accustomed to human disturbance differed significantly compared to the undisturbed group.

[0021] In some specific implementations, the human interference includes environmental pollutants. Exemplarily, the environmental pollutant is a novel environmental pollutant.

[0022] According to embodiments of the present invention, the environmental pollutant includes: a biocide. Exemplarily, the biocide includes at least one of: an insecticide, a disinfectant, an antimicrobial peptide, and a herbicide.

[0023] In another aspect of the invention, a method for assessing the impact of environmental pollutants on the reproductive health of wild animals is proposed. According to embodiments of the invention, fecal samples from sentinel animals are collected for steroid hormone detection and microbial metagenomic sequencing to obtain the types of steroid hormones in the fecal samples and the assembled genomes (MAGs) of the gut microbiota.

[0024] Based on the types of steroid hormones, a first gene set is determined, the first gene set containing a first gene, the first gene being a metabolic enzyme gene, the metabolic enzyme participating in the metabolism of the steroid hormones; based on the MAGs, the sentinel animal gut microbiota is determined; based on the types of environmental pollutants, a second gene set is determined, the second gene set containing a second gene, the second gene being a resistance gene to the environmental pollutant.

[0025] Based on the first gene set, the second gene set, and the MAGs, a first coding sequence (CDS) set, a second coding sequence CDS set, and a third CDS set (PHCs) are determined; wherein the first coding sequence CDS set contains a first CDS, the second coding sequence CDS set contains a second CDS, the first CDS originates from the MAGs, the coding region of the first gene contains the first CDS, the second CDS originates from the MAGs, and the second gene contains the second CDS; the PHCs are the intersection of the first coding sequence CDS set and the second coding sequence CDS set.

[0026] Based on the PHCs, a third set of microorganisms (PHCBs) is determined; wherein, the PHCBs are a collection of host bacteria (PHCBs) of the PHCs, and the PHCBs are selected from the gut microbiota of the sentinel animals;

[0027] Based on the total relative abundance of the PHCBs, it is determined whether environmental pollutants affect the reproductive health of wild animals; the total relative abundance of the PHCBs is calculated according to Formula I:

[0028] Formula I;

[0029] Among them, TA PHCB C represents the total relative abundance of PHCB. i N represents the coverage of the i-th bacterium. i This represents the number of contigs contained in the i-th bacterium, and N refers to the total number of contigs successfully mapped by MAGs of all bacteria in the sample.

[0030] or,

[0031] Based on the ability of the environmental pollutants to affect steroid hormone metabolism, it is determined whether the environmental pollutants affect the reproductive health of wild animals; the ability of the environmental pollutants to affect steroid hormone metabolism is calculated according to Formula II:

[0032] Formula II;

[0033] Among them, Capacity PHCBs PHCB indicates the ability of environmental pollutants to affect steroid hormone metabolism.i PHC represents the abundance of the i-th PHCB. i S represents the number of PHCs in the i-th PHCB. i This indicates the types of steroid hormones whose metabolism is affected by PHC in the i-th PHCB.

[0034] The method according to embodiments of the present invention assesses the impact of human interference on the reproductive health of wild animals by quantitatively evaluating specific gut microbiota characteristics. This method is scientifically reliable and yields highly accurate assessment results.

[0035] For example, information on metabolic enzymes involved in steroid hormone metabolism is obtained from the KEGG database.

[0036] In some specific implementations, the environmental pollutant resistance genes include: antibiotic resistance genes, disinfectant resistance genes, metal resistance genes, and microplastic metabolism genes. For example, information on microbial antibiotic resistance genes is obtained from the CARD (http: / / arpcard.mcmaster.ca; (blastp, evalue: 1e–5), BacMet (http: / / bacmet.biomedicine.gu.se; blastp, evalue: 1e–5), and PlasticDB (https: / / plasticdb.org; blastp, evalue: 1e–5) databases. Antibiotic resistance genes enable bacteria to resist the effects of antibiotics; disinfectant resistance genes confer tolerance to disinfectants or bactericides; metal resistance genes help microorganisms survive in environments polluted by heavy metals (such as arsenic, mercury, and cadmium); and microplastic metabolism genes encode enzymes that can break down plastic polymers and are closely related to the environmental fate of microplastics.

[0037] According to embodiments of the present invention, compared to sentinel animals accustomed to the interference of the environmental pollutants, the total relative abundance of PHCBs in the intestines of sentinel animals accustomed to the interference of the environmental pollutants is increased, which is considered as the environmental pollutants affecting the reproductive health of wild animals; and / or, compared to sentinel animals accustomed to the interference of the environmental pollutants, the ability of the environmental pollutants to affect the metabolism of steroid hormones is increased in the intestines of sentinel animals accustomed to the interference of the environmental pollutants, which is considered as the environmental pollutants affecting the reproductive health of wild animals.

[0038] According to an embodiment of the present invention, the sentinel animal is a golden snub-nosed monkey, and the PHCBs comprise Bacillota and Actinomycetota phyla. According to an embodiment of the present invention, compared with the group of Sichuan golden snub-nosed monkeys accustomed to environmental pollution, the total relative abundance of these two bacteria and their ability to influence steroid hormone metabolism differed significantly in the intestines of Sichuan golden snub-nosed monkeys accustomed to environmental pollution.

[0039] According to embodiments of the present invention, the PHCBs consist of the following phyla: Bacteroidota, Bacillota, and Actinomycetota. According to embodiments of the present invention, compared with the undisturbed Sichuan golden monkey group, the total relative abundance of these three bacteria and their ability to influence steroid hormone metabolism were significantly different in the intestines of Sichuan golden monkeys accustomed to human disturbance.

[0040] According to an embodiment of the present invention, the environmental pollutant is a novel environmental pollutant.

[0041] The environmental pollutants include: biocides.

[0042] According to embodiments of the present invention, the biocidal agent comprises at least one of the following: insecticide, disinfectant, antimicrobial peptide, and herbicide.

[0043] In another aspect of the invention, an apparatus is provided. According to an embodiment of the invention, it includes: a computer-readable storage medium having a computer program stored thereon for performing the aforementioned method; and one or more processors for executing the program in the computer-readable storage medium.

[0044] The computer program integrated into the device according to embodiments of the present invention can provide users with data-driven decision support, helping to formulate more effective conservation strategies and management measures, thereby promoting the improvement of wildlife habitats. By automatically executing the method, the device can significantly improve processing efficiency, reduce human intervention and operational errors, and optimize workflows. The device can analyze the collected data in real time, thereby quickly responding to changes in environmental conditions and wildlife growth status, and making timely adjustments. The device can visualize the analysis results, facilitating researchers and decision-makers to intuitively understand the data and enhance communication. The device design can integrate multiple functional modules, such as data storage, analysis, and simulation, providing comprehensive solutions to adapt to different research needs.

[0045] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0046] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0047] Figure 1 This is a graph showing the results of the analysis of the effects of human interference on the abundance of hormone metabolism microorganisms (HCPs) in the intestine of golden snub-nosed monkeys in an embodiment of the present invention.

[0048] Figure 2 This invention relates to the total relative abundance of biosensitive microorganisms (PHCBs) resistant to novel environmental pollutants in the gut of human-induced habituated golden monkeys, and the capacity of environmental pollutants to influence steroid hormone metabolism. PHCBs The results of the investigation are shown in the figure; where (A) is the total relative abundance of PHCBs; and (B) is the capacity. PHCBs ;

[0049] Figure 3 The results of the investigation of steroid hormone content in the intestines of human-induced habituated golden monkeys in the embodiments of the present invention;

[0050] in, Figures 1-3 In the figure, * and ** represent significant correlation (P < 0.05) and highly significant correlation (P < 0.01), respectively. Detailed Implementation

[0051] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0052] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0053] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0054] In this paper, the term "sentinel animal" refers to a class of animals used to monitor or provide early warning of the levels of toxic or potentially toxic substances in a specific area. Wild animals are diverse, and their types of ecological stress vary. Studies often select an animal capable of monitoring or providing early warning of the levels of toxic or potentially toxic substances in a specific area to investigate the ecological risks posed by pollutants to all animals living in the same or similar environments. Golden snub-nosed monkeys can be used as sentinel animals; by utilizing the ability of their gut microbiota to metabolize novel environmental pollutants and influence steroid hormone metabolism, the impact of novel environmental pollutants on the reproductive capacity of wild animals can be evaluated.

[0055] In this article, the term "emerging environmental pollutants" refers to a specific form of human interference, including but not limited to: persistent organic pollutants (POPs): such as certain pesticides and industrial chemicals, which are highly toxic, can migrate long distances, and accumulate through the food chain, causing harm to health and the ecological environment; endocrine disruptors (EDCs): found in some plastics, pharmaceuticals, and pesticides, which may interfere with the endocrine systems of organisms and humans, affecting reproduction, development, and behavior. Studies have found that estrogen-like substances at levels as low as nanograms can cause hermaphroditism in wild fish; antibiotics: widely used in medicine, animal husbandry, and aquaculture, which, once released into the environment, may promote and spread drug-resistant bacteria, disrupting the original microbial ecosystem balance; microplastics: which may themselves affect human health and can also adsorb other pollutants (such as persistent organic pollutants) and transport them to other places.

[0056] In this paper, the term "Metagenome-Assembled Genomes (MAGs)" refers to a nearly complete single microbial genome "pieced together" from metagenomic sequencing data of the gut microbiome through computational analysis. In traditional microbiology, to study a bacterium, we must first isolate and culture it in a laboratory. However, scientists have long recognized that the vast majority (possibly over 80%) of microorganisms in the natural environment (including the gut) cannot be cultured using current technologies. This means we know very little about their functions; they are referred to as "microbial dark matter." MAG technology completely bypasses the "culturing" step, allowing us to directly peer into the genomes of these "unculturable" microorganisms, thereby revealing their identities and potential functions.

[0057] The construction process of MAGs technology mainly includes: 1. Sample collection and sequencing: Collect intestinal samples such as feces, extract DNA (i.e., metagenomics) from all microorganisms, and perform high-throughput sequencing to generate a large number of short DNA sequence fragments. 2. Sequence assembly: Using specialized bioinformatics software, these short fragments are spliced ​​together according to the overlapping regions of the sequences to form longer continuous sequences (called contigs). 3. Binning: This is the most crucial step. The algorithm "sorts" contigs belonging to the same microorganism into the same "bin" (i.e., a group) based on characteristics such as sequence composition (e.g., GC content) and abundance in the sample. This is because DNA fragments from the same microorganism have similar composition and abundance. 4. Quality assessment and optimization: Each "bin" is evaluated to see if it is complete (containing most of the genes of the microbial genome) and whether it is contaminated (mixed with DNA from other microorganisms). 5. Annotation and Analysis: After obtaining a high-quality MAG, it can be analyzed to predict which classification, class, order, family, genus, and species it belongs to, and to analyze which genes it carries and what potential metabolic functions it has (e.g., whether it can synthesize a certain vitamin, degrade a certain toxin, or metabolize steroid hormones).

[0058] In some embodiments of this invention, the enzyme genes involved in steroid hormone metabolism are identified using the KEGG database. The KEGG (Kyoto Encyclopedia of Genes and Genomes) database is a comprehensive bioinformatics resource designed to facilitate data mining and analysis of genomics, metabolism, and biological systems in scientific research.

[0059] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0060] Example 1

[0061] In this embodiment, golden snub-nosed monkeys were selected as sentinel animals. The influence of the metabolic genes of novel environmental pollutants in their gut microbiota on steroid hormone metabolism was utilized to evaluate the impact of novel environmental pollutants on the reproductive capacity of wild animals.

[0062] 1. Test Methods

[0063] 1.1 Sample Collection

[0064] Golden snub-nosed monkey fecal sample collection: Wild group (W) and human disturbance group (HD) in Baihe National Nature Reserve were selected as research subjects. Fecal samples were collected from W (CK group) and HD (HD group) and temporarily placed in a portable low-temperature vehicle-mounted refrigerator. After all samples were collected, dry ice was added and transported to the testing site.

[0065] The specific sampling method for golden snub-nosed monkey feces is as follows: all feces found when first entering the golden snub-nosed monkey's habitat are disposed of. Every day at 9:00 AM and 5:00 PM, fecal samples of golden snub-nosed monkeys are randomly collected using a fecal sampler. Fifteen fecal samples of golden snub-nosed monkeys are randomly collected and placed in a portable low-temperature vehicle-mounted refrigerator with dry ice for preservation.

[0066] 1.2 Determination of Steroid Hormone Content

[0067] A portion of golden monkey fecal samples were added to dry ice and sent to Metabo-Profile Biotechnology (Shanghai) Co., Ltd. for steroid hormone content determination. In this example, the Targeted Metabolomics Technology Platform (UPLC-TQMS) was used to quantitatively detect specific hormones in the biological sample (Metabo-Profile, Shanghai, PR China). This platform can accurately quantify 20 types of steroid hormones in the sample (Metabo-Profile, Shanghai, PR China). The specific quantitative method is as follows:

[0068] Weigh 15 mg of lyophilized fecal sample into a centrifuge tube, add 10 grinding beads, add 100 μL of deionized water, and homogenize for 3 min (BB24, Next Advance, Inc., Averill Park, NY, USA). Separately, pipette 100 μL of standard curve working solution into a centrifuge tube. Add 50 μL of 20% methanol solution containing internal standard to the standard curve working solution and sample, and extract by shaking at 2000 rpm for 3 min at room temperature (MSC-100, Hangzhou Allsheng Instrument Co., China). Then add 750 μL of methyl tert-butyl ether and extract by shaking at 2000 rpm for 10 min at room temperature. Centrifuge the sample at 18000 g for 10 min at 10°C (Microfuge 20R, Beckman Coulter, Inc., Indianapolis, IN, USA). Pipe 650 μL of the supernatant into a 96-well plate and dry under nitrogen. Then, it was reconstituted with 100 μL of 20% methanol-water solution and shaken at 1450 rpm for 10 min at 10°C. After centrifugation at 4000 g for 10 min (Allegra X-15R, Beckman Coulter, Inc., Indianapolis, IN, USA), it was analyzed. Hormonal substances were detected using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA).

[0069] Reagent blanks and pooled quality control samples were set up before and after the analysis of each batch of samples. The inclusion of these quality controls also serves to monitor potential contamination and data quality during the analysis process. The raw data files generated by UPLC-MS / MS were processed using MassLynx software (V4.1, Waters, Milford, MA, USA) to integrate, calibrate, and quantify the peaks of each metabolite.

[0070] 1.3 Steroid hormone metabolic enzyme pathway

[0071] Each compound is numbered in the KEGG database (https: / / www.genome.jp / kegg). These compounds participate in biochemical reactions, which are catalyzed by corresponding enzymes. Using the KEGG database, we compiled the compound numbers of steroid hormones detected in fecal samples, the numbers of the biochemical reactions they participate in, and the gene numbers of the enzymes associated with these reactions.

[0072] 1.4 Microbial metagenomic sequencing and assembly

[0073] Metagenomic sequencing of the gut microbiota of golden snub-nosed monkeys was performed using Illumina Miseq. The sequencing data were quality checked using FASTP software, removing raw sequencing data with a length less than 50 bp after quality cutting, an average quality score below 20, and containing N bases, retaining high-quality reads. Reads were aligned to the golden snub-nosed monkey host DNA sequence using BWA software, and contaminating reads with high similarity to the golden snub-nosed monkey DNA were removed. The quality-checked sequences were assembled into contigs using MEGAHIT software, and contigs with a length ≥1000 bp were selected and processed using MetaBAT software.

[0074] Gene sequences of golden monkey gut microbiota obtained from metagenomic sequencing were binning to obtain metagenomically assembled genes (MAGs). High-quality MAGs (≥60% integrity and ≤10% contamination) were then reassembled and predicted to obtain scaffolds. The coding sequences (CDS) of the scaffolds were aligned to the KEGG database (https: / / www.genome.jp / kegg; blastp, evalue: 1e–5) to identify MAGs and their CDS that are involved in steroid hormone metabolism. The Scaffolds CDS was compared with the CARD (http: / / arpcard.mcmaster.ca; (blastp, evalue: 1e–5), BacMet (http: / / bacmet.biomedicine.gu.se; blastp, evalue: 1e–5), and PlasticDB (https: / / plasticdb.org; blastp, evalue: 1e–5) databases to obtain information on antibiotic resistance genes, antimicrobial and metal resistance genes, and microplastic metabolism genes.

[0075] 1.5 The impact of emerging environmental pollutants on the metabolism of reproductive-related hormones

[0076] We define host genes (MAGs) capable of metabolizing steroid hormone CDS as steroid-hormone metabolic bacteria (HCBs). If a MAG's CDS gene is involved in both steroid hormone metabolism and the metabolism of novel environmental pollutants, then the genes of the functional enzymes involved in steroid hormone metabolism and the genes involved in the metabolism of novel environmental pollutants are conjugated. We define CDS genes conjugated with those involved in steroid hormone metabolism as PHCs (New-environmental-pollutants and steroid-hormone CDS), and the host genes (MAGs) of PHCs as host bacteria of novel environmental pollutants and steroid-hormone metabolic genes (PHCBs).

[0077] (1) Calculate the total relative abundance of PHCBs.

[0078] Formula I;

[0079] Among them, TA PHCB C represents the total relative abundance of PHCBs. i N represents the coverage of the i-th bacterium. i This represents the number of contigs contained in the i-th bacterium, and N refers to the total number of contigs successfully mapped by MAGs of all bacteria in the sample.

[0080] (2) Count the number of CDS of PHCs and the types of steroid hormones affected.

[0081] (3) Ability to calculate the impact of emerging environmental pollutants on steroid hormone metabolism

[0082] Formula II;

[0083] Among them, Capacity PHCBs PHCB indicates the ability of emerging environmental pollutants to affect steroid hormone metabolism. i PHC represents the abundance of the i-th PHCB. i S represents the number of PHCs in the i-th PHCB. i This indicates the types of steroid hormones whose metabolism is affected by PHC in the i-th PHCB.

[0084] Capacity PHCBsThe numerical value is determined by the abundance and variety of PHCBs, the number of conjugated genes (PHC genes), and the types of steroid hormones affected by them. Higher PHCB abundance and more PHCB varieties, along with a greater number of PHC genes and a higher S... i The larger, the greater the capacity PHCBs The higher the value, the greater the influence of emerging environmental pollutants on steroid hormone levels, and the greater the impact of emerging environmental pollutants on the reproductive health of wild animals.

[0085] 1.6 Statistical Analysis

[0086] Preliminary data processing was performed using Office 2010. Analysis of variance and correlation analysis were performed using IBM SPSS (Version 21, IBM Inc. 2012) software. Bar charts were created using Oringin 2020 Pro software.

[0087] 2. Results

[0088] 2.1 The results of the gene investigation of hormones and their metabolic enzymes in golden snub-nosed monkey fecal samples are shown in Table 1.

[0089] Table 1. Gene information on steroid hormone metabolic enzymes detected in golden snub-nosed monkey feces

[0090]

[0091] The results showed that targeted metabolomics could detect 20 steroid hormones, while only 10 were detected in golden monkey feces. These are estradiol, estradiol, cortisol, testosterone, 17-Hydroxyprogesterone, aldosterone, 11-Deoxycorticosterone, cortisone, androstenedione, and dihydrotestosterone. Comparative analysis in the KEGG database revealed that these 10 hormones directly participate in 84 biochemical reactions, and 40 enzymes are directly involved in their metabolism.

[0092] 2.2 The results of the investigation of microorganisms involved in hormone metabolism and their genes in golden snub-nosed monkey feces are shown in Table 2.

[0093] Table 2. Microbial species (phyla) and CDS quantity involved in steroid hormone metabolism in golden monkey feces.

[0094] Microbial species (phyla) Steroid hormone names Number of CDS (types) Unclassified Testosterone 1 Bacteroidota Testosterone 18 Bacillota Testosterone 1 Bacteroidota Estrone 3 Bacillota Estrone 52 Bacteroidota Estradiol 2 Bacillota Estradiol 1 Actinomycetota Estradiol 7 Spirochaetota Dihydrotestosterone 1 Bacteroidota Dihydrotestosterone 18 Bacillota Dihydrotestosterone 18 Actinomycetota Dihydrotestosterone 6 Bacteroidota Cortisol 2 Actinomycetota Cortisol 4 Bacteroidota Androstenedione 2 Bacillota Androstenedione 143 Actinomycetota Androstenedione 195

[0095] Note: Actinomycetota is the main microorganism involved in hormone metabolism, while Androstenedione is the hormone that is reacted by microorganisms.

[0096] The results showed that 474 CDS from 223 MAGs were involved in hormone-related enzyme genes. At the phylum level, these 223 MAGs were classified into five microbial categories: Spirochaetota, Bacteroidota, Bacillota, Actinomycetota, and Unclassified, with the number of CDS involved in enzyme genes in these bacteria being 1, 45, 215, 212, and 1, respectively. Of the 10 reproductive-related hormones detected, 6 hormones involved in biochemical reactions could be directly catalyzed by enzyme genes from the golden snub-nosed monkey gut microbiota. These 6 hormones were Testosterone, Dihydrotestosterone, Androstenedione, Cortisol, Estrone, and Estradiol, with the number of CDS involved in the metabolism of these hormones in the golden snub-nosed monkey gut microbiota being 20, 43, 340, 6, 55, and 10, respectively.

[0097] Further analysis showed that human interference significantly reduced the total relative abundance of microorganisms involved in hormone metabolism, and significantly reduced the relative abundance of Bacteroidota, Bacillota, and Actinomycetota in the gut of golden snub-nosed monkeys. Figure 1 ).

[0098] 2.3 The results of the investigation into the effects of emerging environmental pollutants on the metabolism of steroid hormones in wild animals are shown in Table 3.

[0099] Table 3. Conjugated characteristics of metabolic genes and steroid hormone response genes for novel environmental pollutants

[0100]

[0101] Note: KO is an abbreviation for KEGG Orthology, which refers to functional annotation by assigning genes and gene products to specific functional categories. Each functional category is assigned a unique KO number so that researchers can better understand the functions of different genes and gene products.

[0102] The results showed that among 474 CDS of 223 MAGs directly involved in the metabolism of reproductive-related hormones, 40 CDS of 32 MAGs contained novel environmental pollutant resistance genes. At the phylum level, the 32 MAGs were classified into three PHCBs: Bacteroidota, Bacillota, and Actinomycetota. These 40 CDS contained the metabolism of three reproductive-related hormones (Estrone, Estradiol, and Dihydrotestosterone) and three novel environmental pollutants (Biocide, Disinfecting agents, and Peptide antibiotics). Further analysis showed that human interference significantly increased the total relative abundance of Bacillota and Actinomycetota in PHCBs. Figure 2 (A) Bacillota and Actinomycetota also significantly increased their metabolic effects on genes involved in reproductive hormone function. Figure 2 (B)).

[0103] The above findings indicate that novel environmental pollutants interfere with the function of steroid hormone metabolism genes through gut microbial resistance genes, thereby significantly reducing the gut microbiota's ability to metabolize reproductive hormones. Therefore, supported by existing data, the method of this invention can scientifically evaluate the impact of human interference on reproductive hormones in wild animals.

[0104] 2.4 Validity Verification

[0105] To verify the effectiveness of this method, the content of 10 types of steroid hormones in golden monkey feces was correlated with the abundance of microorganisms involved in hormone-catalyzed reactions and the metabolic capacity of these microorganisms for emerging environmental pollutants (Table 4).

[0106] Table 4. Correlation between hormone content and the total relative abundance of PHCBs and HCBs

[0107] Hormone categories Total relative abundance of PHCBs Total relative abundance of HCBs Estradiol content -0.367 * ]]> 0.454 * ]]> Estrone content -0.336 <![CDATA[0.424 * ]]> Cortisol content -0.194 <![CDATA[0.516 ** ]]> Testosterone content -0.448* <![CDATA[0.491 ** ]]> 17-Hydroxyprogesterone content <![CDATA[-0.479 ** ]]> <![CDATA[0.403 * ]]> Aldosterone content -0.057 0.109 11-Deoxycorticosterone content <![CDATA[-0.552 ** ]]> <![CDATA[0.388 * ]]> Cortisone content -0.192 <![CDATA[0.436 * ]]> Androstenedione content <![CDATA[-0.700 ** ]]> <![CDATA[0.686 ** ]]> Dihydrotestosterone content <![CDATA[-0.429 * ]]> <![CDATA[0.596 ** ]]> Total relative abundance of PHCBs 1.00 <![CDATA[-0.536 ** ]]> Total relative abundance of HCBs <![CDATA[-0.536 ** ]]> 1.00

[0108] Note: * and ** indicate significant correlation (P < 0.05) and highly significant correlation (P < 0.01), respectively.

[0109] The analysis results showed a significant positive correlation between the levels of reproductive-related hormones (the aforementioned 10 steroid hormones) and the abundance of HCBs (human steroid bacteria) involved in hormone-catalyzed reactions (P < 0.01), and a significant negative correlation between these and the abundance of PHCBs (prophylactic microbes resistant to novel micropollutants) (P < 0.05). This result validates the role of wild animal gut microbiota in the catalytic reactions of reproductive hormones. Therefore, the functions and capabilities of gut microbiota can be used to predict the levels of reproductive hormones in wild animals, thereby assessing the impact of novel environmental pollutants on the reproductive health of wild animals.

[0110] The ability of microorganisms in wild animal feces to catalyze reproductive hormone-related reactions influenced by the metabolic function of novel environmental pollutants is significantly negatively correlated with the content of reproductive hormones (such as estradiol, estrone, testosterone, 17-Hydroxyprogesterone, 11-Deoxycorticosterone, and Androstenedione, and Dihydrotestosterone). This validation analysis indicates that in the gut microbiota of wild animals, the stronger the influence of novel environmental pollutant resistance genes in PHCBs on reproductive hormone metabolism (…), the greater the influence of these genes. Figure 2 A), the lower the abundance of gut microbiota involved in hormone metabolism ( Figure 1 (and Table 4), the lower the level of reproductive-related hormones in feces ( Figure 3 The greater the negative impact of emerging environmental pollutants on the steroid hormones of wild animals, the greater the impact.

[0111] Further testing of steroid hormone levels in golden monkey fecal samples yielded the following results: Figure 3 The results showed that among the 10 hormones detected in golden snub-nosed monkey feces, the levels of Cortisol, Testosterone, 11-Deoxycorticosterone, Cortisone, Androstenedione, and Dihydrotestosterone differed significantly between the CK and HD groups, and the levels of all 6 hormones were significantly lower in the HD group than in the CK group.

[0112] Cortisol plays a crucial role in maintaining vascular tone and enhancing the excitability of the central nervous system, thus contributing significantly to stress responses. 11-Deoxycorticosterone, with aldosterone-like effects, participates in the body's stress response. Cortisone can be used to treat adrenocortical insufficiency and pituitary hypofunction. Cortisol, 11-Deoxycorticosterone, and Cortisone are all stress hormones, bioactive substances secreted by the neuroendocrine system in higher animals under stress. Prolonged exposure to human stress can lead to adrenal fatigue in animals, resulting in decreased stress hormone levels, which may cause emotional paralysis, reduced motivation, and decreased libido. Testosterone, a male hormone, has physiological functions including promoting male sex organ development, maintaining libido, and promoting protein synthesis. Dihydrotestosterone is a steroid hormone secreted by the testes. It is the main androgen in the human body and is involved in the development of male secondary sexual characteristics and the synthesis of estrogen. Androstenedione is a precursor to sex hormones. In females, androstenedione is synthesized from cholesterol.

[0113] The above findings indicate that human interference reduces the sex hormone levels and stress response capacity of golden snub-nosed monkeys, negatively impacting their reproductive capacity.

[0114] Therefore, the abundance of host microorganisms whose metabolic genes of novel environmental pollutants are conjugated with genes catalyzing reproductive hormone reactions can be used to evaluate the impact of human interference on the reproductive capacity and reproductive desire of wild animals, and to evaluate the impact of human interference on the reproductive health of wild animals.

[0115] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for assessing the impact of human disturbance on the reproductive health of wild animals, characterized in that, Fecal samples were collected from sentinel animals for steroid hormone detection and microbial metagenomic sequencing to obtain the types of steroid hormones in the fecal samples and the metagenomic genomes (MAGs) of the gut microbiota. Based on the types of steroid hormones, a first gene set is determined, the first gene set containing a first gene, the first gene being a metabolic enzyme gene, the metabolic enzyme participating in the metabolism of the steroid hormones; based on the MAGs, the sentinel animal gut microbiota is determined. Based on the first gene set and the MAGs, a first coding sequence CDS set is determined, the first coding sequence CDS set contains a first CDS, the first CDS comes from the MAGs, and the coding region of the first gene contains the first CDS. Based on the first coding sequence CDS set, a first microbial set HCBs is determined, wherein the HCBs are a collection of host bacteria HCBs of the first CDS, and the HCBs are selected from the intestinal microbiota of the sentinel animal; Based on the total relative abundance of the HCBs, determine whether human disturbance affects the reproductive health of wild animals; The sentinel animal is the golden snub-nosed monkey, and the HCBs include the phyla Bacteroidota, Bacillota, and Actinomycetota.

2. The method according to claim 1, characterized in that, Compared to sentinel animals that have not experienced the aforementioned human interference, sentinel animals accustomed to human interference showed a lower total relative abundance of HCBs in their intestines, which is considered an impact of human interference on the reproductive health of wild animals.

3. The method according to any one of claims 1 or 2, characterized in that, The HCBs consist of the following phyla: Spirochaetota, Bacteroidota, Bacillota, Actinomycetota, and Unclassified.

4. The method according to claim 1, characterized in that, Human interference includes environmental pollutants.

5. The method according to claim 4, characterized in that, The environmental pollutants include: biocides.

6. The method according to claim 5, characterized in that, The biological control agent includes at least one of the following: insecticide, disinfectant, antimicrobial peptide, and herbicide.

7. A method for assessing the impact of environmental pollutants on the reproductive health of wild animals, characterized in that, Fecal samples were collected from sentinel animals for steroid hormone detection and microbial metagenomic sequencing to obtain the types of steroid hormones in the fecal samples and the metagenomic genomes (MAGs) of the gut microbiota. Based on the types of steroid hormones, a first gene set is determined, the first gene set includes a first gene, the first gene is a metabolic enzyme gene, and the metabolic enzyme participates in the metabolism of the steroid hormones. Based on the MAGs, the gut microbiota of sentinel animals was determined; Based on the types of environmental pollutants, a second gene set is determined, the second gene set containing a second gene, the second gene being a resistance gene to the environmental pollutant; Based on the first gene set, the second gene set, and the MAGs, a first coding sequence CDS set, a second coding sequence CDS set, and a third CDS set PHCs are determined. The first coding sequence CDS set contains a first CDS, and the second coding sequence CDS set contains a second CDS. The first CDS originates from the MAGs, the coding region of the first gene contains the first CDS, the second CDS originates from the MAGs, and the second gene contains the second CDS. The PHCs are the intersection of the first coding sequence CDS set and the second coding sequence CDS set. Based on the PHCs, a third microbial set PHCBs is determined; wherein, the PHCBs are a collection of host bacteria PHCBs of the PHCs, and the PHCBs are selected from the intestinal microbiota of the sentinel animals; Based on the total relative abundance of the PHCBs, it is determined whether environmental pollutants affect the reproductive health of wild animals; the total relative abundance of the PHCBs is calculated according to Formula I: Formula I; Among them, TA PHCB C represents the total relative abundance of PHCBs. i N represents the coverage of the i-th bacterium. i This represents the number of contigs contained in the i-th bacterium, and N refers to the total number of contigs successfully mapped by MAGs of all bacteria in the sample. or, Based on the ability of the environmental pollutants to affect steroid hormone metabolism, it is determined whether the environmental pollutants affect the reproductive health of wild animals; the ability of the environmental pollutants to affect steroid hormone metabolism is calculated according to Formula II: Official II; Among them, Capacity PHCBs PHCB indicates the ability of environmental pollutants to affect steroid hormone metabolism. i PHC represents the abundance of the i-th PHCB. i S represents the number of PHCs in the i-th PHCB. i This indicates the types of steroid hormones whose metabolism is affected by PHC in the i-th PHCB; The sentinel animal is the golden snub-nosed monkey, and the PHCBs include the phyla Bacillota and Actinomycetota.

8. The method according to claim 7, characterized in that, The environmental pollutant resistance genes include: antibiotic resistance genes, disinfectant resistance genes, metal resistance genes, and microplastic metabolism genes.

9. The method according to claim 8, characterized in that, Compared to sentinel animals accustomed to the disturbance of the environmental pollutants, sentinel animals accustomed to the disturbance of the environmental pollutants showed an increased total relative abundance of the PHCBs in their intestines, and / or an increased ability of the environmental pollutants to affect steroid hormone metabolism, which was considered an impact of the environmental pollutants on the reproductive health of wild animals.

10. The method according to any one of claims 7 to 9, characterized in that, The PHCBs consist of the following phyla: Bacteroidota, Bacillota, and Actinomycetota.

11. The method according to claim 7, characterized in that, The environmental pollutants include: biological pesticides.

12. The method according to claim 11, characterized in that, The biological control agent includes at least one of the following: insecticide, disinfectant, antimicrobial peptide, and herbicide.

13. An apparatus for assessing the impact of human disturbance on the reproductive health of wild animals, characterized in that, include: A computer-readable storage medium having a computer program stored thereon, the program being used to perform the method according to any one of claims 1 to 6; And one or more processors, the processors being used to execute programs in the computer-readable storage medium.

14. An apparatus for assessing the impact of environmental pollutants on the reproductive health of wild animals, characterized in that, include: A computer-readable storage medium having a computer program stored thereon for performing the method according to any one of claims 7 to 12; And one or more processors, the processors being used to execute programs in the computer-readable storage medium.

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