Method for analyzing gastrointestinal food residues of euphausia superba by using molecular sequencing technology

Abstract

The invention relates to the technical field of marine biology, in particular to a method for analyzing food residues in gastrointestinal tracts of euphausia superba by a molecular sequencing technology, which comprises the following steps: dissecting the euphausia superba in a low-temperature environment, acquiring total DNA (deoxyribonucleic acid) and total RNA (ribonucleic acid) by a nucleic acid extraction kit, and realizing target gene amplification in combination with specific primer design; then bar code marking and high-throughput sequencing library construction are carried out, double-end sequencing is carried out through an Illumina NovaSeq platform, data quality control and redundancy elimination processing are completed, high-quality sequence data are obtained, accurate species identification is carried out based on multi-stage sequence alignment and a lowest common ancestor algorithm, and the accuracy of species identification is improved. According to the method, a phylogenetic tree and a network analysis method are used for analyzing diversity and nutrition level distribution of euphausia superba ingestion residues, food sources are further traced through environmental DNA comparison, the precision and range of species identification can be greatly improved, and the method has great significance in understanding energy flow and nutrition level structures of an euphausia superba ecological system.

Description

Technical Field

[0001] The invention relates to the technical field of marine biology, and in particular to a method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology. Background Art

[0002] Antarctic krill plays an important and fundamental role in the Antarctic marine ecosystem. It is not only a key food source for upper-level predators such as penguins, seals, and whales, but also plays a core role in energy flow and nutrient circulation. As global climate change has an increasingly significant impact on environmental factors in the Antarctic waters, the number and distribution of Antarctic krill populations have fluctuated to a certain extent, which has a far-reaching impact on the stability of the Antarctic food web and the sustainability of the ecosystem.

[0003] Existing research methods for Antarctic krill food residues mainly rely on microscopic morphological observation, stable isotope analysis or fatty acid labeling. These methods are usually limited in the identification of soft tissues of target organisms, discrimination of micro-predators, determination of food links, and community-level dynamics: First, microscopic observation requires a lot of time and professional experience, and the accuracy of identifying partially digested soft tissue species is insufficient; second, stable isotope and fatty acid methods can provide information on trophic levels or energy transfer pathways, but it is difficult to annotate specific species at the genus or species level; third, when faced with a wide variety of food sources, traditional methods find it difficult to track residues from different sources and compare across databases, making it impossible to systematically and completely analyze the diet of Antarctic krill. Summary of the invention

[0004] Based on the above purpose, the present invention provides a method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology.

[0005] A method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology, comprising the following steps: S1, sample collection and preservation: Antarctic krill individuals were captured from the target sea area, and were quickly dissected in a low-temperature environment (below -20°C), gastrointestinal contents were collected, and fixed with a preservation reagent without nucleic acid contamination; S2, nucleic acid extraction and quality detection: extracting total DNA and total RNA from the gastrointestinal contents using a nucleic acid extraction kit, and detecting the purity and integrity of the total DNA and total RNA using fluorescence quantitative method and gel electrophoresis; S3, PCR amplification and sequencing library construction: Based on the known taxonomic information of the food source, specific primers are designed to perform PCR amplification on the total DNA and total RNA to obtain amplified sequences, wherein the amplified sequences include target regions of nuclear genes and mitochondrial genes; Adding a barcode tag to the amplified sequence to distinguish different samples, and using a sequencing library construction reagent to perform adapter ligation and fragmentation on the amplified sequence to construct a high-throughput sequencing library, and ensuring the uniformity of the high-throughput sequencing library by fragment quality assessment; S4, high-throughput sequencing and data quality control: using the Illumina NovaSeq high-throughput sequencing platform to perform paired-end sequencing on the high-throughput sequencing library to obtain high-quality sequencing data covering the target gene region; Performing quality control on the sequencing data, including removing low-quality sequences and adaptor-contaminated sequences, and performing redundancy removal on the sequencing data after quality control to obtain valid sequencing data; S5, sequence alignment and species annotation: use alignment tools and databases to identify species of the effective sequencing data, and construct species composition data of gastrointestinal food residues based on classification tree analysis; Based on the species composition data and combined with the Antarctic ecosystem food web model, network analysis methods were used to analyze the trophic level distribution and food source diversity of gastrointestinal food residues; S6, Food source traceability: Verify the source of the species composition data of the gastrointestinal food residues through environmental DNA (eDNA) comparison analysis and evaluate the ecological contribution of specific food populations.

[0006] Optionally, the S1 includes: S11, live capture and pretreatment: Use plankton net vertical trawl to capture live Antarctic krill samples in the target waters of Antarctica, immediately transfer them to pre-cooled incubators, and transport the samples to the deck laboratory within 15 minutes; S12, construction of low-temperature dissection environment: a sterile ice bed is laid in an ISO 5 clean bench, the temperature controller is set to -25±2℃, and liquid nitrogen vapor is continuously introduced to maintain the low-temperature environment of the operation area; S13, aseptic dissection operation: using titanium dissection tools treated with DNase / RNase inactivation, the thorax of the Antarctic krill was cut open along the dorsal midline, and the gastrointestinal tissue was completely removed and placed in a pre-cooled sterile culture dish; S14, preservation reagent penetration treatment: immersing the gastrointestinal tissue in a preservation solution precooled at 4° C., and fully releasing the gastrointestinal contents under shaking conditions to obtain a gastrointestinal mixed suspension; S15, solid-liquid separation and subpackaging: filtering the obtained gastrointestinal mixed suspension through a 50 μm nylon filter membrane to obtain a filtrate, subpackaging the filtrate into 2 mL cryopreservation tubes, with the filling volume of each tube not exceeding 1.5 mL to prevent cryopreservation explosion, and obtaining subpackaging filtrate samples; S16, gradient cryopreservation: the subpackaged filtrate samples are sequentially placed in a -20°C environment for 2 hours, then transferred to a -80°C environment for 12 hours, and finally placed in a liquid nitrogen vapor phase for long-term storage; S17, metadata association record: Use QR code labels to mark the cryopreservation tubes for gradient freezing preservation, and write the sample collection location (latitude and longitude), seawater temperature (-1.8℃ to 2.5℃) and krill body length (30-60 mm) information into the blockchain evidence system.

[0007] Optionally, S2 includes: S21, sample pretreatment and lysis: transfer the aliquoted filtrate samples to -20 °C for equilibration for 2 hours, and then thaw in a 4 °C refrigerator for 1 hour; The cryovial was opened in an ultraclean workbench (ISO grade 5), and lysis buffer (pH 7.5, containing 20 mM Tris-HCl, 2% CTAB, 0.8 M NaCl) containing 1% β-mercaptoethanol was added. Zirconia beads with a diameter of 3 mm were placed in a grinder precooled with liquid nitrogen, and the mixture was ground at 1800 rpm for 3 min to obtain a homogenized lysate.

[0008] S22, complex nucleic acid adsorption purification: transfer the homogenized lysate to a DNA / RNA co-extraction centrifuge column, add equal volumes of pre-cooled chloroform-isoamyl alcohol (24:1) in sequence, shake and mix, centrifuge at 12000×g for 15 min (4°C), take the supernatant and combine it with silica-based magnetic beads for 10 minutes, then apply a magnetic field for separation and discard the waste liquid.

[0009] S23, step-by-step elution and concentration: first wash the magnetic beads twice with 80% ethanol containing 10 mM EDTA, then elute the total DNA with nuclease-free water preheated at 35°C (elution volume 50 μL), then enrich the total RNA by 70% ethanol precipitation and dissolve it in 20 μL DEPC water to obtain the purified total DNA / total RNA; S24, verification of double-strand integrity: The total DNA concentration (dsDNA HS channel) and total RNA concentration (RNA HS channel) were measured using a Qubit 4.0 fluorescent quantitative instrument, and electrophoresis analysis was performed using an Agilent 2100 bioanalyzer to evaluate the integrity of the total DNA and total RNA.

[0010] S25, antifreeze protein removal: 0.1 U / μL Antarctic krill antifreeze protease (AFP-III) was added to the total DNA solution, incubated at 37°C for 30 minutes, and then the enzymatic products were purified using AMPure XP magnetic beads.

[0011] S26, aliquot and freeze: The verified qualified nucleic acid samples are aliquoted into pre-cooled low-adsorption EP tubes, marked with batch codes, and stored in a -80°C ultra-low temperature freezer. At the same time, the electrophoresis pattern and concentration data are uploaded to the laboratory information management system and defined as qualified nucleic acid samples.

[0012] Optionally, the S3 includes: S31, Food source classification information summary: Collect and organize references and database information related to Antarctic krill feeding objects, identify common or high-proportion potential food source species, determine the conserved regions of their nuclear gene and mitochondrial gene sequences, and construct an Antarctic target gene library to provide key references for primer design; S32, primer design and verification: Based on the conserved region, use bioinformatics software to predict the specificity and annealing temperature of candidate primer sequences, select primer pairs that meet the requirements of amplification efficiency and specificity, verify the primers in vitro, and perform small-scale PCR tests using target templates and negative controls; S33, PCR reaction system configuration: weigh the qualified nucleic acid sample packaged in S26, add specific primers, DNA polymerase, dNTP mixed solution, buffer and required magnesium ion solution, adjust the concentration and volume of each component, so that the PCR reaction system is in the optimal amplification condition; S34, amplification cycle and preliminary detection: set the temperature and time of the pre-denaturation, annealing and extension steps, set the number of cycles, so that the target fragments of nuclear genes and mitochondrial genes can be effectively amplified, and the size and specificity of the PCR reaction products can be detected by agarose gel electrophoresis to confirm that clear bands are generated in the expected segment, and the product is defined as the "amplified sequence"; S35, amplified sequence purification: Use column purification or magnetic bead purification to remove enzymes, primer residues and other impurities to obtain high-purity amplified sequences, and perform fluorescent quantitative determination on the purified amplified sequences to obtain accurate concentration and quality information.

[0013] Optionally, S3 further includes: S36, dual-index barcode labeling: The purified amplified sequences were divided into independent reaction tubes, and a unique i5 / i7 dual-index combination was added to each sample using the Nextera XT Index Kit; S37, adapter cryoligation: ligating Illumina TruSeq adapters to the dual-indexed labeled products of S36; S38, antifreeze fragmentation treatment: the ligation product of S37 was fragmented using a Covaris S220 sonicator; S39, Antarctic library purification: fragmentation products were purified using AMPure XP beads (bead: sample volume ratio 0.8:1), and 300-500 bp target fragments were screened using the BluePippin system; S40, library homogenization and quality control: The purified library was quantified using Qubit 4.0, and the fragment distribution was detected using Agilent 4200 TapeStation.

[0014] Optionally, the S4 includes: S41, library dilution and denaturation: The high-throughput sequencing library that passed the quality inspection was diluted to 1.8 pM with 0.1 N NaOH solution, and 5% PhiX Control v3 library was added as an internal reference. After denaturation at 95°C for 5 minutes, it was immediately cooled in an ice bath.

[0015] S42, sequencing chip loading: load the denatured library into the designated lane of the Illumina NovaSeq 6000 S4 flow cell.

[0016] S43, paired-end sequencing run: Start the NovaSeq 6000 system and run the 2×150 bp paired-end sequencing program.

[0017] S44, raw data generation and preliminary quality control: After sequencing is completed, the raw data in FASTQ format are exported through the Illumina BaseSpace platform, and basic filtering is performed.

[0018] Optionally, S4 further includes: S45, adapter contamination filtering: Use Cutadapt (v3.4) to remove Illumina adapter residual sequences from the raw data and output the decontaminated sequencing data.

[0019] S46, low-quality sequence trimming: Trimmomatic (v0.39) was used to perform quality trimming on the decontaminated sequencing data and output quality control trimmed data.

[0020] S47, Removal of Antarctic contamination sequences: The quality control trimmed data were compared with the Antarctic eDNA contamination database (AntEco v2.1), and the exogenous contamination sequences were removed using Bowtie2 (v2.4.4). The unaligned reads were retained and defined as Antarctic-specific sequencing data.

[0021] S48, de-redundancy processing: VSEARCH (v2.18.0) was used to perform de-redundancy clustering on the Antarctic-specific sequencing data and output valid sequencing data.

[0022] Optionally, the S5 includes: S51, multi-level sequence alignment: perform BLAST alignment of the output valid sequencing data with the Antarctic target gene library (e value ≤ 1e-5), and simultaneously align to the NCBI nt library to supplement the unannotated sequences; S52, LCA taxonomic assignment: Species taxonomy was assigned using the lowest common ancestor (LCA) algorithm of MEGAN6 (v6.21); S53, classification tree construction: Based on the alignment results, RAxML (v8.2.12) was used to construct a maximum likelihood phylogenetic tree; S54, species composition data generation: Integrate the classification results with the phylogenetic tree topology, count the read abundance by classification level, and generate the species composition matrix of Antarctic krill food residues.

[0023] Optionally, S5 further includes: S55, food web model loading: calling the Antarctic ecosystem food web model from the SCAR-EcoFoodWeb database and integrating the species composition matrix; Energy transfer efficiency: 10% per trophic level; S56, network topology construction: Cytoscape (v3.9.1) was used to construct the feeding relationship network; S57, trophic level index calculation: Calculate the trophic level position of each food source based on network topology; S58, Diversity quantification and visualization: The Shannon-Wiener index and Simpson index were used to quantify the diversity of food sources, and a network heat map was generated using Gephi (v0.9.7).

[0024] Optionally, the S6 includes: S61, environmental DNA sequence matching: cross-comparison of the obtained valid sequencing data with the Antarctic environmental DNA pollution database (AntEco v2.1) and the generated species composition matrix; S62, Geographical traceability analysis: Based on the collection location information stored in S17 blockchain, the environmental DNA reference data of the corresponding sea area is extracted from the SCAR-MarBIN geographic database to build the Antarctic regional environmental DNA traceability database; S63, Quantification of ecological contribution: using linear mixed models to assess the relative contribution of target food populations.

[0025] Beneficial effects of the present invention: The present invention has designed a full chain in terms of the collection of gastrointestinal contents of Antarctic krill, cryo-dissection, nucleic acid extraction, library sequencing, data quality control, species annotation, and network analysis, which not only refines sample collection and cryo-protection, but also takes into account nucleic acid integrity and efficient extraction, ensuring high accuracy and high coverage from sample to sequencing library. Through multi-level sequence alignment and LCA classification algorithms, in-depth and precise food source identification can be achieved at the species annotation level, greatly improving the accuracy and comprehensiveness of the study of Antarctic krill feeding composition.

[0026] In the process of high-throughput sequencing and subsequent data processing, the present invention sets up strict quality control and redundancy removal mechanisms, such as aptamer contamination filtering, low-quality sequence pruning, and Antarctic eDNA contamination database comparison and elimination, to ensure the high quality and specificity of sequencing data; the non-redundant ASV / OTU table generated by VSEARCH clustering can minimize repeated noise. After the species annotation is completed, the geographical and biological distribution of suspicious or contradictory sequences is verified using authoritative databases such as SCAR-EcoFoodWeb and AntEco v2.1, forming a multivariate cross-validation system to ensure the reliability and traceability of the final conclusion.

[0027] The present invention, targeting the species composition information of Antarctic krill food residues, not only uses the maximum likelihood phylogenetic tree to perform phylogenetic hierarchical analysis of the feeding objects, but also integrates it into the SCAR-EcoFoodWeb ecological model. Through network topology construction, trophic level index calculation and diversity quantification, an in-depth analysis of the feeding structure and nutrient transfer efficiency of Antarctic krill is achieved. Further combined with environmental DNA tracing and geographic information tracing, the ecological contribution rate of key food sources can be accurately located, and the impact of marine environmental changes on the food source of Antarctic krill and the balance of the Antarctic ecosystem can be assisted in the assessment, which has important scientific research and protection management value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 A schematic diagram of a method flow of an embodiment of the present invention; Figure 2 Schematic diagram of the S1 process of an embodiment of the present invention. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternatives to implement some known technologies; and the accompanying drawings are only for more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0031] like Figure 1-Figure 2 As shown, a method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology comprises the following steps: S1, sample collection and preservation: Antarctic krill individuals are captured from the target sea area, quickly dissected in a low-temperature environment (below -20°C), gastrointestinal contents are collected, and fixed with a preservation reagent without nucleic acid contamination to ensure the integrity of DNA and RNA in the gastrointestinal contents; S2, nucleic acid extraction and quality testing: Use a nucleic acid extraction kit to extract total DNA and total RNA from the gastrointestinal contents, and use fluorescence quantification and gel electrophoresis to detect the purity and integrity of total DNA and total RNA to ensure that they are suitable for subsequent library construction and sequencing; S3, PCR amplification and sequencing library construction: Based on the known taxonomic information of the food source, specific primers are designed to perform PCR amplification on total DNA and total RNA to obtain amplified sequences, which include the target regions of nuclear genes and mitochondrial genes; Add barcode tags to the amplified sequences to distinguish different samples, and use sequencing library construction reagents to perform adapter ligation and fragmentation on the amplified sequences to construct high-throughput sequencing libraries. The uniformity of the high-throughput sequencing libraries is ensured by fragment quality assessment. S4, high-throughput sequencing and data quality control: Use the Illumina NovaSeq high-throughput sequencing platform to perform paired-end sequencing on the high-throughput sequencing library to obtain high-quality sequencing data covering the target gene region; Perform quality control on sequencing data, including removing low-quality sequences and adaptor-contaminated sequences, and performing redundancy processing on the sequencing data after quality control to obtain valid sequencing data; S5, sequence alignment and species annotation: use alignment tools and databases to identify species of valid sequencing data, and construct species composition data of gastrointestinal food residues based on classification tree analysis; Based on species composition data and combined with the Antarctic ecosystem food web model, network analysis methods were used to analyze the trophic level distribution and food source diversity of gastrointestinal food residues; S6, Food source traceability: Verify the source of species composition data of gastrointestinal food residues through environmental DNA (eDNA) comparison analysis and evaluate the ecological contribution of specific food populations.

[0032] S1 includes: S11, live capture and pretreatment: Use plankton net vertical trawling in the target Antarctic waters to capture live Antarctic krill samples, immediately transfer them to pre-cooled incubators, and transport the samples to the deck laboratory within 15 minutes, including: Trawling was carried out using a plankton net with a pore size of 500 μm, and the trawling depth was 0-200 m; The captured live krill samples were placed in a stainless steel incubator (-80°C) pre-cooled with liquid nitrogen and filled with sterile ice packs to prevent temperature fluctuations; S12, construction of low-temperature dissection environment: a sterile ice bed is laid in an ISO 5 clean bench, the temperature controller is set to -25±2℃, and liquid nitrogen vapor is continuously introduced to maintain the low-temperature environment of the operation area, including: A liquid nitrogen vapor generator (flow rate 2 L / min) was used to maintain temperature stability within the workbench; The operating table surface is covered with sterile aluminum foil and precooled to below -20°C; S13, aseptic dissection operation: Use titanium dissection tools that have been inactivated by DNase / RNase to cut open the carapace along the dorsal midline of the Antarctic krill, completely remove the gastrointestinal tissue and place it in a pre-cooled sterile culture dish, including: After processing each sample, the dissection tools were immersed in 0.1% DEPC aqueous solution for 5 minutes for inactivation; The weight of the exfoliated gastrointestinal tissue was controlled within the range of 10-50 mg to avoid spillage of the contents; S14, preservation reagent penetration treatment: immersing the gastrointestinal tissue in a 4°C precooled preservation solution, and fully releasing the gastrointestinal contents under shaking conditions to obtain a gastrointestinal mixed suspension, specifically comprising: The preservation solution consisted of the following components: 2% guanidine thiocyanate, 25 mM EDTA disodium salt, 1% sodium lauryl sarcosinate, 0.5 M Tris-HCl buffer (pH 8.0), and the volume ratio of the preservation solution to gastrointestinal tissue was 10:1 (1 mL of preservation solution was used to treat 100 mg of tissue); After shaking at 200 rpm for 30 minutes, the mixture was allowed to stand for 5 minutes to allow the undissolved shell fragments to settle to the bottom of the container; S15, solid-liquid separation and subpackaging: Filter the obtained gastrointestinal mixed suspension through a 50 μm nylon filter membrane to obtain a filtrate, and subpack the filtrate into 2 mL cryopreservation tubes. The filling volume of each tube shall not exceed 1.5 mL to prevent cryopreservation explosion. Obtain subpacked filtrate samples, specifically including: Before filtering, precool the nylon filter membrane to 4°C, and immediately freeze the filtrate (i.e., the liquid portion obtained in this step) with liquid nitrogen after filtering; Label each 2 mL cryovial with the batch number and collection time (accurate to the minute). S16, Gradient cryopreservation: The aliquoted filtrate samples are sequentially placed in a -20°C environment for 2 hours, then transferred to a -80°C environment for 12 hours, and finally placed in liquid nitrogen vapor phase for long-term storage, specifically including: When storing in liquid nitrogen vapor phase, cryovials are placed in stainless steel sleeves to avoid direct contact with liquid nitrogen; The temperature transition rate is controlled to drop by 1°C per minute, forming a gradual cooling to protect the integrity of nucleic acids; S17, metadata association record: Use QR code labels to mark the cryopreservation tubes that have been frozen in gradient storage, and write the sample collection location (latitude and longitude), seawater temperature (-1.8℃ to 2.5℃) and krill body length (30-60 mm) information into the blockchain evidence storage system, including: The blockchain uses the Hyperledger Fabric framework, and the data hash value is synchronized to the Antarctic Research Data Center in real time; The QR code label is made of low-temperature resistant polyimide and can withstand extreme environments of -196°C to 100°C.

[0033] S2 includes: S21, sample pretreatment and lysis: transfer the aliquoted filtrate samples to -20 °C for equilibration for 2 hours, and then thaw in a 4 °C refrigerator for 1 hour; Open the cryovial in an ultra-clean workbench (ISO 5), add lysis buffer (pH 7.5, containing 20 mM Tris-HCl, 2% CTAB, 0.8 M NaCl) containing 1% β-mercaptoethanol, place 3 mm diameter zirconium oxide beads in a grinder pre-cooled with liquid nitrogen, grind at 1800 rpm for 3 minutes to obtain a homogenized lysate, specifically including: The thawing process was carried out in a refrigerated environment at 4°C and the time was controlled within 2±0.5 hours to reduce nucleic acid degradation; The volume ratio of lysis buffer to sample was 5:1 (5 mL buffer for 1 mL filtrate) to ensure adequate lysis and cell disruption.

[0034] S22, complex nucleic acid adsorption purification: transfer the homogenized lysate to a DNA / RNA co-extraction centrifuge column, add equal volumes of pre-cooled chloroform-isoamyl alcohol (24:1) in sequence, shake and mix, centrifuge at 12000×g for 15 minutes (4°C), take the supernatant and combine it with silica-based magnetic beads for 10 minutes, then apply magnetic field separation and discard the waste liquid, specifically including: The centrifugal column was precooled to 4°C, and the chloroform-isoamyl alcohol mixture was prepared and used immediately to avoid decomposition of the components; The particle size of silica-based magnetic beads is 50 nm, and the binding time is accurate to 10±0.5 minutes to ensure the directional adsorption efficiency of nucleic acids.

[0035] S23, step-by-step elution and concentration: first wash the magnetic beads twice with 80% ethanol containing 10 mM EDTA, then elute the total DNA with nuclease-free water preheated at 35°C (elution volume 50 μL), then enrich the total RNA by 70% ethanol precipitation and dissolve it in 20 μL DEPC water to obtain the purified total DNA / total RNA, specifically including: The pH value of the DNA elution buffer was 8.5, and the RNA elution buffer contained 1 mM DTT to protect the RNA structure; After ethanol precipitation, centrifugation conditions were 12,000 × g, 4°C, 15 min to ensure sufficient precipitation of RNA; S24, double-strand integrity verification: The total DNA concentration (dsDNA HS channel) and total RNA concentration (RNA HS channel) were measured using the Qubit 4.0 fluorescence quantifier, and the Agilent 2100 bioanalyzer was used for electrophoresis analysis to evaluate the integrity of total DNA and total RNA, and nucleic acid samples that met the following conditions were selected: DNA integrity index (DIN) ≥7.0 and 28S / 18S rRNA ratio ≥1.8; The RNA had no degradation peak and the RIN value was ≥8.0.

[0036] S25, antifreeze protein removal: 0.1 U / μL Antarctic krill antifreeze protease (AFP-III) was added to the total DNA solution, incubated at 37°C for 30 minutes, and then the enzymatic products were purified using AMPure XP magnetic beads, including: The enzymatic reaction system contained 5 mM CaCl 2 To activate protease activity and prevent residual antifreeze proteins from affecting sequencing; After magnetic bead purification, the elution volume was 30 μL (10 mM Tris-HCl, pH 8.0) to ensure appropriate DNA concentration and purity.

[0037] S26, aliquoting and freezing: aliquot the qualified nucleic acid samples into pre-cooled low-adsorption EP tubes, mark the batch codes, and store them in -80℃ ultra-low temperature refrigerators. At the same time, upload the electrophoresis patterns and concentration data to the laboratory information management system, which are defined as qualified nucleic acid samples, including: EP pipe is made of polypropylene and pre-cooled to -20℃ to reduce high and low temperature shock; The LIMS system automatically generates a sample QC report (PDF format) containing DIN, RIN and concentration data for subsequent query and traceability.

[0038] S3 includes: S31, Food source classification information summary: Collect and organize references and database information related to Antarctic krill feeding objects, identify common or high-proportion potential food source species, determine the conserved regions of their nuclear gene and mitochondrial gene sequences, and construct an Antarctic target gene library to provide key references for primer design; S32, Primer design and validation: Based on the conserved regions, use bioinformatics software to predict the specificity and annealing temperature of candidate primer sequences, select primer pairs that meet the requirements of amplification efficiency and specificity, validate the primers in vitro, and perform small-scale PCR tests using target templates and negative controls to ensure that the primers have good amplification efficiency while excluding non-target amplification, including: Use qualified nucleic acid samples packaged in S26 as templates; Three redundant G bases were added to the 5′ end of the primer to compensate for the GC bias of the Antarctic diatom rbcL gene, and the annealing temperature was set at 58 ± 2°C; S33, PCR reaction system configuration: Weigh the qualified nucleic acid sample packaged in S26, add specific primers, DNA polymerase, dNTP mixed solution, buffer and required magnesium ion solution, adjust the concentration and volume of each component, so that the PCR reaction system is in the optimal amplification condition to avoid over-amplification or primer dimer generation, including: Prepare 10× antifreeze buffer (pH 8.3) containing 1.5 M betaine by mixing the following proportions: Antifreeze buffer 5 μL; dNTPs (2.5 mM each) 4 μL; Antarctic-specific primer pair (10 μM), 1 μL each; Hot start Taq enzyme (5 U / μL) 0.5 μL; cDNA / DNA template (≥5 ng / μL) 2 μL; MgCl 2 (25 mM) 3 μL; Make up to 50 μL with DEPC water; S34, amplification cycle and preliminary detection: set the temperature and time of the pre-denaturation, annealing and extension steps, set the number of cycles, so that the target fragments of nuclear genes and mitochondrial genes can be effectively amplified, and the size and specificity of the PCR reaction products can be detected by agarose gel electrophoresis to confirm that clear bands are generated in the expected segment, and the product is defined as the "amplified sequence", which includes: Run program: 95°C pre-denaturation for 5 minutes → 35 cycles (95°C for 30 seconds → 58°C for 30 seconds → 72°C for 45 seconds) → 72°C final extension for 10 minutes; Use nucleic acid samples that have passed S24 verification as positive controls; S35, amplified sequence purification: Use column purification or magnetic bead purification to remove enzymes, primer residues and other impurities to obtain high-purity amplified sequences. Perform fluorescence quantitative determination on the purified amplified sequences to obtain accurate concentration and quality information, providing a basis for subsequent barcode labeling and library construction. Specifically include: Purification was performed using S25 AMPure XP magnetic beads (bead: sample volume ratio 0.8:1); The concentration was determined using the Qubit 4.0 for S24 (≥10 ng / μL) and the A260 / A280 ratio was required to be ≥1.8.

[0039] S3 also includes: S36, dual-index barcode labeling: The purified amplicon sequences were aliquoted into separate reaction tubes and a unique i5 / i7 dual-index combination (8 bp) was added to each sample using the Nextera XT Index Kit, including: Index Add Reaction System: Purified amplified sequence (≥10 ng / μL) 5 μL; i5 index primer (10 μM) 1 μL; i7 index primer (10 μM) 1 μL; T4 DNA ligase (5 U / μL) 0.5 μL; 10× ligation buffer (containing 0.5 M NaCl) 2 μL; Make up to 20 μL with DEPC water; Reaction conditions: incubation at 20°C for 15 minutes, followed by inactivation at 65°C for 10 minutes; S37, low temperature ligation of adapters: Ligation of Illumina TruSeq adapters to the dual indexed labeled products of S36, including: Ligation reaction system: 15 μL of double-indexed labeled product; TruSeq adaptor (15 μM) 2 μL; Antarctic optimized ligase (3 U / μL) 1 μL; 5× low temperature ligation buffer (containing 1 M betaine) 4 μL; Make up to 30 μL with DEPC water; Reaction conditions: 16°C for 20 min, 1:1 molar ratio of aptamer to labeled product; S38, antifreeze fragmentation treatment: The ligation product of S37 was fragmented using a Covaris S220 sonicator, specifically including: Fragmentation parameters: Peak energy: 5 W; Action time: 45 seconds; Number of cycles: 200 times; Precooling of reaction tubes: Precooling with liquid nitrogen for 30 seconds to maintain an operating environment at 4°C; Target fragment length: 350 ± 50 bp; S39, Antarctic library purification: Fragmentation products were purified using AMPure XP beads (bead: sample volume ratio 0.8:1) and screened for 300-500 bp target fragments using the BluePippin system, including: Purification conditions: Washing solution: 80% ethanol (containing 10 mM EDTA, pH 8.0); Elution buffer: 10 mM Tris-HCl (pH 8.5); Elution volume: 30 μL; Screening parameters: BluePippin selected 2% Agarose Cassette, cutoff range 300-500 bp; S40, library homogenization and quality control: The purified library was quantified using Qubit 4.0 and the fragment distribution was detected by Agilent 4200 TapeStation, including: Homogenization Standard: Library concentration: 4 nM ± 5% (error range); The main peak ratio is ≥90%, and the CV value is ≤10%; The proportion of aptamers is less than 1%; Quality inspection failure processing: If the quality inspection fails to meet the standards, repeat steps S38-S39 to adjust the fragmentation parameters.

[0040] S4 includes: S41, library dilution and denaturation: Dilute the qualified high-throughput sequencing library with 0.1 N NaOH solution to 1.8 pM, add 5% PhiX Control v3 library as internal reference, denature at 95°C for 5 minutes and immediately cool in an ice bath, including: Diluent: HT1 buffer (Illumina) containing 0.1% Tween-20; Internal reference ratio: 5% PhiX is used to monitor the sequencing error rate.

[0041] S42, sequencing chip loading: Load the denatured library into the designated lane of the Illumina NovaSeq 6000 S4 flow cell, including: Loading volume: 600 μL per lane (concentration 1.8 pM); Loading temperature: Maintain the flow cell temperature at 20 ± 0.5 °C.

[0042] S43, paired-end sequencing run: Start the NovaSeq 6000 system and run the 2×150 bp paired-end sequencing program, including: Sequencing parameters: Read1 length: 150 cycles; Index1 length: 8 cycles (read i7 index); Index2 length: 8 cycles (read i5 index); Read2 length: 150 cycles; Quality monitoring: real-time tracking of cluster density (target range: 200-300 K / mm²).

[0043] S44, raw data generation and preliminary quality control: After sequencing is completed, the raw data in FASTQ format is exported through the Illumina BaseSpace platform and basic filtering is performed, including: Filter by: Sequences with N content > 5% in reads were removed; Reads with a mean Phred quality score ≥ 30 were retained; Output file: Paired-end sequencing raw data (file naming: SampleID_R1.fastq.gz / SampleID_R2.fastq.gz).

[0044] S4 also includes: S45, adapter contamination filtering: Use Cutadapt (v3.4) to remove Illumina adapter residual sequences from the raw data and output the decontaminated sequencing data, including: Parameter settings: Maximum error rate: 0.1; Minimum match length: 5 bp; Adaptamer sequence: TruSeq Universal / Indexed aptamer (SEQ ID NO: 11-12); Output file: decontaminated sequencing data.

[0045] S46, low-quality sequence trimming: Trimmomatic (v0.39) is used to perform quality trimming on the decontaminated sequencing data and output quality control trimmed data, including: Pruning rules: Sliding window: 4 bp window, average quality ≥ 20; Remove bases with the first and last quality <Q20; Minimum retention length: 50 bp; Output file: QC trimmed data.

[0046] S47, Antarctic contamination sequence removal: The quality control trimmed data were compared with the Antarctic eDNA contamination database (AntEco v2.1), and exogenous contamination sequences were removed using Bowtie2 (v2.4.4). Unaligned reads were retained and defined as Antarctic-specific sequencing data, including: Comparison parameters: Mode: --very-sensitive Maximum mismatch: 3 Unaligned reads were retained and defined as Antarctic-specific sequencing data.

[0047] S48, de-redundancy processing: Use VSEARCH (v2.18.0) to perform de-redundancy clustering on the Antarctic-specific sequencing data and output valid sequencing data, including: Parameter settings: Similarity threshold: 97%; Maximum allowed chimerism rate: 1%; Reference database: Antarctic target gene library of S31; Output file: valid sequencing data (non-redundant ASV / OTU table).

[0048] S5 includes: S51, multi-level sequence alignment: perform BLAST alignment (e value ≤ 1e-5) on the output valid sequencing data and the Antarctic target gene library, and simultaneously align to the NCBI nt library to supplement the unannotated sequences, including: Alignment tools: BLASTn (v2.12.0) for nuclear genes and BLASTx (v2.12.0) for mitochondrial genes; Parameter settings: Nuclear genes (18S / ITS2): maximum number of matches 100, coverage ≥ 90%; Mitochondrial gene (COI / 16S): consistency ≥97%; S52, LCA taxonomic assignment: Assign species taxonomy using the lowest common ancestor (LCA) algorithm of MEGAN6 (v6.21), specifically: Classification threshold: Phylum / class level: support read number ≥ 5 and relative abundance > 0.1%; Genus / species level: support read number ≥ 10 and consistency ≥ 99%; Eliminate species that do not match Antarctic biogeographic distributions (e.g. tropical plankton); S53, classification tree construction: Based on the alignment results, RAxML (v8.2.12) was used to construct a maximum likelihood phylogenetic tree, including: Model selection: GTR+GAMMA model (suitable for highly variable genes in Antarctica); Bootstrap value: 1000 repetitions, node support rate ≥ 70%; Output file: Antarctic food source phylogenetic tree (Newick format); S54, species composition data generation: Integrate the classification results with the phylogenetic tree topology, count the read abundance by classification level, and generate the species composition matrix of Antarctic krill food residues, including: Matrix dimensions: sample (row) × species (column), value is the number of normalized reads (CPM); Data format: CSV file (including species classification information and confidence scores).

[0049] The S5 also includes: S55, food web model loading: call the Antarctic ecosystem food web model from the SCAR-EcoFoodWeb database and integrate the species composition matrix, including: Model parameters: Basic data of trophic levels: phytoplankton (level 1), krill (level 2), penguins / cetaceans (level 3); Energy transfer efficiency: 10% per trophic level; S56, Network topology construction: Use Cytoscape (v3.9.1) to build a feeding relationship network, including: Node definition: Food source node: species in the S54 matrix; Krill node: central node, connecting all ingested species; Edge weight: log10 transformed value of normalized read abundance (CPM); S57, trophic level index calculation: Calculate the trophic level position of each food source based on network topology, including: Calculation formula: ; in, is the trophic level of species i, is the feeding weight of krill on species j; Iteration convergence condition: trophic level change < 0.01 or maximum number of iterations 50 times; S58, Diversity Quantification and Visualization: The Shannon-Wiener index and Simpson index were used to quantify the diversity of food sources, and a network heat map was generated through Gephi (v0.9.7), specifically including: Index formula: Shannon-Wiener: ; Simpson: ; Among them, is the relative abundance of species i; Visualization output: Antarctic krill feeding ecological map (PDF / PNG format).

[0050] S6 includes: S61, Environmental DNA Sequence Matching: The obtained valid sequencing data was cross-compared with the Antarctic environmental DNA pollution database (AntEco v2.1) and the generated species composition matrix, specifically including: Alignment tool: VSEARCH (v2.18.0), parameter settings: Similarity threshold: 97%; Minimum matching length: 100 bp; Sequences matching non-Antarctic sources such as humans and ship equipment were removed; Output file: Antarctic local food source sequence set; S62, Geographic Traceability Analysis: Based on the collection location information stored in the S17 blockchain, environmental DNA reference data for the corresponding sea area was extracted from the SCAR-MarBIN geographic database to construct an Antarctic regional environmental DNA traceability database, specifically including: Data integration: Matching latitude range: ±0.5° (centered on the collected longitude and latitude); Matching depth range: ±50 meters (centered on the trawling depth); Analysis method: Calculate the Bray-Curtis similarity index to quantify the species composition consistency between krill food residues and local eDNA; Use SIMPER analysis (similarity percentage decomposition) to identify key contributing species; S63, Ecological Contribution Quantification: A linear mixed model was used to evaluate the relative contribution rate of the target food population, specifically including: Model parameters: Fixed effects: species abundance (CPM), trophic level position (TL); Random effects: collection sea area, season (metadata from S17); Output results: Report on the contribution rate of key food sources (in CSV / PDF format), marking the ecological weights of species such as diatoms and copepods; Visually generate a heat map of the feeding traceability of Antarctic krill (using R ggplot2, PDF resolution ≥ 300 dpi).

[0051] This invention covers any alternatives, modifications, equivalent methods, and solutions made within the essence and scope of this invention. To enable the public to have a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments of this invention. However, those skilled in the art can fully understand this invention even without the description of these details. Additionally, well-known methods, processes, procedures, components, and circuits, etc., are not described in detail to avoid unnecessary confusion to the essence of this invention.

[0052] The above are only the preferred embodiments of this invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of this invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as within the protection scope of this invention.

Claims

1. A method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology, characterized in that: The following steps are involved: S1, sample collection and preservation: Antarctic krill individuals were captured from the target sea area, quickly dissected in a low-temperature environment, gastrointestinal contents were collected, and fixed with a preservation reagent without nucleic acid contamination; S2, nucleic acid extraction and quality detection: extracting total DNA and total RNA from the gastrointestinal contents using a nucleic acid extraction kit, and detecting the purity and integrity of the total DNA and total RNA using fluorescence quantitative method and gel electrophoresis; S3, PCR amplification and sequencing library construction: Based on the known taxonomic information of the food source, specific primers are designed to perform PCR amplification on the total DNA and total RNA to obtain amplified sequences, wherein the amplified sequences include target regions of nuclear genes and mitochondrial genes; Adding a barcode tag to the amplified sequence to distinguish different samples, and using a sequencing library construction reagent to perform adapter ligation and fragmentation on the amplified sequence to construct a high-throughput sequencing library, and ensuring the uniformity of the high-throughput sequencing library by fragment quality assessment; S4, high-throughput sequencing and data quality control: using the Illumina NovaSeq high-throughput sequencing platform to perform paired-end sequencing on the high-throughput sequencing library to obtain sequencing data covering the target gene region; Performing quality control on the sequencing data, including removing low-quality sequences and adaptor-contaminated sequences, and performing redundancy removal on the sequencing data after quality control to obtain valid sequencing data; S5, sequence alignment and species annotation: use alignment tools and databases to identify species of the effective sequencing data, and construct species composition data of gastrointestinal food residues based on classification tree analysis; Based on the species composition data and combined with the Antarctic ecosystem food web model, network analysis methods were used to analyze the trophic level distribution and food source diversity of gastrointestinal food residues; S6, Food source tracing: Through environmental DNA comparison analysis, verify the source of the species composition data of the gastrointestinal food residues and evaluate the ecological contribution of specific food populations.

2. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 1, characterized in that: The S1 includes: S11, live capture and pretreatment: live samples of Antarctic krill are captured using vertical trawl nets in the target Antarctic waters, transferred to pre-cooled incubators, and transported to the deck laboratory; S12, construction of low-temperature dissection environment: a sterile ice bed is laid in an ISO 5 clean bench, and liquid nitrogen vapor is continuously introduced to maintain the low-temperature environment of the operation area; S13, aseptic dissection operation: using inactivated titanium dissection tools, cut open the thorax along the dorsal midline of the Antarctic krill, completely remove the gastrointestinal tissue and place it in a pre-cooled sterile culture dish; S14, preservation agent penetration treatment: immersing the gastrointestinal tissue in a pre-cooled preservation solution, and fully releasing the gastrointestinal contents under shaking conditions to obtain a gastrointestinal mixed suspension; S15, solid-liquid separation and subpackaging: filtering the obtained gastrointestinal mixed suspension through a nylon filter membrane to obtain a filtrate, and subpackaging the filtrate into cryopreservation tubes to obtain subpackaging filtrate samples; S16, gradient cryopreservation: the subpackaged filtrate samples are sequentially placed in a -20°C environment for 2 hours, then transferred to a -80°C environment for 12 hours, and finally placed in a liquid nitrogen vapor phase for long-term storage; S17, metadata association record: Use QR code labels to mark the cryotubes that have completed gradient freezing preservation, and write the sample collection location, seawater temperature and krill body length information into the blockchain evidence system.

3. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 2, characterized in that: The S2 includes: S21, sample pretreatment and lysis: transfer the aliquoted filtrate samples to -20 °C for equilibration for 2 hours, and then thaw in a 4 °C refrigerator for 1 hour; Open the cryotube in the clean bench, add lysis buffer containing 1% β-mercaptoethanol, place zirconium oxide beads in a grinder pre-cooled with liquid nitrogen, and grind for 3 minutes to obtain a homogenized lysate; S22, complex nucleic acid adsorption purification: transfer the homogenized lysate to a DNA / RNA co-extraction centrifuge column, add equal volumes of pre-cooled chloroform-isoamyl alcohol in sequence, shake and mix, centrifuge for 15 minutes, take the supernatant and combine it with silica-based magnetic beads for 10 minutes, then apply a magnetic field for separation and discard the waste liquid; S23, step-by-step elution and concentration: first wash the magnetic beads twice with ethanol, then elute the total DNA with preheated nuclease-free water, then enrich the total RNA by ethanol precipitation and dissolve it in DEPC water to obtain the purified total DNA / total RNA; S24, double-strand integrity verification: using a fluorescence quantification instrument to measure the total DNA concentration and the total RNA concentration, and using a bioanalyzer to perform electrophoresis analysis to evaluate the integrity of the total DNA and total RNA; S25, antifreeze protein removal: Antarctic krill antifreeze protease was added to the total DNA solution and incubated for 30 minutes, and then the enzymatic products were purified and removed using AMPureXP magnetic beads; S26, aliquoting and freezing: The verified qualified nucleic acid samples are aliquoted into pre-cooled low-adsorption EP tubes, marked with batch codes, and stored in ultra-low temperature refrigerators. At the same time, the electrophoresis spectrum and concentration data are uploaded to the laboratory information management system and defined as qualified nucleic acid samples.

4. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 3, characterized in that: The S3 includes: S31, Food source classification information summary: Collect and organize references and database information related to Antarctic krill feeding objects, identify common or high-proportion potential food source species, determine the conserved regions of their nuclear gene and mitochondrial gene sequences, and construct an Antarctic target gene library to provide key references for primer design; S32, primer design and verification: Based on the conserved region, use bioinformatics software to predict the specificity and annealing temperature of candidate primer sequences, select primer pairs that meet the requirements of amplification efficiency and specificity, verify the primers in vitro, and perform small-scale PCR tests using target templates and negative controls; S33, PCR reaction system configuration: weigh the qualified nucleic acid sample packaged in S26, add specific primers, DNA polymerase, dNTP mixed solution, buffer and required magnesium ion solution, adjust the concentration and volume of each component, so that the PCR reaction system is in the optimal amplification condition; S34, amplification cycle and preliminary detection: set the temperature and time of the pre-denaturation, annealing and extension steps, set the number of cycles, so that the target fragments of nuclear genes and mitochondrial genes can be effectively amplified, and the size and specificity of the PCR reaction products can be detected by agarose gel electrophoresis to confirm that clear bands are generated in the expected segment, and the product is defined as the "amplified sequence"; S35, amplified sequence purification: Use column purification or magnetic bead purification to remove enzymes, primer residues and other impurities to obtain high-purity amplified sequences, and perform fluorescent quantitative determination on the purified amplified sequences to obtain accurate concentration and quality information.

5. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 4, characterized in that: The S3 further includes: S36, dual-index barcode labeling: The purified amplified sequences were divided into independent reaction tubes, and a unique i5 / i7 dual-index combination was added to each sample using the Nextera XT Index Kit; S37, adaptor cryo-ligation: ligating Illumina TruSeq adaptors to the dual-indexed labeled products of S36; S38, antifreeze fragmentation treatment: the ligation product of S37 was fragmented using a Covaris S220 sonicator; S39, Antarctic library purification: fragmentation products were purified using AMPure XP magnetic beads and 300-500 bp target fragments were screened using the BluePippin system; S40, library homogenization and quality control: The purified library was quantified using Qubit 4.0, and the fragment distribution was detected using Agilent 4200 TapeStation.

6. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 5, characterized in that: The S4 includes: S41, library dilution and denaturation: The high-throughput sequencing library that passed the quality inspection was diluted to 1.8 pM with 0.1 N NaOH solution, and 5% PhiX Control v3 library was added as an internal reference. The library was denatured at 95°C for 5 minutes and then cooled in an ice bath. S42, sequencing chip loading: the denatured library was loaded into the designated lane of the Illumina NovaSeq 6000 S4 flow cell; S43, paired-end sequencing run: start the NovaSeq 6000 system and run the 2×150 bp paired-end sequencing program; S44, raw data generation and preliminary quality control: After sequencing is completed, the raw data in FASTQ format are exported through the Illumina BaseSpace platform, and basic filtering is performed.

7. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 6, characterized in that: The S4 further comprises: S45, adapter contamination filtering: Use Cutadapt to remove Illumina adapter residual sequences from the raw data and output the decontaminated sequencing data; S46, low-quality sequence trimming: Trimmomatic is used to perform quality trimming on the decontaminated sequencing data and output quality control trimmed data; S47, Antarctic contamination sequence removal: The quality control trimmed data were compared with the Antarctic eDNA contamination database, and the exogenous contamination sequences were removed using Bowtie2, and the unaligned reads were retained, which were defined as Antarctic-specific sequencing data; S48, de-redundancy processing: Use VSEARCH to perform de-redundancy clustering on the Antarctic-specific sequencing data and output valid sequencing data.

8. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 7, characterized in that: The S5 includes: S51, multi-level sequence alignment: perform BLAST alignment of the output valid sequencing data with the Antarctic target gene library; S52, LCA taxonomic assignment: Assign species taxonomy using the lowest common ancestor algorithm of MEGAN6; S53, classification tree construction: Based on the alignment results, RAxML was used to construct a maximum likelihood phylogenetic tree; S54, species composition data generation: Integrate the classification results with the phylogenetic tree topology, count the read abundance by classification level, and generate the species composition matrix of Antarctic krill food residues.

9. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 8, characterized in that: The S5 further includes: S55, food web model loading: calling the Antarctic ecosystem food web model from the SCAR-EcoFoodWeb database and integrating the species composition matrix; S56, network topology construction: use Cytoscape to build a feeding relationship network; S57, trophic level index calculation: Calculate the trophic level position of each food source based on network topology; S58, Diversity quantification and visualization: The Shannon-Wiener index and Simpson index were used to quantify the diversity of food sources, and a network heat map was generated using Gephi.

10. The method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology according to claim 8, characterized in that: The S6 includes: S61, Environmental DNA sequence matching: cross-matching the obtained valid sequencing data with the Antarctic environmental DNA pollution database and the generated species composition matrix; S62, Geographical traceability analysis: Based on the collection location information stored in S17 blockchain, the environmental DNA reference data of the corresponding sea area is extracted from the SCAR-MarBIN geographic database to build the Antarctic regional environmental DNA traceability database; S63, Quantification of ecological contribution: using linear mixed models to assess the relative contribution of target food populations.

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