Biodiversity and distribution of zoobenthos in the ecological water replenishment area of the Yellow River estuary coastal wetland revealed by eDNA metabarcoding

2.3. Physicochemical parameters analysis The life of zoobenthos is closely related to the surrounding water environment; therefore, the physicochemical indices of the collected water samples were analyzed with emphasis [26]. Part of the physicochemical indices were measured in situ. For example, dissolved oxygen (DO), oxidation-reduction potential (ORP), and temperature (T) were determined using a portable DO meter (HQ40d, Hach, Colorado, USA). Electrical conductivity (EC), total dissolved solids (TDS), salinity (SAL) and pH were measured using an EC meter (DDB-303A, Leici, Shanghai, China), a TDS meter (WQM01H-3, Bit Atom, Shenzhen, China), a SAL meter (WS-100, Bingmu, Shenzhen, China) and a pH meter (pH-100, Lichen, Shanghai, China), respectively. The total organic carbon (TOC) content was measured using a TOC analyzer (TOC-L, Daojin, Kyoto, Japan). The total phosphorus (TP) and ammonium ion (NH4+) contents were deter- mined using a UV-Visible spectrophotometer (UV1800, Shimadzu, Japan) in accordance with standard procedures (APHA, 2005). The sulfate ion (SO42−), sodium ion (Na+), magnesium ion (Mg2+), potassium ion (K+), and calcium ion (Ca2+) concentrations were quantified using an ion chromatography analyzer (HD-TLS51, HORDE, Shandong, China). 2.4. DNA extraction and sequencing Studies have demonstrated that directly extract sediment eDNA can reliably characterize zoo- benthic diversity and community composition [27,28]. Sediment eDNA was extracted from approximately 0.25 g of sediment for each sample using the E.Z.N.A.1 Soil DNA Kit (Omega

Bio-tek, Norcross, GA, USA) according to the manufacturer’s protocols [27]. To ensure robustness, triplicate subsamples were analyzed independently for each site. The V4 regions of the zoobenthos 18S ribosomal RNA gene were amplified by PCR (95˚C for 2 min, followed by 25 cycles of 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s, with a final extension at 72˚C for 5 min) using primers F04mod F: 5’-GCTTGWCTCAAAGATTAAGCC-3’ and F04mod R: 5’-CCT GCTGCCTTCCTTDGA-3’. Each sample was tagged with a unique eight-base barcode sequence [27]. PCR reactions were performed in triplicate in 20 μL mixtures, each containing 4 μL of 5× FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Poly- merase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and puri- fied using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) following the manufacturer’s instructions [24]. To prevent sample DNA contamination, equipment such as forceps, scissors, pipettes, and measuring spoons were cleaned with 10% bleach and rinsed with ultrapure water before and between each use. To avoid laboratory con- tamination, separate rooms were used for pre- and post-PCR processes [29]. Purified PCR products were quantified using a Qubit1 3.0 (Life Technologies, Invitrogen). Twenty-four amplicons with unique barcodes were pooled in equal amounts. This pooled DNA was used to construct an Illumina paired-end library according to Illumina’s genomic DNA library prepa-

ration protocol. The amplicon library was sequenced (2 × 250 bp) on an Illumina MiSeq plat- form (Shanghai BIOZERON Co., Ltd) following standard protocols. Raw FASTQ files were demultiplexed using in-house Perl scripts based on barcode sequences for each sample, following these criteria: (i) reads were truncated at any site with an average quality score below 20 over a 10 bp sliding window, and truncated reads shorter than 50 bp were discarded. (ii) exact barcode matching was required, allowing for a 2-nucleotide mismatch in primer matching; reads with ambiguous characters were removed. (iii) only sequences with overlaps longer than 10 bp were assembled; non-assembled reads were dis- carded. A reference database was generated using ECOPCR by extracting sequences from the EMBL database (version 143) with F04mod primers and the NCBI classification database. Ini- tial taxonomic assignments were made using ECOTAG [30]. Each assigned zoobenthic PLOS ONE Biodiversity and zoobenthos distribution in yellow river estuary wetlands revealed by eDNA metabarcoding PLOS ONE | https://doi.org/10.1371/journal.pone.0315346 December 18, 2024 5 / 19