2.4.4. Taxonomic Diversity of Gut Microbiota

The DNA was isolated from the microorganisms, inhabiting the intestinal tract of *Daphnia* from all replications of the experiment. The isolated DNA was then mixed v/v to obtain a representative sample and was then sequenced. In order to analyse the microbiota, we used a standard Miseq illumine sequencing method used in numerous previous studies, e.g., in [62–65]. The *D. magna*, the digestive tracts were collected in 1.5 mL Eppendorf and stored at −20 ◦C. Afterwards, the DNA extraction was performed in the spin-column-based method using the GeneMATRIX DNA Purification kit (EurX, Gda ´nsk, Poland), according to the manufacturer's procedure. The total DNA was assessed for its quality and quantity by the absorbance measurement in a Synergy H1 microplate reader (Gen5 software, BioTek, Broadview, IL, USA) equipped with a Take3 microvolume plate. We then stored the samples at −20 ◦C for further analysis. We performed the phylogenetic analysis of the bacterial community using Illumina sequencing method [66]. The 16S rRNA genes, V3–V4 hypervariable regions (amplicons of approximately 459 bp), were selected. The PCR amplification was carried out using a Q5 Hot Start High-Fidelity 2X Master Mix, using reaction conditions as recommended by the manufacturer (95 ◦C for 3 min, 25 cycles of 95 ◦C for 30 s, 55 ◦C for 30 s, 72 ◦C for 30 s, and, after the last cycle, 72 ◦C for 5 min) with region-specific (341F and 785R) [67] primers that include the Illumina flow cell adapter sequences. The primer sequence was as follows: the forward primer was 5 CGGGNGGCWGCAG 3 and the reverse primer was 5 GACTACHVGGGTATCTAATCC 3 . The Illumina overhang adapter sequences added to the locus-specific sequences were as follows: the forward overhang was 5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG (locus-specific sequence) and the reverse overhang was 5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (locus-specific sequence).

The amplicons were sequenced using a MiSeq (Illumina, San Diego, CA, USA) platform on a single run using the MiSeq Reagent Kit v2 (Illumina, San Diego, CA, USA) and the paired-end method (2 × 300 bp), according to the standard protocols by Genomed (Warsaw, Poland). A demultiplexing and trimming of the Illumina adapter sequences (cutadapt software [68]) were performed. FastQC (https://www.bioinformatics.babraham. ac.uk/projects/fastqc; acecssed on 4 October 2018) and MultiQC [69] were used to achieve the quality inspection, visualisation, and assessment of the raw FASTQ files. The sequences were processed using the DADA2 plugin within QIIME 2 [70]. We trimmed the sequence at 270 nt, while the first 8 nt were truncated. The alpha rarefaction plots confirmed that

the number of the remaining sequences is sufficient for detecting the current microbial diversity. Taxonomies were assigned to the resulting amplicon sequence variants (ASV) with a q2-feature-classifier plug-in using a pre-trained Naive Bayes classifier based on a 16S rRNA silva 138 SILVA SSU gene database at 99% similarity. The core diversity metrics pipeline was the tool for calculating the phylogenetic and non-phylogenetic core diversity metrics. Data for this purpose were rarefied to a sampling-depth equal to the lowest frequency among the samples (23,500 reads).
