Metabarcoding amplicon of gut microbiome from Daphnia galeata
At the end of the experiment, the D. galeata were pooled (n = 20), and three biological replicates per experimental condition (control and fish kairomones exposed) were used, resulting in a total of 120 guts (20 individuals  three biological replicates  two conditions). The gut extraction from D. galeata was performed following the method of Akbar et al. (2021). The individuals cultured under both conditions were placed in fresh sterile ADaM medium for 24 hours to remove food particles and non-symbiotic bacteria from the gut. Subsequently, individuals were gently washed with sterile water to remove the external bacterial communities and then dissected under a stereoscopic microscope to isolate their guts. Afterward, the DNA was immediately extracted from the guts of all experimental replicates. The DNA extraction was performed using the Qiagen DNeasy blood and tissue kit (Qiagen, Hilden, Germany), according to the manufacturers procedure. Total DNA was stored at 20 C for further analysis. Then, polymerase chain reaction (PCR) was carried out using total DNA samples as the template. The V3-V4 hypervariable regions of partial 16S rRNA gene, with amplicons of around 459 base pairs, were amplified using the primer pairs S-D-Bact-0341-b-S-17 (5'- CCT ACG GGN GGC WGC AG-3') and S-D-Bact-0785-a-A-21 (5'- GAC TAC HVG GGT ATC TAA TCC-3'). PCR reactions consisted of 1 L DNA (50 ng/L), TOPsimple PCR DyeMIX-Tenuto 10 L (0.2 U/L) (Enzynomics, Daejeon, Korea), 1 L each of forward and reverse primers (10 pmol/L), and 7L of distilled water. To amplify sequences from various taxa, gradient PCR was performed by dividing the annealing temperature into 12 temperatures between 40C and 60C. Reaction cycling conditions were as follows: initial denaturation at 95 for 2 min, 35 cycles of 30 s at 95, 45 s at 40 - 60, 45 s at 72, and a final extension of 5 min at 72. PCR negative controls were included in each PCR to check for potential contamination. Amplified PCR products were then visualized by gel electrophoresis to verify band size, and then gradient PCR products were pooled in the same quantity before library construction. The sequencing libraries were prepared using TruSeq Nano DNA High Throughput Library Prep Kit (Illumina, CA, USA). The library was measured for quality and quantity using the Agilent Technologies 4200 TapeStation D1000 screentape (Agilent Technologies, California, USA), and sequenced using an Illumina MiSeq sequencing platform with 300 base pair (bp) paired-end reads.