Control_1
Experimental setup of crayfish exposure to antibiotics was performed as previously reported by Hernndez-Prez et al. (2020) and can be seen in supplementary figure 1A. Briefly, fresh stocks of sulfamethoxazole (SMX) (Sigma-Aldrich, CAS: 723-46-6) was prepared following the standard recommendation from Clinical and Laboratory Standards Institute (CLSI), (2012). Then, test solutions at 100 ng/L and 1ug/L for SMX  were prepared from the stock solutions and used for the exposure assays. For exposure assays, lales freshwater crayfish, Pacifastacus leniusculus (mean weight: 37  2 g) were obtained from Lake Erken in Sweden, transported to our lab facilities, and kept in aquaria with aerated clean tap water at 10-12C . The antibiotic exposure started when the animals had been kept in tanks with aerated water for about 2 months (eg. 6-9 weeks) after arrival from the lake. Then, crayfish were exposed to concentrations of 100 ng/L and also to a higher dose of 1 g/L of SMX. In all experiments, the water was renewed every second day. For the antibiotic-treated groups, a freshly prepared antibiotic solution was added to the water every time the water was changed. During all experiments, the photoperiod was maintained at 12:12 h light:dark, and animals were fed with carrots and potatoes once a week with 3% of its live weight. After antibiotics exposure, crayfish were euthanized by decapitation and the gut from midgut to rectum was collected, rinsed with cold CPBS (10 mM Na2HPO4, 10 mM KH2PO4, 150 mM NaCl, 10 mM CaCl2, and 10 mM MnCl2, pH 6.8), and immediately stored at 80C until DNA extraction. DNA was extracted from each sample using the QIAamp DNA Stool Mini Kit (QIAGEN). PCR inhibitors were removed from purified DNA using the kit DNeasy PowerClean Pro Cleanup (QIAGEN). The purity of DNA obtained was determined by an A260/A280 nm ratio using a spectrophotometer and was within the expected range (1.8  2) for all samples. For microbiota analysis, the 16S rRNA gene was amplified following Vass et al., (2020) approach, using primers 341F and 804NR (Herlemann et al., 2011). The detailed two-step PCR procedure followed is available in the protocols.io repository (Vass and Szkely, 2020) (dx.doi.org/10.17504/protocols.io.468gzhw). Amplicons were sequenced at the SciLifeLab SNP&SEQ Technology Platform hosted by Uppsala University using Illumina MiSeq v3 sequencing chemistry.