Metataxomics of bacterial communties: Steatoda grossa opisthosoma
The study was carried out on adult S. grossa females originating from a multigenerational laboratory experimental colony whose individuals had no prior exposure to contaminants. A simple food chain model was designed, which included an uncontaminated (control) medium or a medium contaminated with metal salts: CdCl2 (0.25 mM) or CuSO4 (0.23 mM)  D. hydei flies  adult S. grossa females. Exposure to metals via the oral route lasted for 4 weeks and 12 months, with sublethal concentrations applied to both the prey and the spider S. grossa. For the study, individuals were starved for a week to clear the digestive tract of lingering food debris and reduce microbial populations present in the victims body. Two spiders from each control group and four spiders from the groups exposed to heavy metals were selected for the microbiome analysis as biological replicates. Totally, 20 DNA libraries were analyzed. After individuals were kept in the fridge for 1 h, their outer body surfaces were sterilized for 2 min in ethanol and washed with sterile water. Limbs and prosoma were then removed, and the entire opisthosoma was homogenized in a mortar. DNA isolation was performed using the QIAamp DNA Microbiome Kit (Qiagen) following the manufacturers protocol, and the DNA was suspended in EB buffer. The samples of genomic DNA (gDNA) were further purified using the Monarch Genomic DNA Purification Kit (NEB) following the manufacturers protocol. The quality of the gDNA was assessed using D1000 Screen Tapes (Agilent) and the bioanalyzer Tape Station 2200 (Agilent) and then stored at 20C until further use. DNA libraries for microbiome sequencing were prepared according to the 16S Metagenomic Sequencing Library Preparation available online https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf. The hypervariable V3V4 region of the 16S rRNA gene was amplified, creating the amplicon of approximately 460 bp using primers with adapter overhang sequences (Klindworth et al., 2013). Dual index tags were then added to these primers to differentiate samples in a single sequencing run. Both amplification reactions were carried out with 2 KAPA HiFi HotStart ReadyMix (Roche) on the thermocycler SureCycler 8800 (Agilent Technologies) following Illuminas protocol. After each step, amplicons and libraries were purified using magnetic beads Agencourt AMPure XP Beads (Beckman Coulter Life Science). The libraries were qualified and quantified using D1000 Screen Tapes on the bioanalyzer Tape Station 2200 (Agilent) and the Qubit dsDNA high sensitivity (Thermo Fisher Scientific) kit on Qubit 3.0 (Thermo Fisher Scientific) according to the respective manufacturers instructions. Libraries from each sample were pooled in equimolar ratios and sequenced (2  250) according to standard Illumina protocol on the Illumina MiSeq platform (Illumina).