Lysing matrix e beads

Small intestinal samples were homogenized in Cell Lytic MT buffer (Sigma) using Lysing Matrix E beads (MP Biomedicals) at a concentration of 50mg/ml. Homogenates were then cleared by centrifugation at 10 000 rpm for 15 minutes before quantification of protein using BCA kit (Pierce). About 250 mg of total protein was then used to probe Murine antibody inflammation arrays (Abcam; ab133999) as per manufacturer's instructions. In brief, membranes were for 30 minutes before application of homogenized whole small intestinal lysates in blocking buffer overnight at 4°C. Following washing, biotin conjugated anti cytokine antibodies were applied for 2 hours before further washing and application of HRP conjugated streptavidin. Chemiluminescent detection was then performed using supplied detection reagents and visualization performed using FluorChem E imaging system (Protein Simple). See Table S1 for plate layout.

A fecal sample was collected at the participant’s home and transported to Nagoya University while maintained at 0 °C with ice packs in a thermal insulation jar as previously described^{8 (link)}. The fecal samples were freeze-dried using a freeze-dryer (FDU-2110, EYELA, Shanghai, China) and DNA was extracted from 20 mg of freeze-dried (FD) feces using the QIAamp PowerFecal DNA Kit (QIAGEN, Hilden, Germany) following the instructions of the manufacturer^{22 (link)}. The protocol was partially modified to use Lysing Matrix E Beads (MP Biomedicals, Irvine, CA, USA) with FastPrep-24 5G (MP Biomedicals) for three cycles at 6.0 m/s for 60 s instead of vortex mixing. Sequencing libraries were generated using NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) according to manufacturer’s protocols, and index codes were added to attribute sequencing fragments to each sample. The library quality was assessed using TapeStation (Agilent, Santa Clara, CA, USA). Metagenomic sequencing was conducted using the HiSeq 2500 sequencing platform, HiSeq Rapid SBS Kit v2 and HiSeq PE Rapid Cluster Kit v2 (Illumina, San Diego, CA, USA) to obtain 3 Gbp per sample by 2 × 150 bp paired end-reads.

To also determine the ARGs bound to the membrane, a 24 h incubation experiment was performed using coated and uncoated single-fiber capillaries. In detail, 50 mL of the ARG working solution (10⁶ gene copies∙µL⁻¹ in ultrapure autoclaved water) was transferred in a sterile 50 mL reaction tube and incubated with 10 cm (cut into 5 pieces of 2 cm, and cut lengthwise) of the membrane at room temperature for 24 h, according to the experiments performed by Latulippe et al. [46 (link)]. Before the incubation, the membranes were cut into 5 equally sized pieces of 2 cm and carved lengthwise. After 24 h, total DNA was extracted directly from the membranes by using the Fast DNA^® SPIN Kit for Soil (MP Biomedicals) according to manufacturer’s instructions. Therefore, the DNA bound to the membranes was mechanically eluted with Lysing Matrix E Beads in a FastPrep-24 instrument (MP Biomedicals). Additionally, the working solution was sampled at 0 and 24 h, to determine whether there is a reduction of gene fragments in the solution due to adsorption to the membrane. A negative control was run in parallel with a tube containing the ARG working solution without a membrane and for each tested membrane and a tube containing the membrane without the ARG fragments, respectively. Quantification of the gene fragments was performed using quantitative real-time PCR (qPCR).

DNA from the WWTP samples was extracted using a hexadecyltrimethylammonium bromide (CTAB)-based method with some modifications (Griffiths et al., 2000 (link)). 1.5 mL of the samples was centrifuged at maximum speed for 5 min to collect biomass. Pellets were then transferred into Lysing matrix E beads (MP Biomedicals, France). Five hundred μL 6% CTAB extraction buffer and 500 μL phenol:chloroform:isoamyl alcohol (25:24:1) were added into the extraction tube. Cells were lysed by vortexing at maximum speed on a Vortex Genie2 (Scientific Industries, NY, United States) for 3 min. Supernatant was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and twice with chloroform:isoamyl alcohol (24:1). The aqueous phase was transferred into a clean 1.5-mL tube. DNA was precipitated with 2.5 volume of 100% ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2) and re-suspended in 50 μL PCR-grade water. Extracted DNA was cleaned up with the DNA Clean and Concentrator-5 kit (Zymo Research, Freiburg, Germany) as per the manufacturer’s instruction. A preliminary survey of microbial communities in WWTP samples was performed using pyrosequencing.

Murine TNF‐ɑ or Murine IL‐10 Ready set go enzyme‐linked immunosorbent assay (ELISA) kits were used (eBioscience). Protocol was performed as per manufacturer's instructions. In brief, Costar 9018 ELISA plates were coated with 100 µL/well of capture antibody diluted in Coating Buffer overnight at 4°C followed by blocking with ELISASPOT diluent. Small intestinal samples were homogenized in Cell Lytic MT buffer (Sigma) using Lysing matrix E beads (MP Biomedicals) and homogenates cleared by centrifugation at 10 000 rpm for 15 minutes. After quantification using BCA kit (Pierce), equivalent amounts of test samples or standard material were applied to wells in duplicate and incubated overnight at 4°C. Detection Antibody, Streptavidin‐HRP and substrate were then applied, and plates were read at 450 nm with 570 nm correction.

Adults or imagoes emerged from larvae were identified and sorted by species and sex. All specimens were dissected individually. Abdomens, wings, and legs of up to ten specimens were grouped in pools for rapid screening. Head-thorax segments were kept frozen at −80°C. Tissue pools were suspended in 400 μl of cold PBS and crushed for 1 min at full speed (25 oscillation/s) in a TissueLyser homogenizer (Qiagen) in the presence of LysingMatrix E beads (MP Biomedicals). Residual tissue fragments were pelleted by spinning the tubes at 10,000g for 5 minutes. One half of the supernatants (200 μl) were used for total nucleic acid extraction using Nucleospin Viral RNA kits (Macherey Nagel) according to manufacturer’s instructions. The rest of the tissue lysates were kept frozen at −80°C for viral isolation assays. Samples were screened for the presence of CHIKV sequences by the real-time RT-PCR method [31 (link)].
Head-thorax segments from positive pools were investigated individually by the same procedure to determine the effective number of infected mosquitoes.

After addition of RLT+ lysis buffer from the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), samples were transferred to Lysing Matrix E beads (MP Biomedicals, California, USA) and heated at 37 °C for 10 min with shaking at 700–800 rpm. This was followed by bead beating on a TissueLyser II (Qiagen) system. The supernatant was then removed from the centrifuged samples and placed on an automated QIAcube (Qiagen) workflow system. DNA was then extracted using the AllPrep DNA/RNA Mini Kit (Qiagen) in accordance with the manufacturer’s protocols. Extracted DNA was stored in elution buffer at − 80 °C.