Lysing matrix e

Before dissection, tweezers were sterilized with 70% ethanol and UV light irradiation for 10 min. Insects were dissected under a binocular microscope. The gut was roughly homogenized using tweezers in 978 µL of sodium phosphate buffer included in FastDNA Spin Kit for Soil (MP-Biomedicals), and transferred into Lysing Matrix E (MP-Biomedicals). DNA extraction was performed according to the manufacturer’s instruction with beads-beating for 1 sec at 5,500 rpm, using Micro Smash MS-100 (TOMY). For V. mandarinia samples collected at AIST, beads-beating was performed twice. Extracted DNA samples were quantified using Qubit dsDNA HS Assay Kit (Molecular Probes), and their integrity were verified by agarose electrophoresis.

Baiting of microbes from the rhizosphere or bulk soil suspensions on the cuticle of P. penetrans was done as previously described [25 (link)]. Briefly, 20,000 surface-sterilized nematodes were incubated in 5 mL of each microbial suspension in 15 mL tubes. The tubes were positioned horizontally on a shaker at a speed of 150 rpm at 20 ± 2 °C for 24 h. The nematodes with attached microbes were recovered on 5-µm sieves and washed with 20 mL of sterile tap water to remove loosely attached microbes. To isolate the attached bacterial strains, nematodes were plated on R2A media (Merck, Germany) supplemented with 10 mg L⁻¹ cycloheximide. The plates were incubated at 28 °C and bacterial strains were collected from the emerged colonies over a 2-week period. A portion of the nematodes with attached microbes was transferred to bead-beating tubes with Lysing Matrix E (MP Bio, Heidelberg, Germany) for DNA extraction.

Thirty milliliters of culture were centrifuged at 4000 rpm for 30 min at RT, keeping both the pellet and the supernatant. The supernatant was removed and filtered onto polycarbonate filters (0.22 μm pore size, Merck Millipore) under gentle vacuum (>30 kPa). Filter pieces and pellet were transferred to bead beating tubes (Lysing Matrix E, MP Biomedicals) with 1 ml of acetonitrile:methanol:water (4,4,2, v,v,v). The tubes were vortexed for 10 min at maximum speed. Beads and debris were pelleted by centrifugation at 10000 rpm for 20 min at RT. The clear supernatant was transferred to glass vials and stored at 4°C.

The concentrate, from above, was split into two 1.5-mL aliquots. One aliquot was treated with PMA (18.25 μL of 2 mM, resulting in a final concentration of 25 μM) to assess cells that were viable or had an intact cell membrane [51 (link)], while the second aliquot was handled in a similar manner but without the addition of PMA. The PMA- and non-PMA-treated aliquots were incubated in the dark at RT for 5 min, followed by 15 min of photoactivation using the PMA-Lite™ LED Photolysis Device, specifically designed for photoactivation of PMA (Biotium, Hayward, CA). The PMA- and non-PMA-treated aliquots were split into two 0.75-mL aliquots. One aliquot was transferred to bead beating tubes containing Lysing Matrix E (MP Biomedicals, Santa Ana, CA), followed by bead beating for 60 s using the vortex sample holder (MO Bio, Carlsbad, CA). The bead-beaten aliquot and the aliquot without bead beating were combined for their corresponding PMA-treated and non-treated samples. DNA extraction was performed with the Maxwell 16 automated system (Promega, Madison, WI), in accordance with the manufacturer’s instructions using the Maxwell 16 Tissue LEV Total RNA purification kit. A Maxwell control without any sample added in its cartridge was run concurrently with the samples. The extracted DNA was eluted in 50 μL of water and stored at − 20 °C until further analysis.

TFA extraction and quantification were executed as described by Breuer et al.
[40 ] with the following adjustments. Around 5 mg of pellet was transferred to bead beating tubes (Lysing Matrix E; MP Biomedicals, Santa Ana, CA, USA) and lyophilized overnight. Freeze-dried cells were disrupted by a 30 min bead beating step in the presence of a chloroform:methanol mixture (1:1.25) to extract the lipids from the biomass. Next, 48.6 μg/mL tripentadecanoin (T4257; Sigma-Aldrich, St Louis, MO, USA) internal standard was added to the extraction mixture to enable fatty acid quantification. Methylation of the fatty acids to FAMEs and the quantification of the FAMEs were performed as described by Breuer et al.[40 ]. TFA concentration was calculated as the sum of all individual fatty acids.

The fecal samples (10–20 mg) were collected until 14–18 weeks of age and suspended in a GTC (4 M guanidium thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA (pH 8.0)) solution and then homogenized in Lysing Matrix E (MP-Biomedicals, Santa Ana, CA, USA) using a FastPrep FP120 (Thermo Savant, Waltham, MA, USA). Thereafter, DNA (300–360 ng mL⁻¹) was extracted from a bead-treated suspension using a phenol-chloroform extraction method and was purified with the FastGeneT Gel/PCR Extraction Kit (NIPPON Genetics Co, Ltd., Tokyo, Japan). All samples were stored at −80 °C until further analysis.