Vortex genie 2

Purified, freeze dried avenin (10 mg) was re-solubilized using 70% (v/v) ethanol and vortexed for 30 min (MO BIO Laboratories, Inc. Vortex-Genie^® 2). Samples were prepared in triplicate and were centrifuged for 20 min at 15870x g in an Eppendorf Centrifuge 5424. The supernatant was filtered using a 0.45 μm filter. The protein extracts were separated using an Agilent 1200 LC system (Agilent Technologies) using a modified method (32 (link)). An aliquot (10 μl) of extract was injected into a C18 reversed-phase Zorbax 300SB-C18 column (4.6 × 150 mm, 5 μm, 300 Å, Agilent Technologies) maintained at 60°C. The eluents used were ultrapure water (solvent A) and acetonitrile (solvent B), each containing 0.1% trifluoroacetic acid (TFA) (HPLC grade, Sigma Aldrich). The separation was carried out using a linear gradient from 33 to 80% solvent B over 65 min at a flow rate of 1 mL/min.

Total genomic DNAs were extracted from pooled (5–30) whole gut samples, depending on size of species, using FastDNA® SPIN kit for Soil (MP Biomedicals, Australia). Termite guts were added to a lysing matrix, treated with lysis buffer, and underwent bead beating in the Vortex-Genie® 2 (MoBio Laboratories, USA). DNA was bound to silica matrix and washed and eluted in DNase-free water. DNA yield was then quantified by the Qubit™ fluorometer and QuantIT ds-DNA BR assay kit (Invitrogen, Australia). DNA concentration varied depending on the biomass of the whole gut. DNA concentrations were standardized across all samples to 20 μg/ml, diluting where necessary in Ultrapure™ distilled water (Invitrogen, Australia). DNA quality was evaluated using gel electrophoresis on 1.0% agarose gels stained with SYBR Safe, visualized on a CCD compact image system (Major Science, USA).

Fecal samples were thawed and extracted using the Qiagen PowerFecal Pro DNA kit (Qiagen #51804) according to the manufacturer’s instructions. Sample homogenization was performed using a Qiagen Vortex adapter coupled to a Vortex Genie 2 for 20 min at maximum speed. Initial deoxyribonucleic acid (DNA) quantification was performed on a NanoDrop (Thermo Scientific, Leicestershire, U.K.) and DNA concentration was adjusted to within the desired mass range for sequencing and plated on a 96 well, low profile, skirted polymerase chain reaction (PCR) plate (4titude, Surrey, U.K.) for sequencing submission. For bacterial 16S rRNA gene amplicon sequencing, the variable V3 and V4 regions of the 16S rRNA gene were amplified from genomic DNA using the primers (standard IUPAC nucleotide nomenclature): Forward Primer = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, Reverse Primer = 5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.
The amplicons were then attached with indices and Illumina sequencing adapters using the Nextera XT index kit. The 16S amplicon libraries were pooled and sequenced in an Illumina MiSeq v3 flowcell as 300 paired-end reads. Library preparation and sequencing was performed at the Oxford Genomics Centre.

Samples were freeze-dried for 24 h using a freeze-dryer (ALPHA 1-2LD, Christ, Germany). 10 g of ground sample was mixed with ultrapure water in a ratio of 1:5 (w/v), shaken for 5 min (Vortex-Genie^®2, QIAGEN, Germany) and centrifuged at 6,800 × g (TGL-16A, Changsha) for 15 min. The supernatant was filtered through a 0.22 μm membrane, acidified with 3 M HCl for cation except NH₄⁺ measurement using ICS-600 (Thermo, United States). The ammonium of the filtrate without acidification was measured by the salicylic acid assay (Kandeler and Gerber, 1988 (link)), and the filtrate without acidification was determined for anion with ICP-OES (iCAP 7,600+) ion chromatography. pH was determined using a multi-parameter water quality tester (HACH, Loveland, CO, United States), and TOC was determined using an elemental analyzer (Vario MACRO cube, Elementar, Germany). The amount of un-ionized NH₃ based on pH was calculated using the following formula (Emerson et al., 1975 (link)):
Where pK_a is the dissociation constant of NH₃ + H⁺/NH₄⁺ pair in solution.