Tissuelyser

The measurement of MAD was performed using the thiobarbituric acid (TBA) method [51 (link)]. In brief, approximately 100 mg fresh samples were immediately homogenized in 1 mL 5% (w/v) trichloroacetic acid (TCA) solution using a Tissuelyser (Jingxin, Shanghai, China). After centrifugated at 4 °C for 10 min at 6000 rpm, 400 μL of the supernatant was taken out and added into 400 μL 10% TCA containing 0.67% (w/v) thiobarbituric acid (TBA) in a new tube, then the mixture was incubated in boiling water for 30 min. The mixture was centrifugated for 5 min at 12,000 rpm after cooling to room temperature and the OD_{450 nm}, OD_{532 nm}, and OD_600nm of the supernatant were determined using a Multimode Plate Reader (PerkinElmer).
The content of free proline in plants was determined according to a reported method [52 (link)]. Briefly, 100 mg fresh samples were homogenized in 1 mL 3% (w/v) sulfosalicylic acid using a Tissuelyser (Jingxin, Shanghai, China), and incubated in boiling water for 10 min. After cooling to room temperature, the samples were centrifugated at 12,000 rpm for 5 min. 400 μL of the supernatant was taken out and mixed with 400 μL acid ninhydrin and 400 μL glacial acetic in a new tube, then mixture was incubated in a boiling water bath for 30 min. After centrifuged at 6000 rpm for 5 min, the proline content was measured at 520 nm using a Multimode Plate Reader (PerkinElmer).

After washing with 75% ethanol and RNA/DNA-free water, pooled ticks in tubes were added with 500 μL Dulbecco’s modified Eagle’s minimum essential medium (DMEM) and two stainless steel beads (3 mm diameter), and crushed using the Tissuelyser (Jingxin, Shanghai, China) at 70 Hz for 2 min. The lysates were centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was further pooled for library construction according to the collection sites and species (S1 Table). After digested with micrococcal nuclease (NEB, USA) in 37°C for 2 h, the pooled samples were used for viral RNA extraction with the TIANamp Virus RNA kit (TIANGEN, Beijing, China).
The extracted RNA was subjected to metagenomic sequencing at Tanpu Biological Technology (Shanghai, China). Briefly, the RNA from each pool was used for library preparation with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s instructions. After adapter ligation, 10 cycles of PCR amplification were performed for target enrichment. The libraries were pooled at equal molar ratio, denatured and diluted to optimal concentration, and sequenced with an Illumina NovaSeq 6000 System.

Briefly, 60 mg of bone tissue (from the ribs and vertebral column) were weighed and subjected to metabolite extraction by directly adding 800 µL of precooled extraction reagent (methanol:acetonitrile:water (2:2:1, v/v/v)), followed by a mixture of internal standards for quality control (QC) of the prepared samples. After homogenizing the bone tissue mixture for 5 min using TissueLyser (JXFSTPRP, JingXin Technology, Shanghai, China), the samples were sonicated for 10 min and incubated at −20 °C for 1 h. The samples were then centrifuged for 15 min at 25,000 rpm at 4 °C, and the supernatants were collected for vacuum freeze drying. The metabolites were resuspended in 200 µL of 10% methanol and sonicated for 10 min at 4 °C, followed by centrifugation for 15 min at 25,000 rpm. Finally, the supernatants were transferred to autosampler vials for liquid chromatography–mass spectrometry (LC-MS) analysis. A QC sample was prepared by pooling identical volumes of each sample to evaluate the reproducibility of the whole LC-MS analysis process.