Master mix

DNA was isolated using QIAGEN® QIAamp DNA Mini Kit Tissue Protocol. Of thirty foxes, a pool of five Trichinella ML was analysed per animal before freezing. The concentrations of extracted DNA in samples were measured with ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE 19810, USA). The Multiplex PCR was directed at the ITS1, ITS2 and ESV genes as described by Zarlenga et al. [29 (link)]. PCR reactions were performed in a total volume of 30 μL, containing 15 μL 2× Master mix (PROMEGA M7505, USA), 1 μL of 10 pmol/μL oligonucleotide mixture, 4 μL of RNAse-free water and 10 μL of DNA. As positive control, T. spiralis, T. britovi and T. nativa DNA was used. The PCR conditions were 95 °C for 4 min followed by 35 cycles of 95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s. PCR products were analysed by QIAxcel ScreenGel 1.1.0 (Qiagen, Hilden, Germany) and identified according to banding pattern as described earlier [23 (link),29 (link)]. This work was performed at BIOR (Riga, Latvia).

The Crispr-IIIA-F/Crispr-IIIA-R and Crispr-IIC-F/Crispr-IIC-R primers were designed to specifically detect the cas1 gene for CRISPR-Cas types IIIA and IIC, respectively (Fig. 1B and Table S1). The primers were designed based on S. lugdunensis whole-genome sequences deposited in the National Center for Biotechnology Information (NCBI) database, including sequences for type IIIA-positive strain VISLISI_33 (accession no. CP020769.1) and type IIC-positive strain C_33 (accession no. CP020768.1). The PCRs were carried out in a total volume of 20 μl containing 2× master mix (Promega, Madison, WI, United States), 10 pmol of each primer, and 1 μl DNA template. The PCR cycling conditions for CRISPR-Cas system detection were 95°C for 3 min; 30 cycles of 30 s at 95°C, 30 s of annealing at 50°C, and 1 min of extension at 72°C; and a final extension for 3 min at 72°C.
The primers Crispr-IIIA-F/Crispr-IIIA-R and Crispr-IIC-F/Crispr-IIC-R were designed for also determining type IIIA and type IIC CRISPR-Cas spacer sequences, respectively (Fig. 1B and Table S2). The PCR cycling conditions for CRISPR-Cas system spacer typing were 95°C for 10 s; 30 cycles of 1 min at 94°C, 1 min of annealing at 55°C, 1 min of extension at 72°C (45 s for CRISPR-Cas IIC), and a final extension for 10 min at 72°C. The Crispr-s-R primer (Table S2) was used to perform downstream sequencing of the type IIIA CRISPR array.

For PCR, the tubes were processed as described previously [18 (link)] using Promega Master Mix (Promega, France) according to the manufacturer’s instructions. Thermal cycling conditions comprised an initial 2 min heating step at 95 °C, followed by 35 cycles of 95 °C for 30 s, 60 °C for 60 s, 72 °C for 120 s, and a final extension at 72 °C for 5 min. Positive samples revealed by visualization on an agarose gel (2%) after electrophoresis were selected for sequencing. A purification using the ExoSAP-IT Product Cleanup reagent (Affymetrix, Cleveland, OH, USA) was realized prior to the sequencing reaction with Big Dye X-terminator v. 3.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Excess labeled nucleotides were removed using a Bid Dye X-terminator purification kit, and sequencing products were run on an ABI PRISM 3130 Genetic Analyzer. Forward and reverse reads were obtained.

From each individual leaf, RNA was extracted using the Zymo RNA miniprep kit (Zymo, Irvine, CA, USA) according to the manufacturers’ specifications. Extractions were then lyophilised and shipped to the University of Western Australia for further processing.
Lyophilised RNA was subsequently reconstituted with nuclease free water. From an aliquot of the RNA, cDNA was prepared using Promega master mix (Promega, Madison, WI, USA) as described by the manufacturer. Subsequently, PCR was carried out using the Bioneer master mix (Bioneer, Daedeok, Republic of Korea) using two sets of primers; universal Potyvirus primers LegPotyF 5-GCWKCHATGATYGARGCHTGGG-3 and LegPotyR 5-AYYTGYTYMTCHCCATCCATC-3 (Webster, 2008 ) and for Carlavirus primers 5-GTTTTCCCAGTCACGAC-3 and 5-ATGCCXCTXAXXCCXCC-3 (Chen, Chen & Adams, 2002 (link)). Bean common mosaic virus was used as the positive control Potyvirus and a non-template control nuclease free water was used as the negative control.