Mediterranea

Hapten-labeled anti-sense RNA probes were generated from in vitro transcription reactions containing either DIG-12-UTP (Roche), DNP-11-UTP (PerkinElmer), or FAM-12-UTP (Roche) according to the manufacturer’s suggested protocol (Roche). DNA template for the in vitro transcription reaction was generated by PCR amplifying sequences from gene clones obtained from either the S. mediterranea EST Database [48 (link)] or from cDNA clones generated using standard methods. Primers and unincorporated nucleotides were removed from PCR products using a DNA clean and concentrator kit (Zymo Research) prior to use as template in transcription reactions. Probes were precipitated using LiCl/ethanol according to the manufacturer’s suggested protocol (Roche) and resuspended in 50 μl RNAse-free water. Probe quality and concentration were assessed on a 1% agarose gel and using a NanoDrop ND-1000 spectrophotometer. Probe concentration was adjusted to 50 ng/μl by adding hybridization buffer (see Additional file 1) and probes were stored at −20°C. In some cases, FAM-labeled probes were further purified using a sephadex G-50 quick spin column (Roche) according to the manufacturer’s recommendations.

Illumina MiSeq reads were trimmed to Q ≥30 and adaptors removed using Sickle and Perl and assembled using Newbler (Roche GS-Assembler v2.6) with flags set for large genome and a heterozygote sample. Mate-pair reads were first mapped to these contigs using Bowtie2 [64 (link)] to remove duplicates and wrongly orientated reads, and scaffolded into contigs using SSPACE [65 (link)]. Gap filling was achieved using GapFiller for 2× 250 bp and 2× 100 bp paired-end reads and run for three iterations (available as ENA accessions LN627018-LN647175). RNAseq data were mapped to scaffolds within the assembled genome greater than 3 Kbp using TopHat2 to identify transcribed regions and splice junctions. These, together with RNAseq data assembled using Trinity and S. mansoni protein sequence, were passed to the MAKER pipeline [66 (link)] to predict genes. Repeatmasker, Windowmasker and Dustmasker were used to identify repetitive regions. CEGMA v2.4 [67 (link)], which searches for 248 highly conserved genes, was used to assess the completeness of genome with the settings for vertebrates to allow long introns. REAPR [68 (link)] was used to assess the quality of scaffolding within the assembly and to produce an alternative, more conservative assembly by splitting scaffolds at locations with lower support (available as ENA accessions LN736597-LN774150). Homologs of F. hepatica predicted protein sequences were identified within UniProt using BLAST and functional domains identified using InterPro. InParanoid and MultiParanoid [69 (link)] were used to identify ortholog clusters from Schistosoma mansoni (v3.1.16), Clonorchis sinensis (v3.7), Schmidtea mediterranea and Echinococcus multilocularis (v29042013) predicted proteins [11 (link)-15 (link),70 (link)].

Asexual (Clone CIW4) and sexual (Clone S2F1L3F2) strains of Schmidtea mediterranea were maintained in Montjuïc water at 20°C.³⁴ (link) Animals were starved for 7–14 days before each experiment. Animals exposed to 6,000 rads of γ rays were used as transplant hosts.⁸ (link) After transplantation, hosts were maintained in Montjuïc water with 50 μg/mL of Gentamicin (GEMINI, 400-100P). For transplant rescue experiments, host animals were kept in 3.5 cm Petri dishes (1 worm/dish), and Montjuïc water was changed every 2–3 days.

An HPLC-FLD from Shimadzu (Prominence-i LC-2030C Plus) was used to conduct the PST analyses. This equipment comprises a quaternary pump, a refrigerator autosampler, a column oven and a spectrofluorometric detector (RF-20A XS). The temperatures used were 10 °C, 25 °C and 30 °C, in the autosampler, column oven and cell detector, respectively. A reversed-phase C18 column (Mediterranea Sea18, Teknokroma), 25 cm × 0.46 cm (5 µm), and an ultraguard^TM column (Sea18 10 × 3.2 mm, Teknokroma) were used. Ammonium formate 0.1 M (adjusted to pH 6 with acetic acid 0.1 M) and acetonitrile (HPLC grade, Honeywell) were applied as mobile phase A and B, correspondingly. All eluents and solvents for HPLC were filtered, using PVDF membrane filters (0.22 μm, 47 mm, Teknokroma). The gradient elution conditions were as follows. First 6 min: 1–5% phase B; between 6 and 13 min: 5–28% phase B; between 13 and 16 min: 28–1% phase B; between 16 and 19 min: 1% phase B. The flow rate was 1.5 mL/min. The injected volume was 30 µL or 100 µL, for standards and samples oxidised with peroxide or periodate, respectively. Three injections of each sample were performed. Fluorescence detection was achieved at 340 nm (excitation) and 395 nm (emission).
To quantify PST, calibration solutions ranging 0.03 and 2.00 µM were prepared from certified reference materials (CRM): dcGTX2&3, C1&2, GTX2&3, GTX5 (or B1), GTX6 (or B2), GTX1&4, C3&4, NEO, dcNEO, STX (from CIFGA laboratory S.A., Lugo, Spain) and dcSTX (from National Research Council of Canada, Halifax, Canada). To prepare the solutions, the following mixtures were considered: (I) dcGTX2&3, C1&2 and dcSTX (for peroxide oxidation); (II) GTX2&3, GTX5 and STX (for peroxide oxidation); (III) GTX1,4 and NEO (for periodate oxidation); (IV) C3&4 and GTX6 (for periodate oxidation); (V) dcNEO (for periodate oxidation); (VI) dcGTX2&3 and dcSTX (for periodate oxidation). The quantification of each toxin in the samples was performed by interpolation of data in the corresponding calibration curve. The contribution of the chromatographic peaks corresponding to the matrix modifier were subtracted (before interpolation) from the chromatograms of the samples and standards pre-oxidized with periodate in the presence of the matrix modifier. The limits of detection and quantification (LOD and LOQ) were determined as (3σ)/m and (10σ)/m, where σ is the residual standard deviation of the regression line and m is the slope of the calibration curve [33 ,34 ]. For the several calibration curves, all determination coefficients (R²) were above 0.9981. LOD and LOQ ranged from 0.003 µM (1 µg STX.2HCl eqv/Kg) to 0.03 µM (169 µg STX.2HCl eqv/Kg) and from 0.01 µM (3 µg STX.2HCl eqv/Kg) to 0.11 µM (615 µg STX.2HCl eqv/Kg), respectively (Table S3). The least concentrated standard solution of each calibration curve was always equal to or greater than the corresponding LOQ. To avoid misinterpretation, the following solutions were also analysed under the same conditions as the standards and samples: a chemical blank of the entire procedure—extraction, cleaning, fractionation, and oxidation (1% acetic acid), samples without the oxidizing agent (using the same volume of ultrapure water instead of the peroxide or periodate oxidant), the matrix modifier oxidised with periodate (the same volume of ultrapure water instead the sample/standard) (some examples in SI, Figures S1–S3).

Lemon byproduct samples (Citrus limon (L.) Osbeck) containing peels, pulp and seeds were supplied by the Agrumaria Reggina company located in Gallico (Reggio Calabria, Italy) after the extraction of lemon juice and essential oils. Lemon byproducts were transported to the Food Technology Laboratory of the University Mediterranea of Reggio Calabria, immediately dried at a temperature of 50 °C up to a final moisture content of 12% and stored in polyethylene bags under a vacuum to avoid rehydration until the subsequent extraction procedures of the bioactive compounds.
Radishes were supplied by a local distributor in the province of Reggio Calabria (Italy) and transported to the Food Technology Laboratory of the University Mediterranea of Reggio Calabria, where leaves were completely removed before subjecting the radish roots to processing.