Materials
Grass Carp (Ctenopharyngodon idellus), weighing 52 ± 5.8 g, were obtained from Ankang Fisheries Experimental and Demonstration Station, Northwest A&F University, located in Shaanxi province, China. The fish, referred as “naïve fish”, were acclimatized for one week at least in several 200-L glass aquariums equipped with aquarium filters and air stones (water temperature 19.5-22.0 °C, pH 7.1 ± 0.3, dissolved oxygen 5.0-7.0 mg/L). All fish were fed once at 2% body weight daily with commercial fish pellet feed.
A local strain of I. multifiliis was isolated from the goldfish obtained from Zhuque Ornamental Fish Market (Xi’an, Shaanxi, China) and maintained by serial transmission on goldfish [18]. The parasitized and naïve fish were held in a static 200-L glass aquarium under the same conditions as described above in order to develop I. multifiliis infection.
Experimental design
Our previous study showed that no theronts were released from the tomonts after 3 h exposure of the infected fish to MG at a concentration of 0.5 mg/L [7]. In present study, several heavily infected fish were exposed to MG at 0.5 mg/L in opaque beakers, then the fish were transferred into a beaker containing distilled water but no MG after 0.5 and 3 h-exposure, respectively. The same number of fish without MG exposure was served as a control group. The protomonts were collected using a method described by Clayton and Price [19]. Briefly, after the infected fish were placed into the beakers for 30 min (100 mL/fish), mature parasites actively exited from the fish by body movements, then were rinsed three times using distilled water in order to discard fish mucus. A total of eight samples (the tomonts of C1, C2, C3 from the control group (C Group), the tomonts of 3H1, 3H2, 3H3 from the fish exposed to MG for 3 h (3MG Group), the tomonts of 0.5H1, 0.5H2 from the fish exposed to MG for 0.5 h (0.5MG Group) were then put in liquid nitrogen for at least 2 min and stored at -80 °C until use.
Proteins preparation, digestion and iTRAQ labeling
The samples were ground into powder with liquid nitrogen, then homogenized in STD buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl, pH 8.0). Protein extraction was performed by cell lysis at 100 °C for 15 min, followed by sonication at room temperature to shear DNA and reduce the viscosity. The lysates were cleared by centrifugation (14, 000 × g for 45 min), the supernatant of each sample with the soluble protein fraction was transferred to a new tube, respectively. The protein concentration was determined by a BCA protein assay reagent (Beyotime Insitute of Biotechnology, China), the quality of the protein sample measured by SDS-PAGE electrophoresis in order to determine whether these samples can be used for subsequent analysis (Supplemental Figure S1).
Protein digestion was performed according to the filter-aided sample preparation (FASP) procedure (Wisniewski 2009). First, 200 μg of each sample was diluted in DTT to yield a final DTT concentration of 100 mM, heated at 100°C for 5 min. After cool-down to room temperature, each sample was loaded onto an ultrafiltration filter (cutoff 10 KDa, Sartorious, Germany). Two hundred microliters UA buffer (8 M Urea, 150 mM Tris HCl pH 8.0) was added into the filter before a centrifugation at 14, 000 g for 15 min. Subsequently, each supernatant was discarded, then 100 μL of iodoacetamide solution (50 mM iodoacetamide in UA buffer) was used for cysteine blocking. After that, the samples were incubated for 30 min in darkness, then centrifuged at 14, 000 g for 10 min. The filters were washed twice with 100 μL UA buffer and 100 μL dissolution buffer at 14, 000 g for 10 min, respectively. Ultimately, the protein suspensions were digested using 40 μL trypsin buffer (2 μg trypsin in 40 μL dissolution buffer) at 37 °C for 16-18 h, transferred to new tubes and then centrifuged at 14, 000 g for 10 min. The resulting peptides were collected and the concentrations were estimated by UV light spectral density at OD280.
An 8-plex iTRAQ reagent was used to label the peptide mixture according to the manufacture’s instruction (AB Sciex, Framingham, MA, USA). Three control samples (C1, C2 and C3) were labeled with mass 113, 114 and 115 isobaric iTRAQ tags, respectively; the remaining treatment samples (3H1, 3H2, 3H3, 0.5H1 and 0.5H2) were labeled with mass 116, 117, 118, 119 and 121 isobaric iTRAQ tags, respectively. The labeling solutions were incubated at room temperature for 2 h before further analysis.
Strong cationic-exchange chromatography (SCX) fractionation
SCX fractionation was performed on a LC-20AB HPLC pump system (Shimadzu, Kyoto, Japan) with a polysulfoethyl column (4.6 × 100 mm, 5 μm, 200 Ǻ, PolyLC Inc., Maryland, USA) at a flow rate of 1 mL/min. Solvent A (10 mM KH2PO4 in 25% (v/v) ACN, pH 3.0) and solvent B (10 mM NaH2PO4 and 500 mM KCl in 25% ACN, pH 3.0) were applied using a gradient of 100% solvent A for 10 min, 5-60% solvent B for 27 min, 60-100% solvent B in 1 min and 100% solvent B for 10 min. The elution was monitored by absorbance at 214 nm, fractions were collected every 1 min. A total of 20 fractions were obtained based on the quantity of peptide and then desalted with a C18 column Cartridge (Sigma) and vacuum-dried.
Liquid chromatography/tandem mass spectrometry (LC–MS/MS) analysis
A Q-Exactive mass spectrometer coupled to an EASY-nLC 1000 system (Thermo Fisher Scientific) was used for the LC-MS/MS analysis. Five microliters of the mixture (5 μL each iTRAQ sample was added into 15 μL 0.1% trifluoroacetic acid) was loaded onto a 2 cm EASY-column (ID 100 μm, 5 μm, C18; Thermo Fisher Scientific). The peptides were eluted with a 45 min linear gradient from 0 to 100% solvent C (84% acetonitrile and 0.1% formic acide) at a flow rate of 250 nL/min. The mass spectrometer was operated in the positive ion mode, with a mass range of 300-1, 800 mass/charge (m/z) and a resolution of 70, 000 at m/z 200. Determination of the target value was based on predictive automatic gain control (pAGC). Dynamic exclusion was set for 40 s. The top 10 most abundant precursor isons were selected for higher-energy collisional dissociation (HCD) fragmentation and MS/MS data were generated at a resolution of 17, 500 at m/z 200 using a data-dependent acquisition method. The normalized collision energy was 30 eV and the underfill ratio was defined as 0.1%. Each iTRAQ sample was analyzed at least three times.
Protein identification, quantification and bioinformatics analysis
All the raw data were analyzed by Proteome Discoverer 1.4 (Thermo Fisher Scientific). Protein identification was performed using the MASCOT v.2.2 search engine (Matrix Science Ltd., London, UK) against I. multifiliis database (the number of sequences: 16, 267; the date of download: October 29, 2015) from NCBI and decoy database. Search parameters were set up as follows: peptide mass tolerance at 20 ppm; MS/MS tolerance at 0.1 Da; trypsin as the enzyme; missed cleavage up to 2. Variable modifications were defined as oxidation of methionine and iTRAQ 8-plex labeled tyrosine, while fixed modifications were specified as lysine and N-term of peptides labeled by iTRAQ 8-plex and carbamidomethylation on cysteine. Unique protein with at least one unique peptide was identified, false discovery rate (FDR) was set to less than 0.01 for the identification of both peptides and proteins. The values of the intensities of the two/three reporter ions for each group were averaged and then the differences between two groups were statistically assessed by Student’s t-test. The fold change (FC) ratio was set to > 1.2 for protein up-regulation and < 0.83 for protein down-regulation, with p <0.05.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository [20] with the dataset identifier PXD019013.
Biological functions of significantly altered proteins are analyzed using Blast2GO software (https://www.blast2go.com/) for gene ontology (GO) annotation. Pathway analysis was performed by the web-based Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.kegg.jp/).
Ultrastructural analysis by transmission electron microscopy (TEM)
Ichthyophthirius multifiliis protomonts collected from the fish exposed to MG for 0, 0.5 and 3 h, were fixed with 2.5% glutaraldehyde in sodium cacodylate buffer (100 mM, pH 7.2) over 12 h at room temperature. The parasites were then washed twice in the same buffer and post-fixed in 2% aqueous osmium tetroxide for 45 min on ice. After three successive washes with 0.1 M sodium cacodylate buffer, the fixed samples were stained with 1% aqueous solution of uranyl acetate for 1 h and dehydrated in acetone at a series of concentrations from 50 to 100% (v/v). The samples were embedded in EMbed 812 (Electron Microscopy Sciences) according to the manufacturer’s instruction. Ultrathin sections were stained with uranyl acetate and lead citrate [21], examined using a JEOL JEM 1010 transmission electron microscope at 75 kV acceleration voltage.
Cellular apoptosis assays
An Annexin V-FITC/propidium iodide apoptosis detection kit (Vazyme Biotech Co., Ltd, China) was used to determine early and late apoptotic changes of protomonts after treatment. The parasites collected as described above were stained with 5 μl Annexin V-FITC and 5 μl propidium iodide for 10 min in the dark room according to the manufacturer’s instruction. The stained parasites were then dispensed onto slides and photographed under an Olympus fluorescence microscope (BX53 + DP2-BSW).
Verification of the proteome data by RT-qPCR
Total RNA was extracted from the protomonts collected as described above by TRIzol reagent (Takara). DNA contamination was removed by DNase I (Takara) following the manufacturer’s instruction. The purified RNA (500 ng/μL per reaction) was reverse-transcribed using HiScript Q Select RT SuperMix for qPCR (Vazyme). For the normalization of gene expression, 18S rRNA was used as an internal reference gene [22]. Real-time qPCR was carried out using CFX96 Real-Time qPCR Detection System (Bio-Rad) with a reaction mixture containing 6.5 µL SYBR Premix Ex Taq II kit (Takara), 1 µL of cDNA template, 0.5 μL of each primer and 6.5 µl of ddH2O according to manufacturer’s protocol. The PCR cycling reactions were initially denatured at 95°C for 5 min followed by 40 cycles of 95°C for 15 s, 58°C for 20 s, 72°C for 20 s. Each individual sample was determined in triplicate. The results of dissociation-curve analysis (95°C for 15 s, 58°C for 1 min, 95°C for 15 s and 58°C for 15 s) showed a single melting curve in all cases. The primers of qPCR are listed in Supplemental Table 2. The relative gene expression data were calculated using 2-ΔΔCt method [23]. Statistical analyses of the data were performed using Student’s test at p< 0.05.