Propagation of A. cantonensis and Caenorhabditis elegans
A. cantonensis ZJ strain was maintained and propagated in Wenzhou Medical University, China by cycling through the Pomacea canaliculata and Sprague-Dawley (SD) rats as described previously . Intermediate hosts Pomacea canaliculata were infected with A. cantonensis L1 through feeding on rat feces. L3 were collected at 20 days p.i. Infected snails were shelled and crushed. The intestines and other organs were removed and the remaining tissue was homogenized. The homogenates were filtered through a 40-meshsieve, deposited for 5 min at 4 °C, and precipitated 2–3 times at room temperature. The sediments were removed and L3 number and viability were determined by direct observation under a light microscope. Three-week-old Sprague-Dawley (SD) rats (weight 100–120 g, grade clean, Certificate SYXK [ZHE] 2005-0061), supplied by the Laboratory Animal Center of Wenzhou Medical University were orally infected with 50 L3/rat. The rats were housed in polypropylene cages with free access to food and water, and then sacrificed by anesthesia at 25 days and 45 days p.i., respectively. The L3 worms were collected from the intermediate hosts Pomacea canaliculata; the L5 harvested from the brains of mice (C57BL/6J (B6), Certificate SYXK (zhe2015-0009)) (non-permissive host same as humans), which were orally infected with 30 L3/mouse; the adult worms were collected from the blood vessels of the hearts and lungs. Individuals of different sexes were separated using morphological criteria: Females are usually longer and thinner than males, and males exhibit typical copulatory bursa. L3, L5, and adults were washed three times with 0.01 mol/L PBS buffer, and stored at -80 °C. These rats were not used for any other part of the study.
Caenorhabditis elegans strains N2, rps-30 (tm6034/nt1) and ced-3 (ok2734) were maintained on Nematode Growth Media (NGM) agar plates at 15 °C as described previously . Worms were fed Escherichia coli strain OP50 unless otherwise stated. The mutant strain ced-3 (ok2734) was obtained from the Caenorhabditis Genetic Center (CGC) (University of Minnesota,USA). The mutant strain rps-30 (tm6034/nt1) was originally provided by Shohei Mitani, M.D., Ph.D., Department of Physiology, Tokyo Women’s Medical University School of Medicine, Japan. The gene rsp-30 is essential for survival of the worms, and if the gene is deleted, worms would be sterile. Therefore, the mutant strain tm6034/nt1 was conducted as trans-heterozygous animals using a balancer nT1, which has fluorescent marker. So, fluorescence positive animals carry nT1 but animals without nT1 are mutation homozygous.
Isolation, purification, treatment and storage of nucleic acids
Total genomic DNA was extracted from A. cantonensis ZJ strain adult worms using a small-scale genomic DNA extraction kit (Takara Biotechnology Co., Ltd, Japan). Total RNA was extracted from worms at different developmental stages employing Trizol reagent (Invitrogen, USA), followed by treatment with 2 U of DNase I (Takara Biotechnology Co., Ltd, Japan). First strand cDNA was obtained using the M-MLV RTase cDNA Synthesis Kit (Takara Biotechnology Co., Ltd, Japan). Both DNA and RNA samples were stored at −80 °C.
Isolation of full-length cDNA and genomic DNA encoding Acan-rps-30 from A. cantonensis
Using two degenerate primers, rps-30DF and rps-30DR (Additional file 1: Table S1), designed based on a relatively conserved S30 domain, with reference to the C. elegans gene (NC_003283.11) and Homo sapiens gene (NC_000011.10), a portion of Acan-rps-30 was amplified by PCR from cDNA synthesized from total RNA extracted from adult worms. PCR products were cloned into the pMD18-T vector (Takara Biotechnology Co., Ltd, Japan) and sequenced. Based on the available sequence, gene-specific primer pairs (Additional file 1: Table S1) were designed. Then, using 5′- and 3′- rapid amplification of cDNA ends (RACE) (Takara Biotechnology Co., Ltd, Japan), two partially overlapping cDNA fragments were obtained. Products were cloned into the pMD18-T vector and sequenced. Based on these sequences, additional primers (Additional file 1: Table S1) were designed to amplify full-length Acan-rps-30.
Full-length genomic DNA of Acan-rps-30 from the ZJ strain of A. cantonensis was obtained by a Genome Walking kit (Takara Biotechnology Co., Ltd, Japan), using primers designed based on the acquired cDNA sequence (Additional file 1: Table S1), following the manufacturer’s instructions. The third-round PCR products were cloned into a pMD18-T vector and sequenced.
A sequence alignment between Acan-RPS-30, Hs-RPS-30 (NP_001988.1) and Ce-RPS-30 (NP_505007.1) was generated using Clustal Omega. Homology models were built by SWISS-MODEL using H. sapiens ribosome (PDB codes 5LKS and 2L7R) as templates. Three-dimensional structural analysis was performed using the PyMOL program. All calculations were carried out under default conditions.
The amino acid sequence inferred for Acan-RPS-30 and 7 selected other homologues sequences were subjected to phylogenetic analyses. The phylogenetic analysis was conducted using the neighbour-joining (NJ) and maximum parsimony (MP) methods, respectively, based on the Jones-Taylor-Thornton (JTT) model . Confidence limits were assessed using a bootstrap procedure with 1000 pseudo-replicates for NJ and MP trees, and other settings were obtained using the default values in MEGA v.5.0. A 50% cut-off value was implemented for the consensus tree.
Quantitative real-time PCR (qRT-PCR) analysis
qRT-PCR was performed to determine the abundance of Acan-rps-30 transcripts in different developmental stages (L3, L5 female,L5 male, Adult female, Adult male) of A. cantonensis.
Gene expression levels were determined by RT-PCR using SYBR®Green PCR Master Mix and a 7500 Real-Time PCR System (Applied Biosystems, USA). Relative gene expression was compared with18S ribosomal RNA gene (GenBank: AY295804) as an internal loading control. The target genes and the primers used are listed in Additional file 1: Table S1. Statistical analysis was conducted using a one-way ANOVA; P < 0.05 was set as the criterion for significance.
RNAi feeding experiments
To generate ced-3 specific RNAi vectors, ced-3 cDNAs was cloned into the L4440 vector. Plasmids were transformed into E. coli strain HT115. Primers used for PCR analysis are listed in Additional file 1: Table S1. RNAi plates and media were prepared according to . Gravid adults of C. elegans were allowed to lay eggs overnight on the RNAi plates and adult worms were picked off. Empty vector-containing E. coli were used on separate plates as negative controls.
About 2000 bp sequence upstream of Acan-rps-30 5’-UTRs was used as putative promoter. To analyze promoter activity of Acan-rps-30, the promoter regions of Acan-rps-30 and Ce-rps30 were amplified and cloned into plasmid pPD95.77 to construct pAcan-rps-30::gfp and pCe-rps30::gfp, respectively (Fig. 1a).
To perform cross-species expression of Acan-RPS-30 in N2 strain and rps-30 (tm6034/nt1) strains, cDNA sequence was amplified and cloned into pPD95.77 using the promoter of Ce-rps30 to construct plasmid pCe-rps-30::Acan-rps-30::rfp (Fig. 1b). All primers used are listed in Additional file 1: Table S1.
Recombinant plasmids were each microinjected into the gonad of young, adult C. elegans hermaphrodites as described [2, 30], together with plasmid pRF4 containing a dominant mutant allele of rol-6 gene, each at a final concentration of 50 μg/mL in the same mixture, using pPD95.77 (pCe-rps30::gfp) and pRF4 plasmid mixture as a control. The F2 and subsequent generations with a roller phenotype were analyzed and selected to examine the expression patterns of GFP or RFP, using a fluorescent microscope (Olympus IX71). A minimum of three independent lines expressing each transgene were evaluated.
Oxidative stress assay
The oxidative stress assay was performed as described previously . Briefly, adult hermaphrodites (30 worms/group) were transferred to a 96-well plate containing M9 buffer with 3mM H2O2. After incubation at 20°C for the specified durations, the number of dead worms was determined. Worms were scored as dead when they no longer responded with movement to light prodding of the head. Three (H2O2) independent experiments were performed. Statistical analysis was performed with Microsoft Excel 2010 software using an unpaired two-tailed t-test.