Transcriptome and expression profiling analysis revealed changes of multiple signaling pathways involved in immunity in mud crab Scylla paramamosain during Vibrio parahaemolyticus infection
The mud crabs used in this study were taken from a local crab farm (Niutianyang, Shantou, Guangdong, China). Samples collection and the pathogen challenge were described previously (Li, 2013). Briefly, forty healthy mud crabs (100 ± 10 g in weight) were collected from Niutianyang area in Shantou, China. Crabs were acclimated for 3 days in 1 m3 tanks (ten crabs per tank) in conditions (salinity: 8‰; temperature: 28oC) similar to those of culture ponds from which the crabs were obtained. Sea water was changed twice a day. Crabs were then fed with shellfishes for one week before sampling. V. parahaemolyticus isolated from diseased crabs (from Shantou crab farms) were cultured in 2216E medium at 28oC. 30 crabs were inoculated individually in the ventral hind legs with 100 L of V. parahaemolyticus (2.4 × 107 cells/mL), while the other 10 crabs were injected with 100 L of saline (0.9%) as the control group. 10 crabs each time of the treatment crabs were performed for haemocyte sampling at 12h, 24h, 48h after the challenge, respectively. Haemocytes were collected from blood samples after mixing with anti-coagulant solution and centrifugation at 800 g at 4oC for 20 min. The haemocytes were immediately frozen in liquid nitrogen and then stored at -80oC prior to RNA extraction.  mRNA was purified from total RNA using polyATtract mRNA isolation systems (Promega). Fragmentation was carried out using divalent cations under elevated temperature in Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and SuperScript II (Promega). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H (Promega). Products were purified and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system. In the final step, the library preparations were sequenced on an Illumina Hiseq 2000 platform and 100bp paired-end reads were generated.