TGGGGCGGAGTGGA-39 to screen for the fragment-3-contain- ing templates in which the sequence of fragment 3 from segment 1 was paired with the complementary sequence of fragment 3 from segment 2. Subsequently the capped PjCaspase mRNA containing fragment 3 was transcribed from the fragment-3-containing templates with a T7 RNA polymerase in vitro transcription kit (TaKaRa Bio, Dalian, Liaoning, China) according to the manufacturer’s instructions. The synthesized PjCaspase mRNA containing fragment 3 (20 mg/ shrimp) was delivered into the shrimp at the juncture area between the third and forth abdominal section at 0.1 ml/shrimp using a syringe with a 29-gauge needle. Twelve hours later, the PjCaspase mRNA containing fragment 3 (20 mg/shrimp) was simultaneously injected into shrimp with WSSV (105 virion copies/ml) or Vibrio parahaemolyticus (106 copies/ml), respectively. As controls, the physiological saline only, WSSV (105 virion copies/ml) only and Vibrio parahaemolyticus (106 copies/ml) only were included in the injections. At various times after injection, the shrimp hemolymph was collected from the ventral sinus by inserting a 26-gauge needle containing heparin sodium (500 U/ml). Three shrimp specimens from each treatment, selected at random, were subjected to Northern blot and apoptosis assays, including detection of caspase-3 activity and DAPI staining of shrimp hemocytes. All assays were biologically repeated three times. Northern blot Total RNAs were extracted from shrimp hemolymph using TRIzol reagent kit (Invitrogen, USA). After separation in a 1.2% agrose gel, total RNAs were transferred to nylon membrane (Amersham Biosciences, USA), followed by probing with DIG- labeled PjCaspase or shrimp b-actin genes. The sequence of the PjCaspase probe (59-CACCTGGATT ACCTCGTCCAT-39) was identical in all PjCaspase molecules, while the sequence of the PjCaspase fragment 3 probe (59-CCTCTTCACAGGCCGAG- GATT-39) was specific for PjCaspase fragment 3 only. DIG labeling and detection were conducted using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Grenzacher- strasse, Basel, Switzerland) and BCIP/NBT (Amersham Biosci- ences, USA). Detection of caspase-3 activity As a classical measurement for evaluation of apoptosis, the shrimp caspse-3 activity was assayed. After washing with cold phosphate-buffered saline (PBS), shrimp hemolymph was disrupt- ed in a chilled lysis buffer (25 mM HEPES, 5 mM MgCl2, 5 mM EDTA, 5 mM dithiothreitol, 2 mM phenylmethylsulphonyl fluo- ride, pH 7.5). The lysate was centrifuged at 13,0006g for 5 min. The protein concentration of the supernatant was determined using Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercu- les, CA, USA) and adjusted to 0.02 mg protein/ml. Subsequently, its caspase-3 activity was detected using the Caspase-3 Color- rimetric Assay Kit (Keygen Biotech. Co., Ltd, Nanjing, Jiangshu, China), as described in the manufacturer’s manual. Briefly, 50 ml protein solution, 50 ml 26reaction buffer (40 mmol/L PIPES, 200 mmol/L NaCl, 2 mmol/L EDTA, 0.2% CHAPS, 20% sucrose, 20 mmol/L DTT, pH 7.2) and 5 ml caspase-3 substrate were mixed and then incubated at 37uC for 4 hours. The enzyme activity of caspase-3 was measured by monitoring the absorbance at 405 nm using a Perkin-Elmer LS50B fluorimeter. All assays were performed in triplicate. DAPI staining of shrimp hemocytes Shrimp hemocytes were smeared onto poly-L-lysine–treated slides and air-dried. Then the hemocytes were fixed by Gene Sequence Diversity for Antiviral Immunity paraformaldehyde solution (4% in PBS) for 30 min. After rinsing with 4, 6 diamidine-2-phenyllindole dihydrochloride (DAPI) solution, the slide was incubated in DAPI solution (1 mg/ml) at 37uC for 15 min. Subsequently the slide was washed with methanol before examining it using a fluorescence microscope. For each sample, 500 hemocytes were counted and scored for condensed and fragmented nuclei. The initial data was produced by NIS-Elements BR (NIKON fluorescence microscope software). Real-time PCR Virion copies of WSSV were quantified using WSSV-specific primers (59-TTGG TTTCA TGCCCGAGATT-39 and 59- CCTTGGTCAGCCCCTTGA-39) and TaqMan fluorogenic probe (59-FAM-TGCTGCCGTCTCCAA-TAMRA-39). The vi- ral genomic DNA was prepared from WSSV-infected shrimp using SQ Tissue DNA kit (OMEGA, America) and subjected to PCR. The PCR reaction mixture (25 ml) included WSSV genomic DNA, 200 nM of each primer, 100 nM of TaqMan probe and 16PCR reaction buffer containing DNA polymerase. PCR amplification was performed for 4 min at 50uC, followed by 45 cycles of 30 s at 94uC, 30 s at 52uC and 30 s at 72uC. Statistical analysis All numerical data from biologically independent experiments were analyzed using a one-way analysis of variance (ANOVA) to calculate the mean and standard deviation of repeated assays. Comparison of differences among treatments was carried out using Student’s t test. Supporting Information Figure S1 The sequence alignments of PjCaspase cDNA from various shrimp organs or tissues (shrimp 1). The shrimp actin gene was used as control. The numbers on the right showed the positions of nucleotide or amino acid, respectively. All point mutations were indicated below the original sequences. The mutations at the same position were shown with numbers on the top. a. The nucleotide sequence alignment and mutation analysis of the PjCaspase gene. b. The amino acid sequence alignment and mutation analysis of the PjCaspase gene. c. The nucleotide sequence alignment and mutation analysis of the shrimp actin gene. d. The amino acid sequence alignment and mutation analysis of shrimp actin. (TIF) Figure S2 The sequence alignments of PjCaspase cDNA from various shrimp organs or tissues (shrimp 2). The numbers on the right showed the positions of nucleotide or amino acid, respectively. All point mutations were indicated below the original sequences. The mutations at the same position were shown with numbers on the top. a. The nucleotide sequence alignment and mutation analysis of the PjCaspase gene. b. The amino acid sequence alignment and mutation analysis of the PjCaspase gene. (TIF) Figure S3 The genomic DNA sequence alignments of the shrimp PjCaspase gene. The shrimp actin gene was used as control. The numbers on the right showed the positions of nucleotide. All point mutations were indicated below the original sequences. The mutations at the same position were shown with numbers on the top. a. The genomic DNA sequence alignment and mutation analysis of the PjCaspase gene. b. The genomic DNA sequence alignment and mutation analysis of the actin gene. (TIF) PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24955