Gene Sequence Diversity for Antiviral Immunity Figure 5. The model for the role of the PjCaspase gene containing fragment 3 in immunity against virus infection. When the shrimp was challenged by various pathogens, such as WSSV or Vibrio parahaemolyticus, the PjCaspase gene containing fragment 3 produced by gene sequence diversification was specifically initiated to induce apoptosis in response to virus infection. Subsequently the virus infection was inhibited by apoptosis in cells carrying the PjCaspase gene containing fragment 3. The apoptosis caused by Vibrio was not included in this model. doi:10.1371/journal.pone.0024955.g005 pumped circulating sea water at 25uC. For the virus injection assay, the tissues of WSSV-infected shrimp (confirmed by PCR) were homogenized in TN buffer (20 mM Tris-HCl, 400 mM NaCl, pH 7.4) at 0.1 g/ml, followed by centrifugation at 2,0006g for 10 min [2]. The supernatant was diluted to 1:100 with filter- sterilized 0.9% NaCl. Subsequently, 0.1 ml of filtrate (104 virus copies/ml) was injected intramuscularly into virus- free shrimp in the lateral area of the fourth abdominal segment using a syringe with a 29-gauge needle. At different time intervals p.i., the hemolymph of three randomly selected specimens was collected, mixed and immediately stored at 280uC. In order to obtain WSSV-resistant shrimp, the virus-free shrimp was challenged with WSSV by intramuscular injections weekly over a four-week period, as described by Wu et al. [2]. The survivors (approximately 1–5%) were collected as WSSV-resistant shrimp (a pre-existing subpopu- lation resistant against WSSV) which were WSSV-free as detected by PCR. The unchallenged shrimp were used as a control. Challenge of shrimp by its pathogenic bacterium Vibrio parahaemolyticus Vibrio parahaemolyticus, a pathogen of shrimp, was grown in LB (Luria-Bertani) medium [27]. The challenge of shrimp by Vibrio parahaemolyticus was conducted by intramuscular injection of 0.1 ml of bacterial suspension (16106 CFU/ml) in the lateral area of the fourth abdominal segment, using a syringe with a 29-gauge needle. At different times p.i., the hemolymph of three specimens randomly selected was collected, mixed and immediately stored at 280uC for later use. Cloning and sequencing of PjCaspase cDNA Total RNAs of different tissues or organs including hemolymph, hepatopancreas, muscle, gill and intestine from a shrimp individual were extracted using TRIzol kit according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), followed by treatment with DNase I (Roche, Grenzacherstrasse, Basel, Switzerland) to remove any genomic DNA contamination. The concentration of total RNAs was determined spectrophoto- metrically at OD260 and the integrity of total RNAs was examined by electrophoresis on a 2% agarose gel. The extracted RNAs were stored at _80uC for future use. For reverse transcription, 1 mg of total RNAs was mixed with 1 ml oligo-d(T)18 primer and diethypyrocarbonate (DEPC)-treated water to a final volume of 12 ml. After incubation at 70uC for 5 min, the mixture was cooled on ice. A reaction mix of 1 ml M-MLV RT PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24955