Fish and sample preparation
A total of 200 E. coioides were obtained from Dayawan Aquaculture Center of Guangdong province, China. The mean body length of these fish was 25 ± 3 cm, and the mean body weight was 250 ± 50 g. All fish were allowed to adapt for 6 d in 100-L opaque tanks (10 fish per tank) supplied with re-circulated seawater. Water temperature was maintained at 27 ± 1°C with an air conditioner. For the tissue-specific expression study, healthy fish were anesthetized with 150 mg/L tricaine methanesulfonate (MS-222, Sigma), and then head kidney, spleen, thymus gland, intestine, gill, blood, heart, muscle, and gonad were collected, immediately frozen in liquid nitrogen and stored at -80°C.
RNA extraction and complementary DNA preparation
Total RNA was extracted from grouper tissues using TRIzol reagent (Invitrogen, Carlsbad, California, USA) according to manufacturer instructions. The RNA was quantified using a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and the first complementary DNA (cDNA) was synthesized with a M-Mulv Reverse Transcriptase cDNA Synthesis Kit (TaKaRa, Japan) following the manufacturer protocol. The first cDNA was stored at -20°C prior to use.
Preparation of V. alginolyticus
The V. alginolyticus strain was obtained from the Institute of Hydrobiology, Jinan University (Guangzhou, China). The strain was recovered at 30°C for 18 h on a medium plate, then a single colony was picked and inoculated into 5 mL broth at 28°C for 18 h in a shaker incubator at 150 rpm. The harvested V. alginolyticus cells were centrifuged at 10000×g for 10 min, the supernatant was removed, and V. alginolyticus was washed three times with 1% sodium chloride (NaCl). The V. alginolyticus was resuspended in 1% sodium chloride with a concentration of 5.0×104 CFU/mL and stored at 4°C prior to use.
To prepare inactivated V. alginolyticus, 10 mL of the bacteria solution was obtained as above. The concentration of V. alginolyticus was determined by a plate count method. Then 0.5% formalin was added to inactivate the bacteria. The inactivated V. alginolyticus was centrifuged and washed as described above. Finally, V. alginolyticus was resuspended in 1% sodium chloride at a concentration of 2.0×105 CFU/mL.
Vibrio alginolyticus stimulation and IgZ expression analysis
To analyze the IgZ expression induced by V. alginolyticus infection, 90 fish were randomly transferred into six 100-L tanks (10 fish per group) each containing 80 L seawater. Fish in the infection treatment were injected with 100 μL V. alginolyticus solution at a concentration of 5.0×104 CFU/mL by intraperitoneal injection. Fish in the control treatment were injected with 100 μL 1% NaCl solution. Five fish were randomly sampled at 0 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, 5 d, 6 d, 14 d, 21 d, and 28 d post-infection. The head kidney, spleen, thymus gland, intestine, gill, blood, and heart were collected after fish were anesthetized with 150 mg/L MS-222. All samples were immediately frozen in liquid nitrogen and stored at −80°C for total RNA extraction.
To analyze the IgZ expression stimulated by the inactivated V. alginolyticus, 90 fish were randomly transferred into seven 100-L tanks (10 fish per group) containing 80 L seawater. Fish in the infection treatment were injected with 100 μL inactivated V. alginolyticus solution at a concentration of 2×105 CFU/mL by intraperitoneal injection. Fish in control treatment were injected with 100 μL 1% NaCl solution. Five fish were randomly sampled at 0 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, 5 d, 6 d, 14 d, 21 d, 28 d, and 35 d post-stimulation. The head kidney, spleen, thymus gland, intestine, gill, blood, and heart were collected after fish were anesthetized with 150 mg/L MS-222. All samples were immediately frozen in liquid nitrogen and stored at -80°C for total RNA extraction.
IgZ expression in head kidney, spleen, thymus gland, intestine, gill, blood, and heart on different days after V. alginolyticus stimulation was analyzed by real-time quantitative PCR (qPCR). All samples had five replications, and the amplification was carried out in iCycler iQ Real-time PCR Detection System (Bio-Rad, Hercules, California, USA) with an initial denaturing step of 95°C for 3 min, followed by 40 cycles of 94°C for 20 s, 60°C for 20 s, 72°C for 30 s. To ensure only one specific-sized single amplicon was amplified, the specificity of the primer was determined by a dissociation curve. The relative quantification of the IgZ gene was normalized with an internal control (b-actin).
In situ hybridization
A digoxigenin (DIG)-labeled RNA probe and control probe for IgZ were prepared according to the method described by Sato et al. . The head kidney, spleen, intestine, gill, and liver samples were directly placed into 4% paraformaldehyde (PFA) to remove the residual blood. The samples were transferred into 4% PFA with 10 times volume and fixed for 24 h at 4°C. Then, the samples were treated with four processes: (1) dehydration and embedding, (2) tissue sectioning, (3) prehybridization, and (4) hybridization. The more detailed process was similar to a previous study .
Immunohistochemistry on paraffin sections
The anti-IgZ polyclonal antibody used in immunohistochemical procedures was obtained as in a previous study .
The head kidney, spleen, intestine, gill, and heart samples were directly placed into 4% PFA to remove the residual blood. The fixation, dehydration, embedding, sectioning, and deparaffinization of tissues were conducted as described above. Antigen retrieval was performed by boiling slides in citrate antigen retrieval solution (sodium citrate 21.01 g, citric acid 26.41 g, H2O 1000 mL, and pH 6.0) in a pressure cooker for 6 min. Endogenous peroxidase activity was quenched by incubating sections in 1.5% H2O2 for 10 min. The sections were incubated in blocking solution for 15 min at room temperature to block non-specific binding of antibodies. Antibodies at a dilution (in blocking solution) of 1:1000 were added to react for 2 h at 37°C. The sections were washed three times in 1×PBS for 3 min. Thereafter the slides were incubated for 15 min at 37°C in secondary antibody (goat anti-rabbit antibody) and then washed times in 1×PBS for 3 min. Bound antibody was visualized by incubating slides in DAB for 3 min at room temperature. All sections were counterstained with Ehrlich hematoxylin for 30 s and subsequently washed with dilute ammonia water, followed by washing with water. All sections were dehydrated in graded ethanol series, followed by treating in xylene. The sections were mounted with neutral balsam. All sections were examined using a Nikon 80i microscope (Japan).
Relative gene expression levels were calculated using the 2-△△Ct method . The assumptions of normality and homogeneity of variances were confirmed using the Shapiro-Wilk test and Levene's test, respectively. One-way ANOVA was used to analyze the data in SPSS 19.0 software. The results were considered statistically significant at p < 0.05 and p < 0.01.