Figure 1.
Experimental design diagram showing 1) primary exposure of white shrimp Litopenaeus vannamei that received heat-killed Vibrio alginolyticus (HVa) and shrimp that received formalin-inactivated V. alginolyticus (FVa), 2) showing secondary exposure of live V. alginolyticus (LVa) in shrimp that had received HVa and FVa after 7 days, and 3) showing phagocytosis and clearance to LVa and live Bacillus subtilis (LBs) in both 7∼35-day-HVa-PE and 7∼35-day-FVa-PE shrimp, and 7∼28-day-HVa-PE shrimp and 7∼28-day-PE shrimp.
Figure 2.
Hyaline cells (HCs) (A), granular cells (GCs) (including semi-GCs) (B), and the total haemocyte count (THC) (C) of control white shrimp Litopenaeus vannamei, and shrimp that received marine saline (MS), heat-killed Vibrio alginolyticus (HVa), formalin-inactivated V. alginolyticus (FVa), and live V. alginolyticus (LVa) after 0, 0.5, 1, 3, 5 and 7 days.
Each bar represents the mean from six shrimp with the standard error (SE). Data at the same exposure time with different letters (a, b, c) significantly differ (p<0.05) among treatments. Data at the same treatment with different letter (x, y, z) significantly differ (p<0.05) among different elapsed time periods (days).
Figure 3.
Phenoloxidase (PO) activity (A), respiratory bursts (RBs) (B), superoxide dismutase (SOD) activity (C) and lysozyme activity (D) of control white shrimp Litopenaeus vannamei, and shrimp that received marine saline (MS), heat-killed Vibrio alginolyticus (HVa), formalin-inactivated V. alginolyticus (FVa), and live V. alginolyticus (LVa) after 0, 0.5, 1, 3, 5 and 7 days.
See Fig. 2 for statistical information.
Figure 4.
Relative immune parameters (%) of white shrimp Litopenaeus vannamei that received heat-killed Vibrio alginolyticus (HVa), formalin-inactivated V. alginolyticus (FVa), and live V. alginolyticus (LVa) after 0.5, 1, 3, 5 and 7 days.
Data (mean ± SE) with different letters (a, b, c) in shrimp that received HVa, with different letters (r, s, t) in shrimp that received FVa, and with diffferent letters (x, y, x) in shrimp that received LVa significantly differ (p<0.05) among different elapsed time periods (days).
Figure 5.
(A) Optical micrographs of H&E-stained haematopoietic tissues (HPTs) of control shrimp (a), and shrimp that received heat-killed Vibrio alginolyticus (HVa) (b) and formalin-inactivated V. alginolyticus (FVa) (c) after 5 days. Higher numbers of mitotic cells (arrows) were observed in shrimp that received HVa and shrimp that receive FVa.
(B) Proliferation cell ratios (%) in HPTs of shrimp that received FVa and shrimp that received HVa were significantly higher than that of control shrimp. (C) Fluorescent micrographs of propidium iodide-stained HPTs of control shrimp (a), shrimp that received HVa (b), and shrimp that received FVa (c) after 5 days. Higher numbers of mitotic cells (arrows) were observed in shrimp that received HVa and shrimp that received FVa. (D) The mitotic index, expressed as a percentage of dividing cells in HPTs of shrimp that received FVa and those that received HVa was significantly higher than that of control shrimp. Each bar represents the mean value from six different images with the standard error. Data with different letters significantly differ (p<0.05) among treatments. Scale = 20 µm.
Figure 6.
Survival rates of unchallenged control white shrimp Litopenaeus vannamei, control shrimp, and shrimp that received heat-killed Vibrio alginolyticus (HVa) and formalin-inactivated V. alginolyticus (FVa) after 7 days, and then challenged with live V. alginolyticus (LVa) at a dose of 8.0×106 cfu shrimp−1.
Each data point represents the mean of three replicates (10 shrimp in each replicate) with the standard error (SE). Data with different letters in the same time period significantly differ (p<0.05) among treatments.
Figure 7.
Hyaline cell (HC) (A), granular cells (GCs) (including semi-GCs) (B), and total haemocyte count (THC) (C) of control white shrimp Litopenaeus vannamei, and shrimp that received heat-killed Vibrio alginolyticus (HVa) and formalin-inactivated V. alginolyticus (FVa) after 7 days, and then were challenged with V. alginolyticus at a dose of 3.8×105 cfu shrimp−1 after 0, 0.5, 1, 3, 5 and 7 days.
See Fig. 2 for statistical information.
Figure 8.
Phenoloxidase (PO) activity (A), respiratory bursts (RBs) (B), superoxide dismutase (SOD) activity (C), and lysozyme activity (D) of control white shrimp Litopenaeus vannamei, and shrimp that received heat-killed Vibrio alginolyticus (HVa), and formalin-inactivated V. alginolyticus (FVa) after 7 days, and then were challenged with V. alginolyticus at a dose of 3.8×105 cfu shrimp−1 after 0, 0.5, 1, 3, 5 and 7 days.
See Fig. 2 for statistical information.
Figure 9.
(A) Relative immune parameter (%) of control white shrimp Litopenaeus vannamei, and shrimp that received heated-killed Vibrio alginolyticus (HVa) and formalin-inactivated V. alginolyticus (FVa) after 7 days and then were challenged with live V. alginolyticus.
Data (mean ± SE) with different letters (x, y, z) in control shrimp, with different letters (r, s, t) in shrimp that received HVa, and with different letters (a, b, c) in shrimp that received FVa significantly differed (p<0.05) among elapsed times (day). (B) Relative values of HVa-receiving shrimp and FVa-receiving shrimp to that of control shrimp. Data with different leters (r, s, t) in HVa-receiving shrimp, and with different letters (a, b, c) in FVa-receiving shrimp significantly differ (p<0.05) among elapsed times (day).
Figure 10.
(A) Optical micrographs of H&E-stained haematopoietic tissues (HPTs) of control shrimp (a), shrimp that received heat-killed Vibrio alginolyticus (HVa) (b), and formalin-inactivated V. alginolyticus (FVa) (c) after 7 days and then received live V. alginolyticus (LVa) after 3 days.
Higher numbers of mitotic cells (arrows) were observed in shrimp that received FVa. (B) The proliferation cell ratio (%) in HPTs of shrimp that received FVa after 7 days and then challenged with LVa after 3 days was significantly higher. (C) Fluorescent micrographs of propidium iodide-stained HPTs of control shrimp (a), and shrimp that received HVa (b) and FVa (c) after 7 days, and then were challenged with LVa after 3 days. Higher numbers of mitotic cells (arrows) were observed in shrimp that received FVa. (D) The mitotic index, expressed as a percentage of dividing cells in HPTs of shrimp that received FVA after 7 days, and then challenged with LVa after 3 days was significantly higher. Scale = 20 µm. See Fig. 5B for statistical information.
Figure 11.
Agglutinative activity of control white shrimp (A), shrimp that received heat-killed Vibrio alginolyticus (HVa) (B), and shrimp that received formalin-inactivated V. alginolyticus (FVa) (C) after 7 days, and then were subjected to live V. alginolyticus (LVa).
Shrimp that received FVa and HVa showed higher agglutinative activities. Agglutinated “bacteria cluster” phagocytized by hyaline cells (HC) (D) and semi-granular cell (SGC) (E) were observed in both 7-day-HVa-PE shrimp and 7-day-FVa-PE shrimp, by both HC (F) and SGC (G) in 14-day-HVa-PE shrimp and 14-day-PE shrimp, and by both HC (H) and SGC (I) in 21-day-Hva-PE shrimp and 21-day-FVa-PE shrimp, and then received SE. The intact of different V. alginolyticus including LVa (J), HVa (K), and FVa (L) were strained with Liu’s straining. Scale = 5 µm (A∼I), and 2 µm (J∼L).
Figure 12.
Phagocytic activity (A), phagocytic index (B), and clearance efficiency (C) of control shrimp, 7∼35-day-HVa-PE shrimp, and 7∼35-day-FVa-PE shrimp and then received live Vibrio alginolyticus (LVa).
Phagocytic activity (D), phagocytic index (E), and clearance efficiency (F) of control shrimp, 7∼28-day-HVa-PE shrimp, and 7∼28-day-FVa-PE shrimp and then received live Bacillus sublitis (LBs). Data at the same exposure time with different letter (a, b, c) significantly differ (p<0.05) among different treatments.