Figure 1.
Fluorescence micrographs and light micrographs of shrimp hemocytes incubated in shrimp salt solution (SSS), lipopolysaccharide (LPS), β-1,3-glucan (βG), peptidoglycan (PG) and percentage of necrotic cell.
(A) Fluorescence micrographs and light micrographs of shrimp hemocytes incubated in SSS (control) for 30 min. (B) Fluorescence micrographs and light micrographs of shrimp hemocytes incubated in 0.1 mg/ml LPS for 30 min. (C) Fluorescence micrographs and light micrographs of shrimp hemocytes incubated in 0.1 mg/ml βG for 30 min. (D) Fluorescence micrographs and light micrographs of shrimp hemocytes incubated in 0.1 mg/ml PG for 30 min. (E) Percentage of necrotic cells of shrimp hemocytes incubated in SSS (control) and different concentrations (0.1, 0.5, and 1.0 mg/ml) of LPS, βG and PG after 30 min. Each bar represents the mean value from six shrimp with standard error. Data with different letters significantly differ (p <0.05) at the same DAMP (LPS, βG, or PG) among different concentrations. Percentages of necrotic cells of hemocytes incubated in 0.1, 0.5, and 1.0 mg/ml LPS, in 0.1, 0.5, and 1.0 mg/ml βG, or in 0.1, 0.5, and 1.0 mg/ml PG were significantly higher than that in controls (SSS). Scale = 20 µm. N, Nucleus; NL, Nuclei lysed (death cell).
Figure 2.
SDS-PAGE analysis of EMs (EM-C, EM-L, EM-β, and EM-P) with Coomassie staining (A) and silver staining (B).
Lane M, Molecular mass markers; Lane 1, EM-C; Lane 2, EM-L; Lane 3, EM-β; Lane 4, EM-P. Lane 5, Relative position. EM-C, endogenous molecules of shrimp hemocytes incubated in shrimp salt solution (SSS); EM-L, endogenous molecules of shrimp hemocytes incubated in 1.0 mg/ml lipopolysaccharide (LPS). EM-β, endogenous molecules of shrimp hemocytes incubated in 1.0 mg/ml β-1,3-glucan (βG); EM-P, endogenous molecules of shrimp hemocytes incubated in 1.0 mg/ml peptidoglycan (PG).
Table 1.
The proteins identified by LC-ESI-MS/MS in the EM-C, EM-L, and EM-P.
Figure 3.
Monolayer of shrimp hemocytes incubated in EM-C, EM-L, EM-β, EM-P.
(A) Monolayer of shrimp hemocytes in EM-C. (B) Monolayer of shrimp hemocytes incubated in EM-L. (C), Monolayer of shrimp hemocytes incubated in EM-β. (D) Monolayer of shrimp hemocytes incubated in EM-P. dg, degranulation; Ps, pseudopodia; Scale = 10 µm. EM-C, endogenous molecules of shrimp hemocytes incubated in shrimp salt solution (SSS); EM-L, endogenous molecules of shrimp hemocytes incubated in 1.0 mg/ml lipopolysaccharide (LPS). EM-β, endogenous molecules of shrimp hemocytes incubated in 1.0 mg/ml β-1,3-glucan (βG); EM-P, endogenous molecules of shrimp hemocytes incubated in 1.0 mg/ml peptidoglycan (PG).
Figure 4.
Changes in cell viability and necrosis of shrimp hemocytes incubated in EM-C, EM-L, EM-β, and EM-P, and percentage of necrotic cell of different EMs.
(A) Viable and nonviable cells are designated in marker 3 (M3) and marker 4 (M4), respectively. Changes in viability over time of shrimp hemocytes showing the kinetics and proportion of viable hemocyte cells incubated in EM-C, EM-L, EM-β, and EM-P. Cell viability of shrimp hemocytes receiving shrimp salt solution (SSS) served as the control group. (B–E) Fluorescence micrographs and light micrographs showing necrosis of shrimp hemocytes incubated in EM-C (B), EM-L (C), EM-β (D), and EM-P (E) for 30 min. (F) Percentage of necrotic cells of shrimp hemocytes incubated in EM-C, EM-L, EM-β, and EM-P for 30 min. Each bar represents the mean value from six shrimp with standard error. Data with different letters significantly differ (p <0.05) among treatments. The percentages of necrotic cells of shrimp hemocytes incubated in EM-L, EM-β, and EM-L were significantly higher than in EM-C. See Fig. 2 for the abbreviation of EM-C, EM-L, EM-β, and EM-P. N, Nucleus; NL, Nuclei lysed (dead cells); Scale = 20 µm.
Figure 5.
Phenoloxidase (PO) activity and respiratory burst (RB, release of superoxide anion) of shrimp hemocytes incubated with EM-C, EM-L, EM-β and EM-P.
(A) PO activity of shrimp hemocytes incubated in heat-inactivated EM-C (HEM-C), EM-C, heat-inactivated EM-L (HEM-L), and EM-L. (B) RB of shrimp hemocytes incubated in HEM-C, EM-C, HEM-L, and EM-L. (C) PO activity of shrimp hemocytes incubated in HEM-C, EM-C, heat-inactivated EM-β (HEM-β), and EM-β. (D) RB of shrimp hemocytes incubated in HEM-C, EM-C, HEM-β, and EM-β. (E) PO activity of shrimp hemocytes incubated in HEM-C, EM-C, heat-inactivated EM-P (HEM-P), and EM-P. (F) RB of shrimp hemocytes incubated in HEM-C, EM-C, HEM-P, and EM-P. CAC served as negative control for the PO activity assay and MCHBSS served as the negative control for the RB assay. Trypsin (Try) served as the positive control for PO activity assay and zymosan served as the positive control for RB assay. See Fig. 2 for the abbreviation of EM-C, EM-L, EM-β, and EM-P. Each bar represents the mean value from eight shrimp with standard error. Data in the same parameter with different letters significantly differ (p <0.05) among treatments.
Figure 6.
The immune parameters of white shrimp Litopenaeus vannamei that received EM-C, EM-L, EM-β, and EM-P after 12 and 24 h.
The immune parameters of shrimp without any treatment served as the control group. (A) hyaline cells (HCs); (B) granular cells (including semigranular cells, GCs); (C) total hemocyte count (THC); (D) phenoloxidase activity; (E) respiratory burst (RB, release of superoxide anion); (F) superoxide dismutase (SOD) activity; (G) lysozyme activity; (H) relative immune parameter (%) of control shrimp, and shrimp receiving EM-C, EM-L, EM-β, and EM-P after 12 and 24 h. See Fig. 2 for the abbreviation of EM-C, EM-L, EM-β, and EM-P. Each bar represents the mean value from eight shrimp with standard error. Data in the same parameter and same time with different letters significantly differ (p <0.05) among treatments.
Figure 7.
Cell proliferation cell ratio (%) and mitotic index (%) in hematopoietic tissues (HPTs).
(A) Optical micrograph of H&E-stained HPTs of control shrimp (a) and shrimp receiving EM-L (b) after 24 h. A higher number of mitotic cells (arrows) were observed in shrimp receiving EM-L. (B) Cell proliferation cell ratio (%) in HPTs of control shrimp and shrimp receiving EM-L. (C) Fluorescence micrograph of propodium iodide-stained HPTs of control shrimp (a), and shrimp receiving EM-L (b) after 24 h. A higher number of mitotic cells (arrows) were observed in shrimp receiving EM-L. (D) Mitotic index (%) in HPTs of control shrimp and shrimp receiving EM-L. Scale = 20 µl. Each bar represents the mean value from ten images with standard error. Data in the same parameter with different letters significantly differ (p <0.05) between treatments. Shrimp received marine saline served as control shrimp. See Fig. 2 for the abbreviation of EM-L.
Figure 8.
Phagocytic activity of shrimp that received EM-C, EM-L, EM-β, and EM-P after 24 h and then received Vibrio alginolyticus challenge.
Phagocytic activity of shrimp receiving marine saline after 24 h and then receiving V. alginolyticus served as the control group. Phagocytic activity of shrimp receiving EM-L, EM-β, and EM-P was significantly higher than in control and shrimp receiving EM-C. Each bar represents the mean value from eight shrimp with standard error. See Fig. 2 for the abbreviation of EM-C, EM-L, EM-β, and EM-P. Data with different letters significantly differ (p <0.05) among treatments.
Figure 9.
Diagram showing the immune activation of endogenous molecules (EMs) induced by lipopolysaccharide (LPS), β-1,3-glucan (βG), and peptidoglycan (PG).
EMs contains DAMPs including HMGBa, HMGB, and other DAMPs (H2a, H2b, H4), signaling transduction molecules (Rab 7 GTPase, Rab 11 GPTase) and other immune molecules.