Chien-Lun Huang

Modulation of the innate immune system in white shrimp Litopenaeus vannamei following long-term low salinity exposure.

Immune parameters, haemocyte lifespan, and gene expressions of lipopolysaccharide and β-glucan-binding protein (LGBP), peroxinectin (PX), integrin β, and α2-macroglobulin (α2-M) were examined in white shrimp Litopenaeus vannamei juveniles (0.48 ± 0.05 g) which had been reared at different salinity levels of 2.5‰, 5‰, 15‰, 25‰, and 35‰ for 24 weeks. All shrimp survived during the first 6 weeks. The survival rate of shrimp reared at 2.5‰ and 5‰ was much lower (30%) than that of shrimp reared at 15‰, 25‰, and 35‰ (76%~86%) after 24 weeks. Shrimp reared at 25% grew faster. Shrimp reared at 2.5‰ and 5‰ showed lower hyaline cells (HCs), granular cells (GCs), phenoloxidase activity (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity, but showed a longer haemocyte lifespan, and higher expressions of LGBP, PX, integrin β, and α2-M. In another experiment, shrimp which had been reared at different salinity levels for 24 weeks were challenged with Vibrio alginolyticus (6 × 10(6) cfu shrimp(-1)), and WSSV (10(3) copies shrimp(-1)) and then released to their respective seawater. At 96-144 h, cumulative mortalities of shrimp reared at 2.5‰ and 5‰ were significantly higher than those of shrimp reared at 15‰, 25‰, and 35‰. It was concluded that following long-term exposure to 2.5‰ and 5‰ seawater, white shrimp juveniles exhibited decreased resistance against a pathogen due to reductions in immune parameters. Increases in the haemocyte lifespan and gene expressions of LGBP, integrin β, PX, and α2-M indicated that shrimp had the ability to expend extra energy to modulate the innate immune system to prevent further perturbations at low salinity levels.

Vaccination Enhances Early Immune Responses in White Shrimp Litopenaeus vannamei after Secondary Exposure to Vibrio alginolyticus

Background Recent work suggested that the presence of specific memory or some form of adaptive immunity occurs in insects and shrimp. Hypervariable pattern recognition molecules, known as Down syndrome cell adhesion molecules, are able to mount specific recognition, and immune priming in invertebrates. In the present study, we attempted to understand the immune response pattern of white shrimp Litopenaeus vannamei which received primary (PE) and secondary exposure (SE) to Vibrio alginolyticus. Methodology Immune parameters and proliferation of haematopoietic tissues (HPTs) of shrimp which had received PE and SE to V. alginolyticus were measured. In the PE trial, the immune parameters and proliferation of HPTs of shrimp that received heat-killed V. alginolyticus (HVa) and formalin-inactivated V. alginolyticus (FVa) were measured. Mortality, immune parameters and proliferation of HPTs of 7-day-HVa-PE shrimp (shrimp that received primary exposure to HVa after 7 days) and 7-day-FVa-PE shrimp (shrimp that received primary exposure to FVa after 7 days) following SE to live V. alginolyticus (LVa) were measured. Phagocytic activity and clearance efficiency were examined for the 7∼35-day-HVa-PE and FVa-PE shrimp. Results HVa-receiving shrimp showed an earlier increase in the immune response on day 1, whereas FVa-receiving shrimp showed a late increase in the immune response on day 5. The 7-day-FVa-PE shrimp showed enhancement of immunity when encountering SE to LVa, whereas 7-day-HVa-PE shrimp showed a minor enhancement in immunity. 7-day-FVa-PE shrimp showed higher proliferation and an HPT mitotic index. Both phagocytic activity and clearance maintained higher for both HVa-PE and FVa-PE shrimp after 28 days. Conclusions HVa- and FVa-receiving shrimp showed the bacteria agglutinated prior to being phagocytised. FVa functions as a vaccine, whereas HVa functions as an inducer and can be used as an immune adjuvant. A combined mixture of FVa and HVa can serve as a “vaccine component” to modulate the immunity of shrimp.

White shrimp Litopenaeus vannamei that received the hot-water extract of Gracilaria tenuistipitata showed protective innate immunity and up-regulation of gene expressions after low-salinity stress

White shrimp Litopenaeus vannamei which had been immersed in seawater (35 per thousand) containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 200, 400, and 600 mg L(-1) for 3 h, were subjected to a salinity transfer to 25 per thousand, and the immune parameters including hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, haemolymph protein concentration, and transcripts of the lipopolysaccharide- and beta-glucan-binding protein (LGBP), peroxinectin (PX), and alpha2-macroglobulin (alpha2-M) were examined 6-96 h post-transfer. Shrimp with no exposure to the hot-water extract and no salinity transfer served as the background control. Results indicated that HCs, GCs, THC, PO activity, RB, SOD activity, and haemolymph protein concentration of shrimp immersed in 600 mg L(-1) extract were significantly higher than those of control shrimp at 6-12 h post-transfer. Results also indicated that these parameters of shrimp immersed in 600 mg L(-1) extract had returned to the background values at 12, 6, 12, 6, 12, 24, and 24 h post-transfer with significant transcripts of LGBP, PX, and alpha2-M at 12 h, whereas these immune parameters in control shrimp had returned to the original values at 96 h post-transfer. It was therefore concluded that the innate immunity of L. vannamei which had been immersed in seawater containing the hot-water extract of G. tenuistipitata exhibited a protective effect against low-salinity stress as evidenced by increases in LGBP, PX, and alpha2-M transcripts, and earlier recovery of immune parameters.

White shrimp Litopenaeus vannamei that had received the hot-water extract of Spirulina platensis showed earlier recovery in immunity and up-regulation of gene expressions after pH stress.

White shrimp Litopenaeus vannamei which had been immersed in seawater (35‰, pH 8.2) containing the hot-water extract of Spirulina platensis at 0 (control), 200, 400, and 600 mg L(-1) for 3 h, were transferred to seawater at pH 6.8, and the immune parameters and transcripts of the lipopolysaccharide- and β-glucan-binding protein (LGBP), peroxinectin (PX), and integrin β (IB) were examined 6-96 h post-transfer. Shrimp with no exposure to the hot-water extract and no pH change served as the background control. Results indicated that the hyaline cells, granular cells (including semi-granular cells), total haemocyte count, phenoloxidase activity, respiratory burst, superoxide dismutase activity, glutathione peroxidase activity, and lysozyme activity of shrimp transferred to seawater at pH 6.8 significantly decreased to the lowest at 6 h post-transfer. These immune parameters of shrimp immersed in 600 mg L(-1) of the extract were significantly higher than those of control shrimp at 24-96 h post-transfer, and had returned to the background values earlier at 48-72 h post-transfer with significant transcripts of LGBP, PX, and IB at 24, 6, and 24 h, respectively, whereas these parameters of control shrimp returned to the original values at ≥96 h post-transfer.

Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) bind to seaweed polysaccharides and activate the prophenoloxidase system in white shrimp Litopenaeus vannamei

Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), important pattern recognition proteins (PRPs), recognize lipopolysaccharide (LPS) and β-1,3-glucan (βG), known as pathogen-associated molecular patterns (PAMPs), and subsequently trigger innate immunity. Several seaweed polysaccharides and seaweed extracts increase immune parameters and resistance to pathogens. Here, we constructed the expression vector pET28b-LvLGBP and transferred it into Escherichia coli BL21 (DE3) for protein expression and to produce the recombinant protein LGBP (rLvLGBP) in white shrimp Litopenaeus vannamei. We examined the binding of rLvLGBP with seaweed-derived polysaccharides including alginate, carrageenan, fucoidan, laminarin, Gracilaria tenuistipitata extract (GTE), and Sargassum duplicatum extract (SDE), and examined the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and each polysaccharide. We also examined the binding of rLvLGBP with LPS and βG, and the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS (rLvLGBP-LPS) or a mixture of rLvLGBP and βG (rLvLGBP-βG). An ELISA binding assay indicated that rLvLGBP binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE with dissociation constants of 0.1138-0.1770 μM. Furthermore, our results also indicated that the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE significantly increased by 328%, 172%, 200%, 213%, 197%, 194%, 191%, and 197%, respectively compared to controls (cacodylate buffer). We conclude that LvLGBP functions as a PRP, recognizes and binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE, and subsequently leads to activating innate immunity in shrimp.

Activation of immunity, immune response, antioxidant ability, and resistance against Vibrio alginolyticus in white shrimp Litopenaeus vannamei decrease under long-term culture at low pH

The growth, activation of immunity, immune parameters, and transcript levels of cytMnSOD, mtMnSOD, ecCuZnSOD, glutathione peroxidase (GPx), catalase, lysozyme, and penaeidin 3a were examined in white shrimp Litopenaeus vannamei reared at pH 6.8 and 8.1 after 24 weeks. No significant difference in growth was observed between the two groups. An in vitro study indicated that phenoloxidase activity and respiratory bursts (RB, release of the superoxide anion) were significantly higher in the haemocytes of pH 8.1 shrimp (shrimp reared at pH 8.1) than in pH 6.8 shrimp (shrimp reared at pH 6.8). An in vivo study indicated that the levels of immune parameters of pH 8.1 shrimp were significantly higher than in pH 6.8 shrimp, and the transcript levels of cytMnSOD, ecCuZnSOD, glutathione peroxidase, lysozyme, and penaeidin 3a were down-regulated in pH 6.8 shrimp. In another experiment, shrimp reared at pH 6.8 and 8.1 for 24 weeks were challenged with Vibrio alginolyticus. The mortality rate of pH 6.8 shrimp was significantly higher than in pH 8.1 shrimp over 12-168 h. Phagocytic activity, phagocytic index, and clearance efficiency to V. alginolyticus were significantly lower in pH 6.8 shrimp. We concluded that shrimp under long-term culture at pH 6.8 exhibited decreased resistance against V. alginolyticus as evidenced by reductions in the activation of immunity and immune parameters together with decreased transcript levels of cytMnSOD, ecCuZnSOD, GPx, lysozyme, and penaeidin 3a.

Crowding of white shrimp Litopenaeus vananmei depresses their immunity to and resistance against Vibrio alginolyticus and white spot syndrome virus.

Immunity parameters and the expression levels of several immune-related proteins, including lipopolysaccharide and β-glucan binding protein (LGBP), peroxinectin (PX), intergin β (IB), prophenoloxidase (proPO) I, proPO II, α2-macroglobulin (α2-M), cytosolic mangangese superoxide dismutase (cytMnSOD), mitochondria manganese superoxide dismutase (mtMnSOD), catalase, glutathione peroxidase (GPx), lysozyme, and penaeidin 3a were examined in white shrimp Litopenaeus vannamei reared at stocking densities of 2, 10, 20, 30, and 40 shrimp L(-1) after 3, 6, and 12 h. All immune parameters including haemocyte count, phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, lysozyme activity, and haemolymph protein were negatively related to density and time. The PO activity, SOD activity, and lysozyme activity of shrimp reared at 10 shrimp L(-1) after 12 h significantly decreased. The transcript levels of these immune-related proteins were down-regulated in shrimp reared at 20, 30, and 40 shrimp L(-1) after 12 h. Phagocytic activity and clearance efficiency to Vibrio alginolyticus were significantly lower in shrimp reared at 30 and 40 shrimp L(-1) after 12 h. The mortality rates of shrimp reared at 20 and 40 shrimp L(-1) were significantly higher than shrimp reared at 2 shrimp L(-1) over 12-144 h and 12-48 h, respectively. Shrimp reared at high densities (>10 shrimp L(-1)) exhibited decreased resistance against pathogens as evidenced by reductions in immune parameters together with decreased expression levels of immune-related proteins, indicating perturbations of the immune system.

White Shrimp Litopenaeus vannamei That Have Received Gracilaria tenuistipitata Extract Show Early Recovery of Immune Parameters after Ammonia Stressing

White shrimp Litopenaeus vannamei immersed in seawater (35‰) containing Gracilaria tenuistipitata extract (GTE) at 0 (control), 400, and 600 mg/L for 3 h were exposed to 5 mg/L ammonia-N (ammonia as nitrogen), and immune parameters including hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, lysozyme activity, and hemolymph protein level were examined 24~120 h post-stress. The immune parameters of shrimp immersed in 600 mg/L GTE returned to original values earlier, at 96~120 h post-stress, whereas in control shrimp they did not. In another experiment, shrimp were immersed in seawater containing GTE at 0 and 600 mg/L for 3 h and examined for transcript levels of immune-related genes at 24 h post-stress. Transcript levels of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), cytMnSOD, mtMnSOD, and HSP70 were up-regulated at 24 h post-stress in GTE receiving shrimp. We concluded that white shrimp immersed in seawater containing GTE exhibited a capability for maintaining homeostasis by regulating cellular and humoral immunity against ammonia stress as evidenced by up-regulated gene expression and earlier recovery of immune parameters.