White shrimp Litopenaeus vannamei that have received mixtures of heat-killed and formalin-inactivated Vibrio alginolyticus and V. harveyi exhibit recall memory and show increased phagocytosis and resistance to Vibrio infection.

Heat-killed Vibrio alginolyticus (HVa), formalin-inactivated V. alginolyticus (FVa), heat-killed Vibrio harveyi (HVh), formalin-inactivated V. harveyi (FVh), live V. alginolyticus (LVa), and live V. harveyi (LVh) were used in this study. White shrimp Litopenaeus vannamei receiving two mixtures (HVa + FVa) or four mixtures (HVa + FVa + HVh + FVh) served as primary exposure, and shrimp receiving LVa or LVh afterward served as secondary exposure. Shrimp receiving marine saline and then receiving either LVa or LVh served as controls. Phagocytic activity and clearance efficiency were examined in shrimp that received two mixtures after 1-8 weeks and then received LVa. Both the phagocytic activity and clearance efficiency of shrimp receiving two mixtures were significantly higher than in control shrimp after 1-8 weeks. In another experiment, phagocytic activity and clearance efficiency were examined in shrimp that received four mixtures after 1-8 weeks and then received LVa and LVh, respectively. The phagocytic activity of shrimp receiving four mixtures was significantly higher than in control shrimp after 1-8 weeks post exposure to LVa and LVh. The clearance efficiency of shrimp receiving four mixtures was significantly higher than in control shrimp after 1-6 weeks post exposure to LVa, and 1-7 weeks post exposure to LVh. In the other experiment, the survival rate of shrimp that received four mixtures after five weeks were challenged with LVa at 6.4 × 107 colony-forming units (cfu) shrimp-1 and LVh at 4.4 × 106 cfu shrimp-1. Shrimp that received marine saline for five weeks and then challenged with LVa and LVh at a same dose served as challenged controls. The survival rate of shrimp that received four mixtures was significantly higher (90%) than that of control shrimp (67%), and significantly higher (73%) than that of control shrimp (53%) after 3-7 days post challenge with LVa and LVh. It is concluded that the mixtures have feature of adjuvant and antigen, and shrimp receiving mixtures of heat-killed and formalin-inactivated V. alginolyticus and V. harveyi even after 5-8 weeks exhibit memory recall and show increased phagocytosis and resistance to Vibrio infections.

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

Lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP), important pattern recognition proteins (PRPs), recognize lipopolysaccharide (LPS) and ß-1,3-glucan (ßG), 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 ßG, and the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS (rLvLGBP-LPS) or a mixture of rLvLGBP and ßG (rLvLGBP-ßG). An ELISA binding assay indicated that rLvLGBP binds to LPS, ßG, 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, ßG, 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, ßG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE, and subsequently leads to activating innate immunity in shrimp.

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 ß-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.

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 ß-glucan binding protein (LGBP), peroxinectin (PX), intergin ß (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.

Endogenous molecules induced by a pathogen-associated molecular pattern (PAMP) elicit innate immunity in shrimp.

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), ß-1,3-glucan (ßG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, ßG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-ß, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-ß, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-ß, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity.

Shrimp that have received carrageenan via immersion and diet exhibit immunocompetence in phagocytosis despite a post-plateau in immune parameters.

The effect of carrageenan on the immune response of white shrimp Litopenaeus vannamei, was studied in vitro and in vivo. Shrimp haemocytes receiving carrageenan at 1 mg ml⁻¹ experienced change in cell size, reduction in cell viability, increase in PO activity, serine proteinase activity, and RB in vitro. Shrimp received carrageenan via immersion at 200, 400 and 600 mg L⁻¹ after 3 h and orally at 0.5, 1.0 and 2.0 g kg⁻¹ after 3 weeks showed higher proliferation of haematopoietic tissues (HPTs) together with increases in haemocyte count and other immune parameters. Shrimp that fed a diet containing carrageenan at 0.5 g kg⁻¹ after 3 weeks significantly up-regulated gene expressions of several immune-related proteins. The immune parameters of shrimp that received carrageenan via immersion and orally increased to a plateau after 3 h and after 3 weeks, but decreased after 5 h and 6 weeks, respectively. Phagocytosis and clearance of Vibrio alginolyticus remained high in shrimp that had received carrageenan via immersion after 5 h and orally after 6 weeks, respectively. Resistances of shrimp against V. alginolyticus and white spot syndrome virus were higher over 24-144 h and 72-144 h, respectively in shrimp that received carrageenan at 600 mg L⁻¹ via immersion after 3 and 5 h. It was concluded that carrageenan effectively triggers an innate immunity in vitro, and increases mitotic index of HPT, immune parameters, gene expressions and resistance against pathogens in vivo. Shrimp received carrageenan via immersion and orally exhibited immunocompetence in phagocytosis and clearance of V. alginolyticus, and resistance to pathogen despite the trend in immune parameters to recover to background values.

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.

Characterization of white shrimp Litopenaeus vannamei integrin ß and its role in immunomodulation by dsRNA-mediated gene silencing.

The full sequence of white shrimp Litopenaeus vannamei integrin ß (LV-B) is 2879bp which encodes 787 amino acids (aa) of the open reading frame (ORF). The mature protein (764 aa) contains (1) an extracellular domain (ED) of 692 aa, (2) a transmembrane domain (TD) of 23 aa, and (3) a cytoplasmic domain (CD) of 49 aa. The cloned LV-B grouped together with crayfish Pacifastacus leniusculus integrin ß (PL-B1), but was far away from vertebrate integrin ß1, ß3, ß5, ß6, ß7, and ß8, and another L. vannamei integrin ß (LV). A Southern blot analysis indicated that the cloned LV-B was a single copy of genomic DNA. LV-B mRNA was expressed in all tissues, and was highly expressed in haemocytes. LV-B was downregulated in shrimp 24 and 96h after having received white spot syndrome virus (WSSV). LV-B expression by haemocytes of shrimp was higher in the postmoult (A and B) stage, and lower in the premoult (D2/D3) stage. LV-B expression was significantly higher by shrimp reared in 2.5‰ and 5‰ salinities. Shrimp injected with integrin ß dsRNA showed gene silencing of integrin ß after 36h. LV-B-silenced shrimp showed decreased hyaline cells (HCs), granular cells (GCs, including semi-granular cells), the total haemocyte count (THC), respiratory bursts (RBs), and lysozyme activity, but showed increased RB/HC, superoxide dismutase (SOD) activity/HC, and the phenoloxidase (PO) activity/GC. LV-B-silenced shrimp showed upregulated expressions of lipopolysaccharide- and ß-glucan-binding protein (LGBP), peroxinectin (PX), prophenoloxidase I (proPO I), proPO II, proPO-activating enzyme (ppA), α2-macroglobulin (α2-M), cytMnSOD, mtMnSOD, and heat shock protein 70 (HSP70). It was concluded that integrin ß plays important roles in proPO activation, phagocytosis, and the antioxidant system for immunomodulation in shrimp.

Fucoidan effectively provokes the innate immunity of white shrimp Litopenaeus vannamei and its resistance against experimental Vibrio alginolyticus infection.

In this study, we examined the effect of fucoidan on the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus infection. Fucoidan induced degranulation, caused changes in the cell morphology, and increased activation of prophenoloxidase (proPO) and the production of superoxide anions in vitro. Shrimp that received fucoidan via immersion at 100, 200, and 400 mg l(-1) after 3 h showed haemocyte proliferation and a higher mitotic index of haematopoietic tissue. In another experiment, the haemocyte count, phenoloxidase (PO) activity, and respiratory bursts (RBs) were examined after the shrimp had been fed diets containing fucoidan at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-21 days. Results indicated that these parameters directly increased with time. The immune parameters of shrimp fed the 1.0 g kg(-1) diet were significantly higher than those of shrimp fed the 2.0 g kg(-1) diet after 14 and 21 days. Phagocytic activity and the clearance efficiency against V. alginolyticus were significantly higher in shrimp fed the 1.0 g kg(-1) diet compared to those of shrimp fed the 0, 0.5 and 2.0 g kg(-1) diets. In a separate experiment, shrimp that had been fed diets containing fucoidan for 21 days were challenged with V. alginolyticus at 10(6) colony-forming units shrimp(-1). Survival rates of shrimp fed the 1.0 and 2.0 g kg(-1) diets were significantly higher than those of shrimp fed the 0 and 0.5 g kg(-1) diets for 96-120 h. We concluded that fucoidan provokes innate immunity of shrimp as evidenced by haemocyte degranulation, proPO activation, and the mitotic index of haematopoietic tissue, and that dietary administration of fucoidan at 1.0 g kg(-1) enhanced the immune response of shrimp and their resistance against V. alginolyticus infection.

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 ß-glucan-binding protein (LGBP), peroxinectin (PX), integrin ß, 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 ß, 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 ß, 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.

Modulation of innate immunity and gene expressions in white shrimp Litopenaeus vannamei following long-term starvation and re-feeding.

The survival rate, weight loss, immune parameters, resistance against Vibrio alginolyticus and white-spot syndrome virus (WSSV), and expressions of lipopolysaccharide- and ß-glucan-binding protein (LGBP), peroxinectin (PX), prophenoloxidase-activating enzyme (ppA), prophenoloxidase (proPO) I, proPO II, α2-macroglobulin (α2-M), integrin ß, heat shock protein 70 (HSP70), cytosolic manganese superoxide dismutase (cytMnSOD), mitochondrial manganese superoxide dismutase (mtMnSOD), and extracellular copper and zinc superoxide dismutase (ecCuZnSOD) were examined in the white shrimp Litopenaeus vannamei (8.18 ± 0.86 g body weight) which had been denied food (starved) for up to 14-28 days. Among shrimp which had been starved for 7, 14, 21, and 28 days, 100%, 90%, 71%, and 59% survived, and they lost 3.2%, 7.3%, 9.2%, and 10.4% of their body weight, respectively. Hyaline cells (HCs), granular cells (GCs, including semi-granular cells), the total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), and SOD activity significantly decreased in shrimp which had been starved for 1, 1, 1, 5, 14, and 3 days, respectively. The expression of integrin ß significantly decreased after 0.5-5 days of starvation, whereas the expressions of LGBP, PX, proPO I, proPO II, ppA, and α2-M increased after 0.5-1 days. Transcripts of all genes except ecCuZnSOD decreased to the lowest level after 5 days, and tended to background values after 7 and 14 days. Cumulative mortality rates of 7-day-starved shrimp challenged with V. alginolyticus and WSSV were significantly higher than those of challenged control-shrimp for 1-7 and 1-4 days, respectively. In another experiment, immune parameters of shrimp which had been starved for 7 and 14 days and then received normal feeding (at 5% of their body weight daily) were examined after 3, 6, and 12 h, and 1, 3, and 5 days. All immune parameters of 7-day-starved shrimp were able to return to their baseline values after 5 days of re-feeding except for GCs, whereas all parameters of 14-day-starved shrimp failed to return to the baseline values even with 5 days of re-feeding. It was concluded that shrimp starved for 14 days exhibited three stages of modulation of gene expression, together with reductions in immune parameters, and decreased resistance against pathogens.

Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei.

The haemogram, phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, and the mitotic index of haematopoietic tissue (HPT) were examined after the white shrimp Litopenaeus vannamei had been fed diets containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-35 days. Results indicated that these parameters directly increased with the amount of extract and time, but slightly decreased after 35 days. RBs, SOD activity, and GPx activity reached the highest levels after 14 days, whereas PO and lysozyme activities reached the highest levels after 28 days. In a separate experiment, white shrimp L. vannamei, which had been fed diets containing the extract for 14 days, were challenged with Vibrio alginolyticus at 2 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1), and then placed in seawater. The survival rate of shrimp fed the extract-containing diets was significantly higher than that of shrimp fed the control diet at 72-144 h post-challenge. We concluded that dietary administration of the G. tenuistipitata extract at ≤1.0 g kg(-1) could enhance the innate immunity within 14 days as evidenced by the increases in immune parameters and mitotic index of HPT in shrimp and their enhanced resistance against V. alginolyticus and WSSV infections. Shrimp fed the extract-containing diets showed a higher and continuous increase in the humoral response indicating its persistent role in innate immunity.

An immersion of Gracilaria tenuistipitata extract improves the immunity and survival of white shrimp Litopenaeus vannamei challenged with white spot syndrome virus.

The innate immunity and resistance against white spot syndrome virus (WSSV) in white shrimp Litopenaeus vannamei which received the Gracilaria tenuistipitata extract were examined. Shrimp immersed in seawater containing the extract at 0 (control), 400 and 600 mg L(-1) for 3 h were challenged with WSSV at 2 × 10(4) copies shrimp(-1). Shrimp not exposed to the extract and not received WSSV challenge served as unchallenged control. The survival rate of shrimp immersed in 400 mg L(-1) or 600 mg L(-1) extract was significantly higher than that of challenged control shrimp over 24-120 h. The haemocyte count, phenoloxidase activity, respiratory burst, superoxide dismutase activity, and lysozyme activity of shrimp immersed in 600 mg L(-1) extract were significantly higher than those of unchallenged control shrimp at 6, 6, 6, 6, and 6-24 h post-challenge. In another experiment, shrimp which had received 3 h immersion of 0, 400, 600 mg L(-1) extract were challenged with WSSV. The shrimp were then received a booster (3 h immersion in the same dose of the extract), and the immune parameters were examined at 12-120 h post-challenge. The immune parameters of shrimp immersed in 600 mg L(-1) extract, and then received a booster at 9, 21, and 45 h were significantly higher than those of unchallenged control shrimp at 12-48 h post-challenge. In conclusion, shrimp which had received the extract exhibited protection against WSSV as evidenced by the higher survival rate and higher values of immune parameters. Shrimp which had received the extract and infected by WSSV showed improved immunity when they received a booster at 9, 21, and 45 h post-WSSV challenge. The extract treatment caused less decrease in PO activity, and showed better performance of lysozyme activity and antioxidant response in WSSV-infected shrimp.

White shrimp Litopenaeus vannamei immersed in seawater containing Sargassum hemiphyllum var. chinense powder and its extract showed increased immunity and resistance against Vibrio alginolyticus and white spot syndrome virus.

This study was to examine the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus and WSSV when shrimp received the Sargassum hemiphyllum var. chinense powder and its hot-water extract. Both powder and extract showed activation of prophenoloxidase and generation of superoxide anion in the shrimp in vitro. The haemocyte count, phenoloxidase (PO) activity, respiratory burst, and lysozyme activity were examined after the shrimp were immersed in seawater containing S. hemiphyllum var. chinense powder or its extract at 0, 100, 300, and 500 mg L⁻¹ for 1, 3, and 5 h. These immune parameters of shrimp immersed in 300 and 500 mg L⁻¹ powder, and 100 and 300 mg L⁻¹ extract were significantly higher than those of control shrimp after 3 h, but slightly decreased after 5 h. In another experiment, shrimp immersed in seawater containing the powder or the extract at 0, 100, 300, and 500 mg L⁻¹ after 3 h were challenged with V. alginolyticus at 8 × 105 colony-forming unit (cfu) shrimp⁻¹, or challenged with WSSV at 1 × 105 copies shrimp⁻¹, and then placed in seawater. Survival rate of shrimp immersed in 500 mg L⁻¹ powder was significantly higher than that of control shrimp after 24-120 h in the V. alginolyticus-challenge test, and after 72 h in the WSSV-challenge test, respectively. Survival rate of shrimp immersed in 300 mg L⁻¹ extract was significantly higher than that of control shrimp after 72-120 h in both V. alginolyticus-challenge and WSSV-challenge tests. It was concluded that the shrimp immersed in seawater containing the powder at 500 mg L⁻¹, and the extract at 300 mg L⁻¹ had increased immunity and resistance against V. alginolyticus infection, and the shrimp that received extract at 300 mg L⁻¹ showed resistance against WSSV infection.

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 ß-glucan-binding protein (LGBP), peroxinectin (PX), and integrin ß (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.

The protective immunity of white shrimp Litopenaeus vannamei that had been immersed in the hot-water extract of Gracilaria tenuistipitata and subjected to combined stresses of Vibrio alginolyticus injection and temperature change.

White shrimp Litopenaeus vannamei which had been immersed in seawater (35 per thousand) containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 400, and 600 mg L(-1) for 3 h, were subjected to temperature transfer (28 degrees C), or combined stresses of Vibrio alginolyticus injection (2.4 x 10(6) colony-forming unit shrimp(-1)) and temperature transfer (28 degrees C) from 24 degrees C, 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, and haemolymph protein concentration were examined 6-144 h post-transfer. Shrimp with no exposure to the extract and no temperature transfer served as the background control. Results indicated that these parameters of shrimp subjected to temperature transfer, or subjected to combined stresses significantly decreased to the lowest at 12 h post-transfer. Results indicated that these parameters of shrimp immersed in 600 mg l(-1) extract had returned to the background values at 24-144 h post-transfer, whereas these parameters of control shrimp returned to the background values at > or =144 h post-transfer. It was therefore concluded that the immunity of L. vannamei which had been immersed in seawater containing the hot-water extract of G. tenuistipitata exhibited a protective effect against temperature transfer, and combined stresses of V. alginolyticus injection and temperature transfer as evidenced by the earlier recovery of immune parameters.

Administration of the hot-water extract of Spirulina platensis enhanced the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus.

White shrimp Litopenaeus vannamei which had been injected with the hot-water extract of Spirulina platensis at 6, 10, and 20 microg g(-1), or immersed in aerated seawater containing extract at 200, 400, and 600 mg L(-1) were challenged with Vibrio alginolyticus at 1.5 x 10(6) or 1.4 x 10(6) colony-forming units (cfu) shrimp(-1), and then placed in seawater. Survival rates of shrimp that received the extract of S. platensis at 6-20 microg g(-1), and those of shrimp immersed in seawater containing the extract at 400 and 600 mg L(-1) were significantly higher than those of control shrimp after 24-96 and 48-96 h, respectively. In a separate experiment, the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, and lysozyme activity were measured when shrimp were injected with the extract at 6, 10, and 20 microg g(-1), and immersed in seawater containing the extract at 200, 400, and 600 mg L(-1). These parameters directly increased with the concentration, and significantly increased when shrimp were immersed in the seawater containing the extract at 0.5-4 h. L. vannamei that received all doses of the extract via injection or via immersion all had increased phagocytic activity and clearance efficiency to V. alginolyticus at 12-72 h and 3-4 h, respectively. It was concluded that L. vannamei that received the hot-water extract of S. platensis had enhanced innate immunity and increased resistance against V. alginolyticus infection.

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.

Molecular cloning and characterization of a cytosolic manganese superoxide dismutase (cytMnSOD) and mitochondrial manganese superoxide dismutase (mtMnSOD) from the kuruma shrimp Marsupenaeus japonicus.

A cytosolic manganese superoxide dismutase (cytMnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from the kuruma shrimp Marsupenaeus japonicus using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of cytMnSOD is 861 bp and encodes a 287 amino acids (aa) protein with a 61 aa leader sequence, whereas the ORF of mtMnSOD is 663 bp and encodes a 221 aa protein with a 21 aa mitochondrial-targeting sequence in the N-terminus. The calculated molecular mass of translated protein of cytMnSOD and mtMnSOD is 31.4 kDa and 24.3 kDa with an estimated pI of 5.62 and 7.27, respectively. The deduced amino acid sequence of cytMnSOD has similarity of 50.2% to that of mtMnSOD. Both cytMnSOD and mtMnSOD contain a manganese superoxide dismutase domain (DVWEHAYY), and four conserved amino acids responsible for binding manganese. Both cytMnSOD and mtMnSOD of M. japonicus were expressed in haemocytes, eyestalk, muscle, intestine, gill, and hepatopancreas. Both cytMnSOD and mtMnSOD transcripts in haemocytes of M. japonicus significantly increased 6 h after injection of Vibrio alginolyticus, and 12 h after injection of beta-glucan, indicating induction of SOD system response in a short time.