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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Identification of the extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene of the mud crab Scylla serrata and its expression following beta-glucan and peptidoglycan injections.

An extracellular copper-zinc superoxide dismutase (ecCuZnSOD, or EC-SOD) gene was cloned from haemocytes of mud crab Scylla serrata. The ecCuZnSOD cDNA consists of 939 bp with an open reading frame (ORF) of 585 bp that encodes 195 amino acids (aa) with 31 residues of a signal peptide sequence. The predicted molecular mass of the mature protein (164aa) is 17.1 kDa with an estimated pI of 6.89. Amino acids responsible for binding Cu (His84, 86, 101, and 163) and Zn (His101, 109, and 121, and Asp 124), two cysteines (95 and 189) that form a disulfide bond, two CuZnSOD signatures (GFHVHAEGDLS) from 82 to 92 and (GNAGGRAGCGLI) from 181 to 192, as well as a N-linked glycosylation site (NVSG) were conserved. The deduced amino acid sequence of S. serrata ecCuZnSOD showed similarity of 84% and 54% with the ecCuZnSOD of blue crab Callinectes sapidus, and crayfish Pacifastacus leniusculus, respectively. The phylogenetic analysis revealed that the ecCuZnSOD of S. serrata grouped together with ecCuZnSODs of crustaceans and insects, but was far way from the intracellular (ic)CuZnSODs of invertebrates and vertebrates. The ecCuZnSOD transcript in haemocytes significantly increased in 6-48 h, and had returned to the original value by 72 h after the beta-glucan (betaG) injection, whereas the ecCuZnSOD transcript significantly increased 6-72 h after the peptidoglycan (PG) injection indicating induction of the immune system within a short time.

Identification and phylogenetic analysis on lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) of kuruma shrimp Marsupenaeus japonicus.

A lipopolysaccharide (LPS) and beta-1,3-glucan binding protein (LGBP) gene was cloned from hemocytes of kuruma shrimp Marsupenaeus japonicus by reverse-transcription polymerase chain reaction (RT-PCR), cloning and sequencing of overlapping PCR, and rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of M. japonicus LGBP is 1062 bp and encodes a 354 amino acid (aa) sequence with a 23 aa signal peptide. The calculated molecular mass of the mature protein (331 aa) is 40.15 kDa with an estimated pI of 4.78. The M. japonicus LGBP sequence contains (1) two putative N-linked glycosylation sites, (2) two putative integrin-binding motifs, (3) a kinase C phosphorylation site (KCPS), (4) a glucanase motif (GM), and (5) two potential polysaccharide recognition motifs (polysaccharide binding motif (PsBM) and beta-glucan recognition motif (GRM)), and with features of tryptophan-rich, slight homology to lysozyme, and slight homology to lectin. A sequence comparison showed that the deduced amino acids of M. japonicus LGBP has an overall high similarity to penaeid LGBP and betaGBP (85.6-89.9%), lobster Homarus gammarus betaGBP (77.0%), and crayfish Pacifastacius leniusculus LGBP (67.8%). The phylogenetic analysis revealed that M. japonicus LGBP grouped together with other crustacean LGBP and betaGBP, and was close to termite GNBP, but was far way from moth betaGBP, betaGRP, fly GNBP, and mosquito betaGRP. The LGBP of M. japonicus was strongly expressed in hemocytes. The LGBP mRNA transcript in hemocytes of M. japonicus was significantly upregulated 12-48 h after a LPS injection, indicating activation of the innate immune system through the binding of the LGBP and LPS complex.

Molecular cloning and phylogenetic analysis on alpha2-macroglobulin (alpha2-M) of white shrimp Litopenaeus vannamei.

The full-length sequence of alpha2-macroglobulin (alpha2-M) was cloned from the hemocytes of white shrimp Litopenaeus vannamei by reverse transcription polymerase chain reaction (RT-PCR), cloning and sequencing of overlapping PCR, and the rapid amplification of cDNA ends method. The alpha2-M cDNA consists of 4682bp with an open reading frame of 4479bp, a 52-bp 5'-untranslated region, and a 132-bp 3'-untranslated region. The predicted molecular mass of the mature protein (1475 amino acids) is 167.1kDa, with an estimated pI of 5.70. The L. vannamei alpha2-M sequence contains putative functional domains including a bait region, a GCGEQNM (985-991) thiol ester domain, and a receptor-binding domain. Sequence comparison and phylogenetic analyses show that alpha2-M deduced amino acid sequence of L. vannamei has a high level of similarity to that of tiger shrimp Penaeus monodon and kuruma shrimp Marsupenaeus japonicus. The alpha2-M transcript was mainly expressed in hemocytes, and was significantly higher in premoult stage than in intermoult and postmoult stages.

Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from the haemocytes of tiger shrimp Penaeus monodon.

An alpha 2-macroglobulin (alpha2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5'-untranslated region, and a 327 bp 3'-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated pI of 5.30. The P. monodon alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species alpha2-M showed that the overall structure is evolutionarily conserved and phylogenetic analysis revealed that P. monodon alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.

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.