The immune response of white shrimp Litopenaeus vannamei following Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei injected with saline, and injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 1.0 x 10(5) and 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) were examined for hyaline cell (HC) counts, granular cell (GC) counts, total haemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after 1-168 h. Shrimp that received no injection served as the control. The shrimps which received V. alginolyticus at both doses showed significant decreases in these parameters after 6-96 h. The values for HC and SOD activity decreased earlier and then RB. The time to cause maximum depletion of haemocytes (haemocytopenia), PO activity, RB, and SOD activity were 12, 72, 48, and 24 h post-injection, respectively. The HC, GC, and RB returned to the original values earlier at 72 h, followed by SOD activity at 96 h, and then PO activity at 168 h post-infection. It was concluded that an injection of V. alginolyticus rapidly reduced the shrimp's immunity by decreasing HC, GC, SOD activity, RB, and PO activity within 3-24 h, followed by a slow recovery during 72-168 h post-injection. Furthermore, white shrimp L. vannamei which received V. alginolyticus showed a 6-9 h later response in PO activity, and a 72-96 h later recovery of PO activity, compared to the responses in RB and SOD activity indicating their roles in shrimp defence and immunity.     

Hemocytic immune responses triggered by CpG ODNs in shrimp Litopenaeus vannamei

CpG oligodeoxynucleotides (CpG ODNs), also called bacterial DNA or synthetic oligodeoxynucleotides, can induce apparent immunity protection against various pathogens, and they are widely used as functional immunostimulant or vaccine adjuvant in mammals. In the present study, CpG-rich plasmid pUC57-CpG was constructed and employed to stimulate the shrimp Litopenaeus vannamei, and the total hemocyte count, percentage of apoptotic hemocytes, regeneration of circulating hemocytes, the ability of phagocytosis and generation of reactive oxygen species (ROS) were measured to reveal the possible protection mechanism of CpG ODNs. After the injection of pUC57-CpG, the total hemocyte count significantly decreased (p < 0.01) to 2.56 × 10⁷ cell/mL at the first day post stimulation, while the apoptosis increased (p < 0.01), which was 1.72-fold of that in control group. At the same time, the regeneration of circulating hemocytes fluctuated in a similar trend, and a significant increase was observed at the first day post stimulation. The phagocytotic activity including the percentage of phagocytosis and phagocytotic index, experienced an upward tend during the whole experimental period and the ROS level increased by 22% (p < 0.05) compared to that in the control group at first day post stimulation. These results together suggested that pUC57-CpG could promote the apoptosis and regeneration of circulating hemocytes, and enhance the phagocytosis and ROS production, which might contribute to the boosted immunity against the infection of pathogens.     

The immune stimulatory effect of sodium alginate on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (9.4-11.3 g) were injected individually with sodium alginate at 10, 20 or 50 microg g(-1). No significant differences in THC, DHC and superoxide dismutase activity were observed among the shrimp injected with saline and those injected with sodium alginate at 10, 20 or 50 microg g(-1). However, L. vannamei injected with sodium alginate at 20 microg g(-1)increased its phenoloxidase activity and respiratory burst after 2 days and one day, respectively. L. vannamei injected with sodium alginate at 50 microg g(-1)maintained a higher phagocytic activity and clearance efficiency to V. alginolyticus after 4 days. In another experiment, L. vannamei which had been injected with sodium alginate, were challenged with V. alginolyticus at 2x10(5)colony-forming units (CFU) shrimp(-1)and then placed in seawater of 34 per thousand. The survival of shrimp that received sodium alginate at either dose was significantly higher than that of control shrimp at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei received sodium alginate at 10 microg g(-1)or more and increased its immune ability and resistance from V. alginolyticus infection.     

Immunostimulation of white shrimp (Litopenaeus vannamei) following dietary administration of Ergosan

Ergosan an algal product containing 1% alginic acid, developed for use in aquaculture and reported to have immunomodulatory activity, was administered orally to intermoult adult white shrimp (Litopenaeus vannamei) for 15 days. Examination of haemolymph proteins using SDS-PAGE did not reveal any obvious differences between control and Ergosan treated shrimp. Similarly, total haemocyte counts were found to be roughly equivalent for both the control and experimental samples. However, differential analysis of haemocyte populations revealed marked changes in terms of the relative levels of hyaline, semi-granular, and particularly granular haemocytes between the two groups. Moreover, enhancement of the in vitro antimicrobial activity of haemolymph towards two shrimp pathogenic Vibrio isolates was recorded for shrimp fed with Ergosan. Finally, shrimp fed with Ergosan showed a significant increase in relative growth when compared with control groups.     

Noradrenaline modulates the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (18.4 +/- 1.2 g) were injected individually with noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1). For the shrimp that received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1), the THC decreased by 15%, 21% and 32%, phenoloxidase activity decreased by 15%, 31% and 31%, respiratory burst decreased by 13%, 21% and 32%, and SOD activity decreased by 46%, 56% and 55%, respectively, after 2 h. The phagocytic activity and clearance efficiency of shrimp that received noradrenaline at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 4, 4, 8, 24, 16 and 8 h, respectively, in the shrimp that received noradrenaline at either dose. In another experiment, L. vannamei which had received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1) were challenged after 1h by injection with V. alginolyticus at 1.0 x 10(5) colony-forming units (cfu)shrimp(-1) and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received noradrenaline at either dose was significantly higher than that of shrimp that received saline after 4 h, and at the termination of the experiment (48 h after the challenge). It is therefore concluded that noradrenaline administration at 10(-6) mol shrimp(-1) or less causes immune modulation of L. vannamei.     

The immunostimulatory effect of hot-water extract of Gracilaria tenuistipitata on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were examined in the white shrimp Litopenaeus vannamei (10.3+/-1.5 g) injected individually with hot-water extract of Gracilaria tenuistipitata at 4 or 6 microg g-1. L. vannamei receiving hot-water extract of G. tenuistipitata at either dose increased significantly its THC, phenoloxidase activity, and respiratory burst after 2 days. L. vannamei received hot-water extract of G. tenuistipitata at 6 microg g-1 increased its phagocytic activity and clearance efficiency to V. alginolyticus after 1 day. In another experiment, L. vannamei which had been injected with hot-water extract of G. tenuistipitata were challenged with V. alginolyticus at 2x10(6) colony-forming units (cfu) shrimp-1 and then placed in seawater of 34 per thousand. The survival of shrimp that received hot-water extract of G. tenuistipitata at 6 microg g-1 was significantly higher than that of shrimp that received saline and the control shrimp after 3 days, and at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei receiving the hot-water extract of G. tenuistipitata at 6 microg g-1 or less increased its immune ability and resistance to V. alginolyticus infection.     

虾头提取物对凡纳滨对虾生长和免疫因子的影响

以添加不同量虾头提取物(0、1.0%、1.5%、2.0%、2.5%、3.0%)的饲料投喂初体质量为6.35±0.38 g的凡纳滨对虾49 d,研究其对对虾的生长及免疫因子的影响。各组凡纳滨对虾的生长及体成分组成没有显著差异。与对照组相比,1.5%~3.0%添加量组对虾血清中的ACP活力明显提高;2.0%~3.0%添加量组对虾血清中的AKP活力明显提高;各添加量组对虾血清中的PO活力都有明显提高。实验结果表明,添加适当剂量的虾头提取物可以增强凡纳滨对虾的免疫能力。     

Effects of dopamine on the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (20.0+/-1.5 g) were injected individually with dopamine at 10(-8), 10(-7) and 10(-6)mol shrimp(-1), respectively. For the shrimp that received dopamine at 10(-7) and 10(-6)mol shrimp(-1), the THC decreased by 25% and 39%, phenoloxidase activity decreased by 15% and 32%, respiratory burst decreased by 21% and 36%, and SOD activity decreased by 50% and 63%, respectively, after 4 h. The phagocytic activity and clearance efficiency of shrimp that received dopamine at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 16, 8, 8, 24, 16 and 4 h, respectively, for the shrimp that received dopamine at either dose. In another experiment, L. vannamei which had received dopamine at 10(-8), 10(-7) and 10(-6)mol shrimp(-1) were challenged after 1 h by injection with V. alginolyticus at 1.0x10(5) colony-forming units (cfu) shrimp-1 and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received dopamine at either dose was significantly higher than that of shrimp that received saline after 8 h, and of shrimp that received saline at the termination of the experiment (48 h after the challenge). It is therefore concluded that dopamine administration at 10(-6)mol shrimp-1 or less causes immune modulation of L. vannamei.     

Chronic effect of nitrite on the rearing of the white shrimp Litopenaeus vannamei in two salinities

The aim of this study was to determine the effects of nitrite on the growth and survival of the white shrimp L. vannamei in two different salinities. Nitrite concentrations tested in salinity 8 g/L were 0 (control), 2.5, 5.0, 10.0, and 20.0 mg NO₂^?-N/L, and in salinity 24 g/L were 0 (control), 5.0, 10.0, 20.0, and 40.0 mg NO₂^?-N/L. For these experiments, 30 experimental units with 30?L of useful volume were stocked with 20 juvenile L. vannamei (8.0 ± 0.50 g), corresponding to a stocking density of 100 shrimp/m², and cultivated for an experimental period of 30 days. A significant difference was found between the control and treatment groups with respect to growth and survival. The 2.5 mg NO₂^?-N/L treatment showed the best performance indexes in salinity 8 g/L, while the best growth performance indexes were found in the control and 5.0 mg NO₂^?-N/L treatments in salinity 24 g/L. Total mortality was observed in the 10 and 20 mg NO₂^?-N/L treatment groups from salinity 8 g/L and in the 40 mg NO₂^?-N/L treatment group in salinity 24 g/L. This study determined that concentrations of nitrite of up to 2.5 and 10 mg/L are acceptable for the rearing of L. vannamei in salinities of 8 and 24 g/L, respectively.     

The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei

White shrimp, Litopenaeus vannamei, which had been injected with chitin at 4, 6 and 8 microg g(-1) or chitosan at 2, 4 and 6 microg g(-1), were challenged with pathogen Vibrio alginolyticus at 2 x 10(6) colony-forming units (cfu) shrimp(-1) and then placed in seawater of 34 per thousand. The survival of shrimp that received chitin or chitosan at either dose was significantly higher than that of control shrimp after 1 day, and at the termination of the experiment (6 days after the challenge). In another experiment, the total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, and phagocytic activity to V. alginolyticus were measured when L. vannamei (10.4 +/- 0.7 g) were injected individually with chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1). L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased significantly its THC and respiratory burst after 2 days. L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) still maintained significantly higher phenoloxidase activity after 6 days. L. vannamei received chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased its phagocytic activity against V. alginolyticus after 1 day, respectively. It is therefore concluded that L. vannamei that received chitin at 6 microg g(-1) or chitosan at 4 microg g(-1) or less increased its immune ability and resistance to 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 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.     

Reconsideration of phenoloxidase activity determination in white shrimp Litopenaeus vannamei

Though phenoloxidase (PO) activity has been used as an important index in immunological research of crustaceans, methods for the determination of PO activity are not consistent even for the same species. Plasma, the major location of PO activity, should be the most reasonable sample, instead of hemocytes or serum, for the determination of PO activity of shrimp. The current study provided a thorough characterization and reconsideration for PO activity assay in the plasma of Litopenaeus vannamei. Results show that the final concentration of l-dihydroxyphenylalanine (l-DOPA) for PO activity assay should be no less than 1.5 mg ml^?1, and pH 6.6 should be used to maintain the stability of l-DOPA solution. This study provides direct evidence that PO activity is significantly inhibited by EDTA, and it is suggested to use EDTA-free anticoagulant in separating plasma for PO activity assay in future studies. Repeated measurements indicated that the assayed PO activities are significantly affected by preservation conditions, and plasma is quite unstable with spontaneous activation when put in ice or stored at ?20 °C. Thus samples need to be measured immediately or preserved at ?80 °C with assay as soon as possible after it is thawed, and should not be preserved for a second time for measuring PO activity.     

Superoxide dismutase as modulator of immune function in American white shrimp (Litopenaeus vannamei)

The immunomodulatory action of superoxide dismutase (SOD) and its possible use as an indicator of immune responses in American white shrimp (Litopenaeus vannamei) were studied. Juvenile shrimp were immersed in aerated beta-glucan and sulfated polysaccharide solutions for 6 h. SOD activity in haemocytes and muscle was quantified to evaluate whether beta-glucan and sulfated polysaccharide induce immunostimulatory activity. Haemocytes and muscle showed similar increased levels of SOD activity (1.5- and 1.4-fold that of control, respectively). Total haemocyte count decreased within the first 24 h after challenge with immunostimulants, but total haemocyte count and total soluble haemocyte protein increased over normal values after 48-120 h. Single immunostimulation with beta-glucan and sulfated polysaccharide is sufficient to generate an increase in the antioxidant activity of L. vannamei SOD.     

Effects of phosphatidyl serine on immune response in the shrimp Litopenaeus vannamei

Phosphatidyl serine plays an important role in animal innate immunity. Given its important functions, numerous investigations have been carried out on its immunological function in many animals. However, studies of phosphatidyl serine in the white shrimp Litopenaeus vannamei, an economically important animal, are rare. In this paper, we demonstrated influences of injecting phosphatidyl serine (PS) on immune response including some parameters from pro-phenol oxidase activating system (pro-PO system) and hemocyanin-derived phenol oxidase activity (Hd-PO) along with antibacterial and bacteriolytic activities in the white shrimp Litopenaeus vannamei with different PS concentrations (5, 10 and 20 μg mL^?1). The results showed that PS could affect immune response of L. vannamei significantly (P<0.05), including total hemocyte counts (THC), PO activity from hemocyte, phenol oxidase (PO) activity from plasma, hemocyanin concentration, Hd-PO activity as well as antibacterial and bacteriolytic activities in the plasma. Among the lines, 20 μg mL^?1 PS had the strongest effect on the above parameters, whereas 5 μg mL^?1 had the least effect. The experimental results indicated that PS was able to activate exocytosis of pro-PO and formation of Hd-PO in white shrimp after injection, further regulating the immune process reflected by variation of antibacterial and bacteriolytic activities in a certain way.     

Administration of hot-water extract of brown seaweed Sargassum duplicatum via immersion and injection enhances the immune resistance of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, and respiratory burst were examined when the white shrimp Litopenaeus vannamei (10.42+/-1.39 g) were immersed in seawater (34 per thousand) containing hot-water extract of brown alga Sargassum duplicatum at 100, 300 and 500 mg l(-1), or injected with hot-water extract of S. duplicatum at 2, 6, 10 and 20 microg g(-1). These parameters increased significantly when the shrimp were immersed in seawater containing hot-water extract at 300 and 500 mg l(-1) after 1 h, or when the shrimp were injected with hot-water extract at 10 and 20 microg g(-1) after 1 day. L. vannamei that were injected with hot-water extract at 6, 10 and 20 microg g(-1) had increased phagocytic activity and clearance efficiency to V. alginolyticus after 1-6 days. In another experiment, L. vannamei which had been immersed in seawater containing hot-water extract at 100, 300 and 500 mg l(-1), or injected with hot-water extract at 2, 6, 10 or 20 microg g(-1) were challenged with V. alginolyticus at 1 x 10(6), or 1.4 x 10(6) colony-forming units (cfu) shrimp(-1), and then placed in seawater. The survival of shrimp that received hot-water extract at either dose was significantly higher than that of control shrimp after 2 days, as well as at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei that were immersed in hot-water extract of S. duplicatum at 300 mg l(-1), or the shrimp that were injected with hot-water extract at 10 microg g(-1) or less had increased immune ability as well as resistance to V. alginolyticus infection.     

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress

The white shrimp Litopenaeus vannamei were challenged with tryptic soy broth (TSB)-grown Vibrio alginolyticus at a dose of 1 x 10(6) colony-forming units (cfu) shrimp(-1), and then placed in water containing concentrations of nitrite-N at 0 (control), 1.12, 5.15, 11.06 and 21.40 mg l(-1). Mortality of shrimp in 5.15, 11.06 and 21.40 mg l(-1) was significantly higher than those in the control solution after 48-168 h. L. vannamei that had been exposed to control, 0.98, 4.94, 9.87 and 19.99 mg l(-1) nitrite-N for 96 h were examined for THC (total haemocyte count), phenoloxidase activity, and respiratory burst (release of superoxide anion). The THC and phenoloxidase activity decreased when the shrimp were exposed to 4.94, 9.87 and 19.99 mg l(-1) nitrite-N, whereas, the respiratory burst increased significantly at 9.87 and 19.99 mg l(-1) nitrite-N after 96 h. It is therefore suggested that nitrite in water caused a depression in the immune ability of L. vannamei and an increased susceptibility to V. alginolyticus infection, together with an increase of superoxide anion production, possibly to cytotoxic levels for the host.