Su-Tuen Yeh

The immune response of the white shrimp Litopenaeus vannamei and its susceptibility to Vibrio infection in relation with the moult cycle

The white shrimp Litopenaeus vannamei (8.0-14.4 g) was examined for haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity, and clearance efficiency to the pathogen Vibrio alginolyticus in relation with moult cycle (postmoult, A, B; intermoult, C; premoult, D(0)/D(1)D(2)/D(3)). Granular cells were the highest at C and D(0)/D(1)stage, and the lowest at A stage. Hyaline cells and THC (total haemocyte count) were higher at C stage, but lower at postmoult stages. Phenoloxidase activity was the highest at C stage, and the lowest at A stage. Respiratory burst was lower at A stage. Phagocytic activity of shrimps against V. alginolyticus decreased significantly at postmoult and premoult stages. Additionally, the clearance efficiency of shrimps to V. alginolyticus was significantly lower for shrimps at A stage than those at C stage. In another experiment, L. vannamei at different moult stages were injected with tryptic soy broth (TSB)-grown V. alginolyticus (1x10(5)cfu shrimp(-1)) and then held in 34% seawater. After 10 h, the mortality of V. alginolyticus-injected shrimps was significantly higher for shrimps at postmoult stage than those at intermoult stage. Over 48-120 h, the mortality of V. alginolyticus-injected shrimps was 50.0%, 33.3% and 40.0% at postmoult, intermoult and premoult stage, respectively. It is concluded that L. vannamei showed a decrease in resistance at A stage through a reduction of its haemocyte count, phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency against V. alginolyticus.

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 shrimps 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.

White shrimp Litopenaeus vannamei that received the hot-water extract of Gracilaria tenuistipitata showed earlier recovery in immunity after a Vibrio alginolyticus injection.

White shrimp Litopenaeus vannamei which had been immersed in seawater containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 200, 400, and 600 mg L(-1) for 3 h, were challenged with Vibrio alginolyticus at 4.6 x 10(6) colony-forming units (CFU) shrimp(-1) and then placed in normal seawater (34 per thousand). The survival rates of shrimp immersed in 200, 400, and 600 mg L(-1) of the hot-water extract were significantly higher than those of control shrimp over 48-120 h. In another experiment, L. vannamei which had been immersed in the hot-water extract at 0, 200, 400, and 600 mg L(-1) for 3 h, were challenged with V. alginolyticus at 4.0 x 10(6) CFU shrimp(-1), and the immune parameters examined included the haemocyte count, phenoloxidase (PO) activity, respiratory burst (RB), and superoxide dismutase (SOD) activity at 12-120 h post-challenge after shrimp had been released into normal seawater. Shrimp not exposed to the hot-water extract or V. alginolyticus served as the background control. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of shrimp immersed in 600 mg L(-1) were significantly higher than those of control shrimp at 12-72 h post-challenge. Results also indicated that total haemocyte count (THC), PO activity, RB and SOD activity of shrimp immersed in 400 and 600 mg L(-1) of the hot-water extract returned to the background values at 96, 48, 48, and 72 h, whereas these parameters of control shrimp returned to the original values at >120, >120, 96, and 96 h post-challenge, respectively. It was therefore concluded that L. vannamei that had been immersed in seawater containing the hot-water extract of G. tenuistipitata exhibited protection against V. alginolyticus as evidenced by the earlier recovery of immune parameters.

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.

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.

Innate immunity of the white shrimp Litopenaeus vannamei weakened by the combination of a Vibrio alginolyticus injection and low-salinity stress.

White shrimp Litopenaeus vannamei were reared at a salinity of 35 per thousand without a Vibrio alginolyticus injection (unchallenged group), and other shrimp were reared at 35 per thousand, injected with tryptic-soy broth (TSB)-grown V. alginolyticus at 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) (challenged group), and then examined for the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after transfer to 35 per thousand (control), 25 per thousand, 20 per thousand, and 15 per thousand for 1, 6, 12, 24, 72, and 120 h. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of unchallenged shrimp and challenged shrimp that were transferred to low-salinity levels all began to significantly decrease at 6, 6, 6, and 1 h, respectively, and reached the lowest levels at 12 h. HC, GC, the THC, PO activity, RB, and SOD activity of unchallenged shrimp that were transferred to 15 per thousand decreased by 53%, 41%, 49%, 68%, 39%, and 62%, whereas those parameters of challenged shrimp that were transferred to 15 per thousand decreased by 79%, 78%, 79%, 82%, 54%, and 72%, respectively after 12 h compared to control shrimp. These immune parameters began to recover after 24-72 h for both unchallenged shrimp and challenged shrimp. We concluded that the innate immunity was weakened in white shrimp L. vannamei that received combined stresses of a V. alginolyticus injection, and low-salinity transfer. It was also concluded that shrimp with respectively 21%, 18%, 46%, and 28% lower THC, PO activity, RB, and SOD activity of the original values would be killed due to decreases in their immunity, and resistance to V. alginolyticus infection. Shrimp farming should be maintained at a constant high salinity level to prevent exacerbated decreases in innate immune parameters of shrimp when infected by a pathogen coupled with low-salinity stress leading to mortality.

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.

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.

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.

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.