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.

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.

The effects of a sudden salinity change on cortisol, glucose, lactate, and osmolality levels in grouper Epinephelus malabaricus.

Grouper Epinephelus malabaricus (weighing 46.37 ± 5.10 g) previously maintained in 24‰ seawater were transferred to 14, 19, 24 (control), 29, and 34‰ seawater. Serum cortisol, glucose, lactate, and osmolality levels were measured at 7 time points during 240 min. Serum cortisol and glucose levels of fish transferred to 29 and 34‰ seawater significantly increased to the highest after 10 and 20 min, respectively. No significant differences in serum cortisol and glucose levels were observed for the fish after 30 min among all treatments. Serum lactate level of fish transferred to 14, 19, 29, and 34‰ seawater was significantly lower than that of the control fish after 10-30 min. However, no significant differences in serum lactate were observed 60 min among five treatments. The serum osmolality of the fish following 240-min transfer increased directly with salinity, whereas the osmoregulatory capacity value (medium osmolality-plasma osmolality) of the fish following 240-min transfer was inversely related to salinity. It is concluded that grouper showed strong osmoregulation in 14-34‰ seawater. Serum cortisol and glucose levels of fish transferred to 29 and 34‰ seawater increased rapidly in 10-30 min, indicating an early stress response.

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.