Semigranular Cells Research Articles

Tyramine's modulation of immune resistance functions in Litopenaeus vannamei and its signal pathway

Tyramine (TA), a neuroactive chemical, plays various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs). In this study, we investigated the effects of by pharmacological injection of TA on immune resistance regulation and its signal pathway in white shrimp Litopenaeus vannamei. Results showed significant increases in the total haemocyte count (THC), semigranular cells (SGCs), granular cells (GCs), phenoloxidase (PO) activity per 50 μL of haemolymph and respiratory bursts (RBs) at 0.5, 1, 2 and/or 4 h; hyaline cells (HCs) at 0.5 h, as well as phagocytic activity (PA) and clearance efficiency (CE) at 2, 4 and/or 8 h, but significantly decreased PO activity per granulocyte at 0.5–2 h for shrimp injected with TA at 100 and 1000 pmol shrimp−1. Plasma lysozyme activities of TA-injected shrimp were significantly higher than those of the saline control at 1 h. All of the immune parameters had returned to control levels by 8 h after receiving TA except the clearance efficiency, which had returned to its control value by 16 h. The TA injection also significantly decreased the mortality of shrimp challenged with Vibrio alginolyticus. Furthermore, immune parameters of shrimp that received TA at 1000 pmol shrimp−1 for 1 h were higher than those of shrimp that received the saline. The upregulating effect of TA was blocked by co-injection with phentolamine (Phe) in terms of the THC, HC, SGCs, PO activity, PA and CE; by co-injection with prazosin (Pra) in terms of the THC, HC, SGCs, PO activity, PA and CE; by co-injection with propranolol (Prop) in terms of the PA and CE; and by co-injection with metoprolol (Meto) in terms of the THC and SGCs. The most potent effect in immunocompetence of tested antagonists was Pra, and except for circulating haemocyte, it was Phe. These results suggest that ≤1000 pmol shrimp−1 of a TA injection mediates transient upregulation of immunity, which in turn promotes the resistance of L. vannamei to V. alginolyticus, and the active effects are mediated via octopamine/tyramine (OA/TA) receptors.

Transient enhancement of immune resistance functions in Litopenaeus vannamei through a low-dose octopamine injection

Octopamine (OA) is known to play an important role in regulating invertebrate immune responses. In this study, we determined the effects of OA on immunity and physiological regulation in the white shrimp Litopenaeus vannamei. The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme, glucose, and lactate levels in plasma, and phagocytic activity and clearance efficiency in response to the pathogen, Vibrio alginolyticus, were measured when shrimp (11.1–13.0 g) were individually injected with saline or OA at 100 and 1000 pmol shrimp−1. Results showed significant increases in THC, semigranular cells (SGCs), and PO activity per 50 μL of haemolymph at 0.5–4 h; granular cells at 0.5–2 h; respiratory bursts (RBs) at 0.5–1 h; phagocytic activity at 2–4 h; and clearance efficiency at 2–8 h, but PO activity per granulocyte at 0.5–2 h significantly decreased after the OA injection. All of the immune parameters had returned to control values by 8 h after receiving OA except granular cells at 4 h, RBs at 2 h, clearance efficiency at 16 h, and PO activity per granulocyte at 4 h. However, no significant differences were observed in hyaline cells, RBs per haemocyte, lysozyme and SOD activities, glucose, or lactate during the experimental period. An injection of OA also significantly decreased the mortality of shrimp challenged with V. alginolyticus. In another experiment, the immune-related genes of transglutaminase-I, lipopolysaccharide- and β-1,3-glucan-binding protein, prophenoloxidase-II, and peroxidase of shrimp that received 1000 pmol OA shrimp−1 for 1 h were significantly higher than those of shrimp that received the saline control. These results suggest that OA administration at ≤1000 pmol shrimp−1 mediates transient upregulation of immunity, which in turn promotes the resistance of L. vannamei to V. alginolyticus.

Litopenaeus vannamei heat shock protein 70 (LvHSP70) enhances resistance to a strain of Vibrio parahaemolyticus, which can cause acute hepatopancreatic necrosis disease (AHPND), by activating shrimp immunity

Heat shock protein 70 (HSP70) acts as a molecular chaperone and a stress protein, but also plays important roles in innate and adaptive immune responses. Previous studies have reported that non-lethal heat shock (NLHS) could enhance the resistance of Pacific white shrimp Litopenaeus vannamei to a specific strain of Vibrio parahaemolyticus, which carried a toxin-producing plasmid (VPAHPND), via the induction of LvHSP70 transcription. Here, we further investigated the specific function of LvHSP70 in shrimp immunity. The upregulation of LvHSP70 at the protein level was detected during recovery time after NLHS treatment, using both western blot analysis and immunofluorescence microscopy. We found that NLHS immediately activated the production of LvHSP70 in shrimp hemocytes and that such induction was observed in all three types of hemocytes: hyaline; granular and semi-granular cells. Furthermore, the role of LvHSP70 in bacterial defense was investigated using the heterologous expression of recombinant LvHSP70 (rLvHSP70) in Escherichia coli. Shrimp receiving rLvHSP70 by injection showed an increased survival rate (75%) to VPAHPND infection compared to just 20% survival in the control group injected with bovine serum albumin (BSA). We also demonstrated that the injected rLvHSP70 accumulated in shrimp hemocytes and was detected in the intracellular space of hemocyte cells leading to the induced expression (P<0.05) of several immune-related genes (LvMyD88, LvIKKβ, LvIKKε, LvCrustin I, LvPEN2, LvPEN3, LvproPO1, LvproPO2 and LvTG1). Collectively, these results suggest that LvHSP70 plays a crucial role in bacterial defense by activating the shrimp immune system.

Modulation of growth performance and nonspecific immunity of red swamp crayfish Procambarus clarkia upon dietary fulvic acid supplementation

An eight-week experiment was conducted to determine the effects of dietary fulvic acids (FAs) on the growth performance, digestive enzymes and nonspecific immunity of red swamp crayfish Procambarus clarkia. Three diets supplemented with three different levels of FAs (0.1, 0.5 and 1 g kg−1) were formulated and tested for the growth performance, digestive enzymes and nonspecific immunity of the crayfish, and a diet without FAs served as control. After eight weeks of feeding, survival rate, phenoloxidase activity, superoxide dismutase activity, glutathione peroxidase level, total haemocyte count and number of hyaline cells, semigranular cells and granular cells and resistance to Aeromonas hydrophila of crayfishes fed with FA-containing diets were higher than those of the control. Moreover, based on the efficiency of FAs on the growth performance and nonspecific immunity of crayfish, the optimum dose of FAs was found to be 0.5 g (kg diet)−1. A high level of FA administration (1 g kg−1) did not further increase the efficiency of FAs compared with those in the moderate group (0.5 g kg−1, p > 0.05). Results indicated that oral administration of FA-containing diets can enhance the growth performance, intestinal digestive enzymes, immune responses and resistance of crayfish to infection by A. hydrophila. Thus, FAs may be utilized as a diet supplement for crayfish

WITHDRAWN: Efficacy of two commercial and indigenous probiotics, Bacillus subtilis and Bacillus licheniformis on growth performance, immuno-physiology and resistance response of juvenile white shrimp (Litopenaeus vannamei)

This study aimed to assess efficacy of commercial and indigenous sources of Bacillus subtilis and Bacillus lichenformis as probiotic dietary supplementation on the growth performance, immuno-physiological variables and resistance response to some stressors in white leg shrimp (Litopenaeus vannamei) weighing 50 ± 6.54 mg at 28 °C for 60 days. Shrimps were fed with four different diets: control (without probiotic); diets supplemented with commercial probiotics(CP) each at 1.5 × 10⁶ CFU/g; commercial +indigenous probiotics(CIP) each at 1.5 × 10⁶ CFU/g (four strains each at 7.5 × 10⁵ CFU/g) and indigenous probiotics (IP) each at 1.5 × 10⁶ CFU/g. Growth and feeding parameters including final weight (FW), specific growth rate (SGR) and feed conversion ratio (FCR) were significantly influenced by all probiotics compared to control one (P < .05), with a better growth performance and survival observed in CP group than other treatments. Glucose and cortisol values in all treated shrimps were lower than control one but was only significantly different for CP group (P < .05). Values of albumin, total protein, and lysozyme in CP group was mostly insignificantly higher than other two treated groups, except for lysozyme value that was significantly different (P < .05). In addition, total haemocyte count (THC), large granular cells (LGC), semi-granular cells (SGC) and hyaline cells (HC) significantly increased in CP group compared to other treatments and control but were only significantly different with control one. However, the values of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly lower in all treatment groups compared with control shrimps, except for ALP. Shrimps fed with all types of probiotics showed a higher resistance to environmental stressors including low and high salinities, formalin, chlorine, high and low water temperatures and ammonia but such resistance was more prominent in CP group. The load of Bacillus bacteria in gut of CP treatment was significantly higher than other two groups and control, and that of CIP group was also significantly higher than IP group (P < .05). These data show that there are some differences in efficacy and potency between the commercial and indigenous probiotic sources in growth and health condition of L. vannamei, and thus requires further attention and research works.

A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain

The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated SpcSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of SpcSP was 1823 bp, containing a 5′ untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3′ UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, SpcSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His156, Asp226, Ser321). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain, with hemocytes and skin expressing the most. The transcriptional level of SpcSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of SpcSP resulted in increased hemolymph clotting time and decreased the mRNA expression of SpproPO mRNA in hemocytes. These findings therefore suggest that SpcSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of SpproPO and hemolymph clotting in S. paramamosain.

Different roles of crayfish hemocytes in the uptake of foreign particles

Crustacean hemocytes are known to remove invading pathogens by phagocytosis. In this study, we investigated how the semigranular cells (SGCs) and granular cells (GCs) of crayfish Cherax quadricarinatus participated in this process. By injecting the animals with excessive amounts of fluorescent microspheres (FMs), we showed that only a small portion of the circulating hemocytes were phagocytic cells, and they took up FMs in a size-dependent manner. The 0.2 μm FMs were internalized almost entirely by SGCs, while GCs and SGCs both contributed to the uptake of 2 μm FMs. Further analysis of the hemocytes from the animals injected with a mixture of FMs suggested that there were a subpopulation of SGCs specifically ingesting 0.2 μm FMs. The size-dependent manner was also applied to biological particles. Escherichia coli was internalized by both SGCs and GCs, whereas white spot syndrome virus (WSSV) was mostly ingested by SGCs. However, the bacterial cells were rapidly taken and cleared from the circulation by the hemocytes, while the WSSV virions were gradually internalized and remained in the cells for a relatively longer period of time. These findings provide basic information of the phagocytic hemocytes of crayfish and how they respond to different foreign particles.

PI3K signaling pathways modulated white spot syndrome virus (WSSV) replication in Procambarus clarkii

The PI3K/AKT signaling pathway is commonly exploited to regulate viral replication and affect the fate of infected cells. In the present study, a PI3K-specific inhibitor (LY294002) was employed to pretreat crayfish to evaluate the effects of PI3K/AKT signaling pathway in WSSV replication. The results showed that the WSSV copy numbers in crayfish pretreated with LY294002 were significantly lower than those in Tris-HCl pretreatment crayfish on the sixth and tenth day after WSSV infection. In semigranular cells, the apoptosis rates were up-regulated on the third day post-WSSV infection, and a significantly lower proportion of apoptosis cells were observed in LY294002-pretreatment group. The expression level of Bax, Bax inhibitor-1 and lectin mRNA in haemocytes of crayfish were increased after WSSV infection. After the secondary stimulation with Tris-HCl, the Bax expression level in LY294002-pretreatment crayfish was significantly higher than that of crayfish pretreated with Tris-HCl on the third or sixth day, but the Toll and lectin mRNA expression decreased significantly on the third, sixth and tenth day. The Bax mRNA expression levels in LY294002-WSSV group were significantly higher than those in Tris-HCl-WSSV group on the third and tenth day. The Bax inhibitor-1 mRNA expression levels in LY294002-WSSV group were significantly lower than those in Tris-HCl-WSSV crayfish on the third day. These results together indicated that the hosts PI3K/AKT signaling pathway play positive roles in WSSV replication through the balance between host cell apoptois and innate immune responses. This information is helpful to further understand the role of PI3K/AKT signaling pathway on WSSV replication in Decapoda crustaceans.

L-3,4-Dihydroxyphenylalanine (l-DOPA) induces neuroendocrinological, physiological, and immunological regulation in white shrimp, Litopenaeus vannamei

L-3,4-Dihydroxyphenylalanine (l-DOPA) is a precursor for dopamine (DA) synthesis. Assessments were conducted to analyze the effects of l-DOPA on mediating regulation of neuroendocrinological, immunological, and physiological parameters in the shrimp, Litopenaeus vannamei when they were individually injected with 0.01 N HCl or l-DOPA at 0.5 or 1.0 μmol shrimp−1 for 60, 120, and 240 min. For catecholamine synthesis evaluation, tyrosine hydroxylase (TH) and DA beta hydroxylase (DBH) activities, l-DOPA, DA, and norepinephrine (NE) levels in hemolymph were determined. The total hemocyte count (THC), differential hemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency in response to the pathogen, Vibrio alginolyticus were assessed for immune responses, and plasma glucose and lactate levels were for physiological response. Results showed that the TH activity, THC, hyaline cells (HCs), and semigranular cells (SGCs) at 120 min, DA levels at 60–240 min, PO activity in hemocytes per 50 μL of hemolymph at 60–120 min, and PO activity per granulocyte (granular cells (GCs) + SGCs) at 60 min significantly increased, but TH activity, l-DOPA levels, GCs, SGCs, and respiratory bursts in hemocytes per 10 μL of hemolymph at 60 min, respiratory bursts per hemocyte and SOD activity at 120 min, phagocytic activity at 60–240 min, and the clearance efficiency at 60–120 min significantly decreased in shrimp injected with l-DOPA at 1.0 μmol shrimp−1. In another experiment, 60 min after shrimp had received l-DOPA at 0.5 or 1.0 μmol shrimp−1, they were challenged with an injection of V. alginolyticus at 2 × 105 colony-forming units (cfu) shrimp−1. The injection of l-DOPA at 1.0 μmol shrimp−1 also significantly increased the cumulative mortality of shrimp by 16.7%, compared to the HCl-challenged control after 120 h. These results suggest that l-DOPA administration at 1.0 μmol shrimp−1 can mediate the transient regulation of neuroendocrinological, immunological, and physiologic responses resulting in immunosuppression, which in turn promoted the susceptibility of L. vannamei to V. alginolyticus.

Stimulating effects of polysaccharide from Angelica sinensis on the nonspecific immunity of white shrimps (Litopenaeus vannamei)

Angelica sinensis polysaccharide (ASP) was prepared by hot water extraction. Then, high-performance liquid chromatography and ion chromatography analyses were conducted, and the results indicated that ASP is a heteropolysaccharide, has a molecular mass of 82,000 Da and consists of arabinose, galactose and glucose (molar ratio of 6:1:1). The effects of ASP on the nonspecific immunity of white shrimps (Litopenaeus vannamei) were investigated by feeding them with ASP-containing diets (0.5, 1 and 1.5 g/kg) during a 12-week breeding experiment. Oral ASP administration significantly improved the survival rate, phenoloxidase activity, superoxide dismutase activity, glutathione peroxidase level, disease resistance against V. alginolyticus, total haemocyte count and number of hyaline cells, semigranular cells and granular cells (p < .05). ASP exhibits immunostimulatory effects on Pacific white shrimps (L. vannamei) and may thus be used as a diet supplement for them.

Đáp ứng miễn dịch và sức đề kháng với Vibrio harveyi của tôm sú (Penaeus monodon) ăn thức ăn có bổ sung chất chiết từ rong mơ (Sargassum microcystum)

Feeding trial was conducted to evaluate the effects of hot water-extract from the Sargassum microcystum supplementation in diet of black tiger shrimp (Penaeus monodon). Shrimps were fed the diets supplemented with different levels of S. microcystum hot-water extract (0%, 0.5%, 1%, 2% of hot-water extract) for 30 days. The challenge experiment was conducted in 60 L plastic container with 30 shrimps for each treatment. Immune parameters including total haemocyte counts (THC), total granular cells (including semi-granular cells) (LGC), total hyaline cells (HC), phenoloxidase activity (PO) and resistance to Vibrio harveyi were evaluated. Results showed that (i) THC, LGC, HC, PO activities were significantly increased in the shrimps fed with 1% hot-water extract supplemented diet; (ii) the highest survival rate (80%) was significantly recorded in the group that was fed with 1% of hot-water-extract. The results suggested that feeding 1% hot-water extract from S. microcystum could enhance immune responses and resistance against V. harveyi in black tiger shrimp P. monodon.

Flow cytometic analysis of Penaeus monodon haemocyte responses to poly I:C

This study was aimed at investigating the cellular responses of Penaeus monodon haemocytes to poly I:C stimulation using flow cytometric assay. Total haemocyte count (THC), percentages of different haemocyte subpopulations [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)], non-specific esterase activity (EA), total reactive oxygen species/reactive nitrogen species (ROS/RNS) production, nitric oxide (NO) production, apoptotic haemocyte ratio and plasmic phenoloxidase (PO) activity were determined in poly I:C-injected shrimp. Results showed that poly I:C at a low dose (5 μg shrimp−1) caused obvious increases in THC, GC proportion, ROS/RNS production and NO production, but had no significant effect on EA, apoptosis and PO activity. In the early stage of poly I:C injection at a high dose (20 μg shrimp−1), THC and GC proportion improvements could also be observed, suggesting that GC might be induced to release from hemocytopoietic or other tissues to participate in immune response, and this subpopulation might be the main cell type involved in the cellular defence against virus. In the later period, proportions of both GC and SGC reduced paralleled by THC reduction, indicating that depletion of GC and SGC was mainly contributed to the reduced count of circulating haemocyte. Obvious increases in ROS/RNS production and NO production were induced in haemocyte of shrimp under a high dose of poly I:C stimulation, but only slight rise of EA and suppression of PO activity could be observed in poly I:C-stimulated shrimp, suggesting that ROS/RNS-dependent system was vital in the immune defence of shrimp against virus. On the other hand, increase of apoptotic haemocyte ratio and THC reduction were presented after the drastic increases of ROS/RNS and NO productions, implying that the stimulated ROS/RNS might be excess and harmful, and was the major factor for the haemocyte apoptosis and depletion. THC recovered after 48 h injection, while haemocyte apoptosis also returned to the control level, suggesting that apoptosis might be contributed to eliminate damaged, weak or infected haemocytes to renew the circulating haemocytes, and it could be considered as an important defending strategy against virus.

Oxidative burst activity in haemocytes of the freshwater prawn Macrobrachium rosenbergii

Oxidative burst, release of reactive oxygen species/reactive nitrogen species (ROS/RNS) contributed to microorganisms killing, is a vital immune response of crustacean haemocyte. Three morphologic haemocyte types (hyaline cells, HC; semigranular cells, SGC; granular cells, GC) have been defined in crustaceans, and found to play different roles in immune defense. However, oxidative burst activities of different haemocyte subpopulations in crustaceans are currently not documented. In the present study, we investigated the oxidative burst activities of the three haemocyte types in the freshwater prawn Macrobrachium rosenbergii using the common ROS fluorescent probe dichlorofluorescin-diacetate (DCFH-DA). Nitric oxide (NO) donor sodium nitroprusside (SNP) improved the DCF fluorescence in haemocytes, while NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO-synthase inhibitor NG-monomethyl-l-arginine (L-NMMA) reduced the fluorescence, suggesting that DCF fluorescence intensity could also be modified by intracellular NO level and activity of NO-synthase pathway. ROS/RNS was also produced in the untreated haemocytes. GC contained most non-induced ROS/RNS production, while oxidative activity of HC was rather weak. No significant impact of PMA could be observed on ROS/RNS level in all the three cell types. Both zymosan A (ZA) and lipopolysaccharide (LPS) significantly triggered the production of ROS/RNS in SGC and GC, whereas they had no effect on those of HC, suggesting that SGC and GC were the primary cell types involved in pathogens killing by ROS/RNS pathway. Cytochalasin B (Cyt B) inhibited the ZA-induced ROS/RNS production, but could not change the ROS/RNS level stimulated by LPS. For unstimulated haemocytes, ROS/RNS productions decreased 29.6%, 44.1% and 48.6% in SGC, and decreased 44.5%, 28.4% and 57.3% in GC, in the presence of L-NMMA, Fccp and DPI respectively, whereas apocynin could not modulate DCF fluorescence in both SGC and GC, suggesting that mitochondrial oxidative pathway was relatively more dominant in SGC, and NO-synthase (NOS) pathway appeared more active in GC. For LPS-stimulated haemocytes, oxidative activities decreased 22.9%, 42.9%, 29.6% and 60.0% in SGC, and reduced 40.6%, 25.2%, 26.7% and 70.6% in GC with the presence of L-NMMA, apocynin, Fccp and DPI respectively, suggesting that NADPH-oxidase (NOX) pathway in both SGC and GC was activated by LPS, and it became the predominant oxidative pathway in stimulated SGC, while NOS pathway was the relative main source for ROS/RNS production in stimulated GC.

Octopamine enhances the immune responses of freshwater giant prawn, Macrobrachium rosenbergii, via octopamine receptors

Octopamine (OA) is known to play an important role in regulating insect immune responses. In Macrobrachium rosenbergii (18.0 ± 1.7 g), OA at 25.0 and 250.0 pmol/prawn significantly increased THC, semigranular cells (SGCs) and PO activity in hemocytes per 50 μL hemolymph, hyaline cells, granular cells (GCs) and RBs in hemocytes per 10 μL hemolymph, and RBs per hemocyte, and however, significantly decreased PO activity per granulocyte (GC + SGC), which returned to control levels after 4 h of injection. The significantly increased phagocytic activity and clearance efficiency of prawn received OA for 8 h returned to control levels after 16 h of injection. In addition, the significantly increased glucose and decreased lactate were observed within 1 h of OA injection. In the susceptibility test, prawn received OA at 25.0 or 250.0 pmol/prawn for 2 h then challenged with Lactococcus garvieae at 105 colony-forming units/prawn significantly increased the resistance of prawns by 23.3% and 30.0%, respectively, compared to the saline-challenged control after 144 h of challenge. In addition, the changes on immunocompetence induced by OA were observed to be blocked by adrenoceptors antagonists. These results suggest that OA administration at 250.0 pmol/prawn or less causes the mediate a transient up-regulation in immune and physiologic responses to promote the resistance of M. rosenbergii to L. garvieae, which are thought to be mediated by α- and β-adrenergic-like octopamine receptors.

Various cellular responses of different shrimp haemocyte subpopulations to lipopolysaccharide stimulation

Different haemocyte types have been reported to play diverse roles in immune defense of shrimp. To investigate the roles of the three haemocyte types [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)] of shrimp in immune responses against lipopolysaccharide (LPS), percentage, non-specific esterase activity (EA), reactive oxygen species (ROS) production and nitric oxide (NO) production of the three haemocyte subpopulations were analyzed in LPS-injected Penaeus monodon using flow cytometry. Results showed that percentage of HC increased after 3 h injection, and returned to the original level after 48 h. Proportion of SGC and GC reduced after 6–36 h and 3–12 h respectively, and recovered to the initial level after 48 and 24 h respectively. Loss of SGC and GC might be related to degranulation to release proPO system, and degranulation of GC seemed more sensitive to LPS stimulation. EA of both HC and SGC improved after 3–6 h injection, while EA of GC was induced after 3–24 h. No significant effect of LPS injection could be found in ROS production and NO production of HC. Enhanced ROS levels was observed in SGC and GC after 3–24 h and 3–36 h respectively, and NO production of SGC and GC improved after 3–48 h injection. These results demonstrated that SGC and GC possessed strong capabilities for LPS-induced EA, ROS production and NO production, while HC only displayed EA response to LPS, suggesting that GC and SGC play the main role in immune defense of shrimp against Gram-negative bacteria.

Evaluation of three non-genetically modified soybean cultivars as ingredients and a yeast-based additive as a supplement in practical diets for Pacific white shrimp Litopenaeus vannamei

Two growth trials and a physiology assessment were conducted to evaluate three non-genetically modified (GM) soybean cultivars as ingredients in practical diets for Pacific white shrimp, Litopenaeus vannamei. In addition, a commercially available fermented yeast product was evaluated as dietary supplement. For the growth trials (46 and 35 days, respectively, for trials 1 and 2), the basal diet was primarily composed of soybean meal (SBM), fishmeal (FM), whole wheat, corn protein concentrate, poultry meal (PM, pet food grade) and corn starch. Non-GM cultivars were processed with novel methodologies to produce Navita™ ingredients (N1, N2 and N3) which were incorporated at low (L) or high (H) levels into the experimental diets, in partial replacement of FM and full replacement of conventional SBM. The last two formulations incorporated the fermented yeast for a total of nine experimental diets (Table 1). Results from the growth trials indicate that shrimp fed diet 5 (HN2) exhibited significantly lower (p < .05) weight gain as compared to shrimp fed diets 1 and 2 (basal and LN1, respectively) in trial 1, as well as compared to animals fed diets 1, 2, 3 and 8 (basal, LN1, HN1 and basal + yeast) in trial 2. The feed conversion ratio significantly increased for shrimp fed diet 5, in contrast with shrimp fed diets 1, 2, 3 and 8 in trials 1 and 2, as well as compared to shrimp fed diets diet 6 (LN3) in trial 2. For the physiological assessment (stress and immune responses), only the effects of diets 1, 3, 8 and 9 (basal, HN1, basal + yeast and HN1 + yeast, respectively) were investigated. Granular cell counts were significantly higher for shrimp fed the yeast-containing diets. Haemolymph glucose and haemolymph packed cell volume were significantly reduced for shrimp fed diets 3, 8 and 9. No significant differences were observed in total haemocyte counts, hyaline cells counts, semi-granular cells counts, haemolymph protein, haemocyte phagocytic capacity and haemocyte respiratory burst activity. Results of this work indicate that selective soy breeding technology coupled with novel processing options has the potential to increase the nutritional value of conventional SBM for shrimp feeds. Trends on immune responses were more difficult to elucidate possibly due to the limited length of the feeding trial.

Ultrastructural and immunocytochemical analysis of circulating hemocytes from Cherax quadricarinatus (von Martens, 1868)

Transmission electron microscopy (TEM) and immunocytochemistry were used to assay the circulating hemocytes characteristics in Cherax quadricarinatus (von Martens, 1868). Three haemocyte types could be identified in circulating hemocytes by TEM, i.e. hyaline cells (HC), semigranular cells (SGC), or granular cells (GC). The circulating hemocytes from C. quadricarinatus could be separated into layers by density gradient centrifugation. The CD34+ cells which are believed to be stem cell-like cells has been discovered in the first layer via immunocytochemical assay, which has not been reported in previous studies. These CD34+ cells may be immature cells derived from a single undifferentiated precursor cell line in specialized hematopoietic tissue (HPT).

The upregulation of immune responses in tyrosine hydroxylase (TH) silenced Litopenaeus vannamei

Catecholamines (CAs) play a crucial role in maintaining physiological and immune homeostasis in invertebrates and vertebrates under stressful conditions. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in CA synthesis. To develop an effective CA-related immunological defense system against stress and pathogen infection, various criteria, were evaluated in TH double-stranded (ds) RNA-injected white shrimp, Litopenaeus vannamei. Specifically, the relative transcript quantification of TH, dopamine β-hydroxylase (DBH), crustacean hyperglycemic hormone (CHH), and other immune-related genes; TH activity in the haemolymph; and the estimation of l-dihydroxyphenylalanine (l-DOPA), glucose, and lactate levels in the haemolymph were examined. TH depletion revealed a significant increase in the total haemocyte count; granular cells; semigranular cells; respiratory bursts (RBs, release of superoxide anion); superoxide dismutase (SOD) activity; phagocytic activity and clearance efficiency; and the expression of lipopolysaccharide and β-1,3-glucan-binding protein and peroxinectin, SOD, crustin, and lysozyme genes. In addition, the reduction of TH gene expression and activity was accompanied by a decline of phenoloxidase (PO) activity per granulocyte, lower glucose and lactate levels, and significantly low expression of DBH and CHH genes. However, the number of hyaline cells, activity of PO, RBs per haemocyte, and expression of POI and POII genes were not significantly different in the LvTH-silenced shrimp. Notably, the survival ratio of LvTH-silenced shrimp was significantly higher than that of shrimp injected with diethyl pyrocarbonate–water and nontargeting dsRNA when challenged with Vibrio alginolyticus. Therefore, the depletion of TH can enhance disease resistance in shrimp by upregulating specific immune parameters but downregulating the levels of carbohydrate metabolites.

Cellular responses of the tiger shrimp Penaeus monodon haemocytes after lipopolysaccharide injection.

This study was aimed at investigating the invivo effects of lipopolysaccharide (LPS) injection on Penaeus monodon haemocytes at a cellular level. Cellular responses of LPS-injected shrimp were analysed using flow cytometry. Results showed that LPS injection caused total haemocyte count (THC) and count of large cells (semigranular and granular cells) decline. In LPS-injected shrimp, percentage of large cells decreased at the initial stage, and returned to the original level later. After LPS infection, non-specific esterase activity, reactive oxygen species (ROS) production and nitric oxide (NO) production in haemocytes were significantly induced, while apoptotic cell ratio of haemocytes increased. PO activity in plasma increased in shrimp received LPS at 2μgg(-1) after 3-12h and at 8μgg(-1) after 3-6h, and then returned to the initial levels. These results demonstrated that LPS induced immune responses on haemocytes, including production of ROS and NO, and release of esterase and PO. On the other hand, THC reduction might be due to the ROS/NO-induced apoptosis. Haemocyte apoptosis which would eliminate damaged or weak cells and contribute to haemocyte renewal, may be a defending strategie against pathogens.

Effects of dietary taurine, carnitine and cholesterol supplementation on growth performance and immunological status ofLitopenaeus vannameiunder cold exposure

The aim of this study was to investigate effects of dietary feed additives on growth and immunological status of Litopenaeus vannamei under cold exposure. Four experimental diets with 0.2% taurine, 0.01% carnitine, 0.1% cholesterol supplementation and control were fed to L. vannamei (IBW 0.86 ± 0.03 g) for 30 days at 30 ± 1°C before water temperature was dropped to 16°C with a rate of −4°C h−1. Growth performance parameters including weight gain rate (WGR), feed conversion ratio (FCR) and survival rate before the cold exposure, as well as immunological parameters related to haemocytes including total haemocyte count (THC), percentages of hyaline cells (SC%), granular cells and semigranular cells (SC + GC%), non-specific esterase (NSE) activity and apoptotic cell ratio before and after the cold exposure were determined. The results showed that: (1) WGR, FCE and survival rate were not significantly different among groups before the cold exposure. Most of immunological parameters did not differ either, except the apoptotic cell ratio of carnitine and cholesterol groups was significantly lower than that of control and taurine groups (P < 0.05). (2) After the cold exposure, THC was decreased by 48.1% (P < 0.05), 54.1% (P < 0.05), 30.1% and 2.2%; NSE activity was decreased by 31.7% (P < 0.05), 21.4%, 7.0% and 3.5% in control, taurine, carnitine and cholesterol respectively. (3) Conversely, ROS production was increased by 23.8% (P < 0.05), 15.1%, 7.2% and 13.0%; apoptotic cell ratio was increased by 10.4%, 12.8%, 6.4% and 7.2% in control, taurine, carnitine and cholesterol respectively. (4) An increase in HC%, decrease in SC + GC% in all groups was observed after cold exposure, and was significant in cholesterol group. This study showed that cholesterol, carnitine and taurine affected immunological status of L. vannamei under cold exposure to different degrees, possibly by modulating some immunological parameters, as discussed. Present study provides theoretical data for nutritional strategies to enhance cold resistance of shrimp.