Effectiveness of various cacao pod husk extraction byproducts in promoting growth and immunocompetence in Litopenaeus vannamei.

Extracts from plant products can promote growth, can act as immunostimulants, and have antibacterial and antiparasitic properties. These extracts can be used as alternatives to the chemical treatments commonly used to prevent and control disease in aquatic species. Research on the subject has focused on identifying invasive plants or agricultural waste products that can be used as immunostimulants. The present study further identified an optimal means of extracting pectin from cacao pod husks to promote growth performance and immunocompetence in Litopenaeus vannamei that would both reduce production costs and enable waste recycling. The byproducts of pectin extraction from cacao pod husks, that is, dried cacao pod husk powder (DCP), steamed DCP (sDCP), hot water-treated cacao pod husk powder (HCP), hot water-treated cacao pod husk supernatant (HCS), and cacao pod husk pectin (CPH pectin), were used to create five experimental diets, which were administered to five groups. The control group was fed a basal diet. The growth and immunocompetence of the shrimp were determined after 30, 60, 90 and 120 days of feeding. To identify the most cost-effective means of obtaining dried cacao pod husks, this study firstly determined the costs and effectiveness of the sun-drying, dehumidification, and heated-wind drying techniques. According to the results of growth performance, the CPH pectin group had higher survival but lower weight gain than the DCP, sDCP, HCP, and HCS groups did. At 30, 60, and 90 days, the clearance efficiency of the experimental groups was higher than that of the control group. At 60 days, the experimental groups had significantly higher phagocytic activity than the control group did. However, at 30 and 90 days the HCP, HCS and CPH pectin group had higher phagocytic activity. The total hemocyte count, differential hemocyte count, phenoloxidase activity, and respiratory bursts of the CPH pectin group were higher at 30 days but the same as those of the control group at 60 and 90 days. After 120 days of feeding trial, the resistance of L. vannamei fed with diets containing byproducts of pectin extraction from cacao pod husks significantly enhanced compared to that in BD group when they were infected with Vibrio aliginolyticus for 168 h, and the related higher survival rate can be observed in HCP, HCS and CPH pectin groups. The study findings suggest that diet-administered HCP and HCS have long-term immunostimulant potential and that CPH pectin has potential in the early stages of feeding. In addition, when heated air drying was employed, a moisture level of below 10% was obtained within 12 h. The results of this study indicate that adding HCP obtained from heated air-dried cacao pod husks to the feed of L. vannamei is the most cost-effective and sustainable means of promoting long-term growth performance and immunocompetence in the species.

In vivo study of a novel protein kinase C that mediates immunocompetence and catecholamine biosynthesis in hemocytes of Litopenaeus vannamei by using its potential competitive inhibitor, bisindolylmaleimide I.

This study applied bisindolylmaleimide I (BSM), a pharmacological competitive inhibitor of protein kinase C (PKC) enzymatic activity, at 1.25 pmol shrimp-1 for 60 min to investigate the potential involvement of PKC in signal transduction pathways in the hemocytes of Litopenaeus vannamei. A novel PKC in L. vannamei (LvnPKC) was identified and characterized and was determined to be involved in mediating the neuroendocrine-immune regulatory network. The hemocytes of L. vannamei that receive BSM exhibit significantly decreased PKC activity and LvnPKC gene and protein expression levels. Furthermore, the total hemocyte count, hyaline cells, and semigranular cells increased significantly along with significant decreases in granular cells, and meanwhile, the significantly increased phenoloxidase activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and neutrophil extracellular trap were observed; however, phagocytic activity decreased significantly. In a molecular model, the gene expressions of lipopolysaccharide- and ß-1,3-glucan-binding protein, peroxinectin, cytosolic manganese SOD, mitochondrial manganese SOD, and copper/zinc SOD in the hemocytes of L. vannamei that had received BSM decreased significantly, but prophenoloxidase I increased significantly. In catecholamine biosynthesis, tyrosine, dopamine, and norepinephrine decreased significantly in the hemocytes of L. vannamei that had received BSM, and l-dihydroxyphenylalanine increased. Moreover, tyrosine hydroxylase (TH) activity increased significantly, whereas TH and dihydroxyphenylalanine decarboxylase gene expression decreased significantly. These findings suggest that BSM inhibits PKC activity in hemocytes in which LvnPKC gene and protein expression are also inhibited. Additionally, the hemocytes' immunocompetence, including their prophenoloxidase and antioxidant systems, phagocytic activity, and catecholamine biosynthesis, was disrupted, confirming the roles of LvnPKC in mediating the neuroendocrine-immune regulatory network in hemocytes.

Synbiotic combination of prebiotic, cacao pod husk pectin and probiotic, Lactobacillus plantarum, improve the immunocompetence and growth of Litopenaeus vannamei.

To reach the sustainable development goals on waste recycling, cacao pod husk (CPH), produced as an agricultural waste byproduct during the cacao bean processing was applied to manufacture CPH pectin for developing the potential for diverse application in aquaculture, minimizing CPH impact to the environment and bringing benefits to the agriculture and aquaculture industries. In this study, CPH pectin (5 g/kg diet) and Lactobacillus plantarum (LP; 1010 cfu/kg diet) were separately introduced to the diets of Litopenaeus vannamei for a 56-day feeding trial, and two synbiotic combinations of CPH pectin and LP (CPH pectin at 5 g/kg diet + LP at 107 cfu/kg diet or at 1010 cfu/kg diet) were also conducted. After the 56-day feeding trial, significantly elevated percent weight gain, percent length gains and feeding efficiency in L. vannamei were only observed in synbiotic combination of CPH pectin at 5 g/kg diet and LP at 107 cfu/kg diet treatment, and the remainder of the treatments remained consistently similar to the control. Significantly increases in total haemocyte count, granular cells, phenoloxidase activity, and respiratory bursts were observed in L. vannamei fed with synbiotics at 7-28 days of feeding, accompanied by significant promotion of phagocytic activity and clearance efficiency in response to V. alginolyticus challenge during 56 days of feeding trial. Furthermore, at the end of the 56 days of feeding trial, shrimp receiving CPH pectin and/or LP treatments showed a significantly higher survival ratio against V. alginolyticus infection and hypothermal stress. It was therefore concluded that CPH pectin or LP was confirmed as an immunostimulant for L. vannamei to trigger immunocompetence through oral administration without negative effects within 56 days of feeding trial, and the synbiotic combination of CPH pectin and LP exhibited complementary and synergistic effects on growth performance and immunocompetence in L. vannamei.

Pectin of cacao pod husk, an efficient immunostimulant for white shrimp, Litopenaeus vannamei.

The disposal of cacao pod husk, a byproduct of cacao bean processing, can cause serious adverse environmental impacts, motivating scientist to explore and develop potential beneficial applications of this resource. Dried cacao pod husk was extracted with ethanol to obtain a 10.6% pectin of cacao pod husks (pCPH), and its effects on the immunocompetence of Litopenaeus vannamei were estimated. Measured variables included total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, as well as phagocytic activity and clearance efficiency against Vibrio alginolyticus after receiving pCPH at 0, 1.5, 3, and 6 µg shrimp-1 for 0, 1, 3 and 7 days via injection, and their resistance to thermal stress and V. alginolyticus infection were further evaluated. No significant differences were observed in total haemocyte count, differential haemocyte count, and respiratory bursts in shrimp receiving pCPH at 1.5 µg shrimp-1 for 1 day; however, these variables were significantly elevated after 3 days of injection, compared to the control group. The significantly increased phenoloxidase activity was assessed in shrimp receiving pCPH at 1.5, 3 and 6 µg shrimp-1 within 3 days, and activity returned to the baseline after 7 days. Furthermore, the reduced phenoloxidase activity per granulocytes or respiratory bursts per haemocytes maintained homeostasis following the variation of haemogram. For gene expression assessments in haemocytes, the immune-related genes of the lipopolysaccharide and ß-1,3-glucan binding protein, prophenoloxidase II and anti-lipopolysaccharide factor as well as innate immune signaling pathway-related genes of toll-like receptors 1 and 3 significantly increased after shrimp received pCPH for 1 day. The increases in phagocytic activity and clearance efficiency were only detected in shrimp receiving pCPH at 6 µg shrimp-1 within 7 days, compared to the control. There was no significant difference in the mortality ratio of shrimp against hyperthermal stress when they received pCPH for 1 day, and the significant higher resistance to hypothermal stress and V. alginolyticus infection were found in shrimp received pCPH at 6 µg shrimp-1 for 1 days than those in the other treatments. It is therefore found that pCPH triggers immune responses serving as an immunostimulant capable of enhancing resistance against V. alginolyticus and hypothermal stress.

Role of novel protein kinase C in neuroendocrine-immune regulatory network in haemocytes of Litopenaeus vannamei: An in vitro approach.

Shrimp lack adaptive immune systems and mainly rely on the cellular and humoral defences, involving the haemocytes (functionally analogous to vertebrate leukocytes) in non-self matter recognition, elimination, and in downstream coagulation. Furthermore, the linkage between stress-induced catecholamine (CA), a class of biogenic amines (BAs), releasing and immunological responses has been detected in shrimp. Varied isotypes of protein kinase C (PKC) regulate multiple cellular processes following their specific location and distribution within the cells, and a novel PKC identified in Litopenaeus vannamei (termed as LvnPKC) is proposed to mediate signaling transduction of immunocompetence and BA biosynthesis. In the present study, we analyzed the effects of the LvnPKC-silenced haemocytes by co-incubating with its dsRNA on the immune responses specific to prophenoloxidase (proPO) and antioxidant systems as well as phagocytic activity. In addition, the capability of haemocytes to produce BAs was assessed. The results revealed that LvnPKC-silenced haemocytes can induce interference in phenoloxidase and superoxide dismutase activities, respiratory bursts, and phagocytic activity; meanwhile, the disturbed gene expressions of proPO activating enzyme, proPOII, lipopolysaccharide- and ß-1,3-glucan-binding protein, and cytosolic manganese superoxide dismutase were detected. The same deviated pattern was observed in tyrosine, dopamine, and norepinephrine levels, and in dopamine ß-hydroxylase (DBH) activity and gene expressions of tyrosine hydroxylase, DOPA decarboxylase, and DBH involving in BA biosynthesis. Taken together, these results suggest that the immunocompetence and BA biosynthesis of haemocytes can be mediated via LvPKC signaling transduction, which proved the presence of a neuroendocrine-immune regulatory network in haemocytes.

Injection of an extract of fresh cacao pod husks into Litopenaeus vannamei upregulates immune responses via innate immune signaling pathways.

Immunostimulation is a novel method and a promising development in aquaculture. Products derived from plants exhibit various biological activities. In this study, the hot-water extract isolated from fresh cacao (Theobroma cacao) pod husks (CPHs) was administered by injection to evaluate cellular signaling pathways of innate immunity, the immunostimulating potential, disease resistance, and hypothermal tolerance of white shrimp, Litopenaeus vannamei. Results showed significant increases in the total hemocyte count, semigranular cells, granular cells, phenoloxidase activity, and respiratory bursts (RBs) of hemocytes per unit of hemolymph at 1 day, and in phagocytic activity toward and the clearance efficiency of Vibrio alginolyticus at 1-3 days after shrimp were injected with fresh CPH extract at 40 µg shrimp-1. However, only RBs per hemocyte had significantly decreased at 1 day after the injection. All immune parameters had returned to control levels by 3 days after receiving fresh CPH extract except for RBs, phagocytic activity, and the clearance efficiency, which had returned to control values by 7 days. Furthermore, at 1 day after the injection, the peroxinectin, prophenoloxidase (proPO) II, toll-like receptors (TLR) 3, signal transducer and activator of transcription (STAT), and crustin in shrimp receiving fresh CPH extract at 20 µg shrimp-1, and the lipopolysaccharide and ß-1,3-glucan-binding protein, proPO II, TLR1, and STAT in shrimp receiving fresh CPH extract at 40 µg shrimp-1 were significantly higher than those of shrimp receiving saline. After injecting fresh CPH extract at 10-40 µg shrimp-1 and 40 µg shrimp-1, the mortality of shrimp challenged with V. alginolyticus and exposed to 14 °C for 96 h significantly decreased, respectively. These results suggest that fresh CPH extract can be used as an immunostimulant and a physiological regulator for shrimp through injection administration to enhance immunological and physiological responses, which can elevate the resistance against V. alginolyticus and tolerance against hypothermal stress in L. vannamei.

The temporary modulation of tyramine on immune responses, carbohydrate metabolism, and catecholamines in Macrobrachium rosenbergii.

Tyramine (TA), a biogenic monoamine, plays various important physiological roles including immunological regulation in invertebrates. In this study, the effects of TA on the regulation of immune resistance, carbohydrate metabolism and biogenic monoamine, as well as its signaling pathway in Macrobrachium rosenbergii were determined. Results showed that total haemocyte count, hyaline cells, semigranular cells, and phenoloxidase activity per 50 µL of haemolymph and per granulocyte (the sum of semigranular and granular cells) at 0.5 h as well as phagocytic activity and clearance efficiency to Lactococcus garvieae at 1 h of prawn injected with TA at 1 nmol prawn-1 significantly increased, but the significantly decreased plasma lysozyme activity, phagocytic activity, clearance efficiency, and haemolymph glucose and dopamine were observed in prawn injected with TA at 10 nmol prawn-1 for 0.5 h. Respiratory bursts and haemolymph lactate in two TA-injection treatments at 0.5 h and 0.5-1 h, respectively, were significantly higher than those of the saline control, and in addition, TA depressed dopamine release in a dose-dependent manner after 0.5 h of TA injection. All the examined parameters returned to control levels after prawn injected with TA for 2 h. The inhibited effect of TA (at 10 nmol prawn-1 injection) on the phagocytic activity and clearance efficiency to pathogens was blocked by prazosin (an α1 adrenoceptors antagonist). For prawn received TA for 1 h then challenged with Lactococcus garvieae at 2 × 105 colony-forming units prawn-1, the survival ratio of TA 1 nmol prawn-1-injected prawn significantly increased by 20%, compared to the saline-challenged control or TA 10 nmol prawn-1-injected prawn after 144 h of challenge. These results suggested that the level of dopamine release suppression regulated by TA resulted in the immunoenhancing or immunosuppressive effects in prawn, and the signaling pathways of TA in mediating immune function were through octopamine (OA)/TA receptors.

Novel protein kinase C participates catecholamine biosynthesis and immunocompetence modulation in haemocytes of Litopenaeus vannamei.

The catecholamine biosynthesis is required for physiological and immunological responses against stress, and the neuroendocrine-immune regulatory network plays a crucial role in immunocompetence of shrimp. A novel protein kinase C of Litopenaeus vannamei (LvnPKC) is involved in immune defense and signaling transduction in haemocytes, and in the present study, the gene silence technique is conducted to identify the role of LvnPKC on catecholamine biosynthesis and immunocompetence modulation in haemocytes of L. vannamei. The results show that tyrosine significantly increases in haemocytes of LvnPKC-silenced shrimp, and in the meantime, the obvious decrease of L-3, 4-dihydroxyphenylalanine and increase of dopamine as well as the consistent norepinephrine levels are detected. Tyrosine hydroxylase and dopamine ß-hydroxylase activities are significantly reduced in haemocytes of LvnPKC-silenced shrimp. Total haemocyte count, hyaline cells and granulocytes insignificantly differ among treatments, and the obvious increase of phenoloxidase activity, respiratory bursts, superoxide dismutase and glutathione peroxidase activities are observed in haemocytes of LvnPKC-silenced shrimp, and furthermore, the downregulated phagocytic activity was observed. It is therefore concluded that the LvnPKC mediates catecholamine biosynthesis and immunocompetence in haemocytes, and plays a crucial role in the neuroendocrine-immune regulatory network.

The intracellular signaling pathway of octopamine upregulating immune resistance functions in Penaeus monodon.

Octopamine (OA), a biogenic monoamine, is known to mediate several immune responses. This study analyzed the effects of OA on immunological regulation in the tiger shrimp Penaeus monodon. The immune parameters including total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and phagocytic activity and clearance efficiency in response to the pathogen, Photobacterium damselae, were determined when shrimp were individually injected with saline or OA at 100 or 1000 pmol shrimp-1. In addition, the intracellular second messengers in haemocyte such as Ca2+ and adenosine 3',5'-cyclic monophosphate (cAMP) were examined in shrimp receiving saline or OA at 1 or 10 nmol shrimp-1. Results showed that all of the immune parameters significantly increased at 2-4 h in OA-injected shrimp except hyaline cells in 100 pmol shrimp-1-injected shrimp at 4 h, but phenoloxidase activity per granulocyte significantly decreased at 2-4 h. However, these had returned to saline control levels after receiving OA for 8 h except differential haemocyte count and phenoloxidase activity per granulocyte for 16 h. An injection of OA also significantly increased the survival rate of shrimp challenged with Pho. damselae. Shrimp receiving OA at 1 and 10 nmol shrimp-1 significantly increased the intracellular Ca2+ concentration ([Ca2+]i) at 30-60 min and 30 min, and cAMP concentration [cAMP]i) at 5-15 min and 15 min, respectively. However, [Ca2+]i at 50-60 min, and [cAMP]i at 30-60 min returned to saline control when the shrimp received OA at 10 nmol shrimp-1, and at 1 and 10 nmol shrimp-1, respectively. These results suggest that OA administration by injection at ≤1000 pmol shrimp-1 mediates transient upregulation of immunity together with the increased resistance of P. monodon to Pho. damselae, which are modulated through intracellular Ca2+ and cAMP second messenger pathways.

Involvement of a novel protein kinase C (nPKC) in immunocompetence in hemocytes of white shrimp, Litopenaeus vannamei.

Complementary (c)DNA encoding novel protein kinase C (PKC) messenger (m)RNA of the white shrimp Litopenaeus vannamei, consisted of 2454-bp cDNA containing an open reading frame (ORF) of 2232 bp, belonging to the novel (n)PKC family of proteins characterized by their containing two phorbol ester/diacylglycerol-binding domains (C1 domain), a C2 domain, and a catalytic domain of the serine/threonine kinase, designated LvnPKC. A comparison of amino acid sequences showed that LvnPKC was closely related to arthropod nPKC. LvnPKC cDNA was detected in all tested tissues with a real-time PCR including the hepatopancreas, gills, muscles, subcuticular epithelium, abdominal nerve, thoracic nerve, brain, the stomach, heart, and especially in hemocytes and the intestines. Moreover, significantly upregulated LvnPKC expression was only observed in the eyestalk, brain, and hepatopancreas of shrimp transferred from 28 °C to 18 °C for 30 min. Induction of LvnPKC expression in hemocytes of L. vannamei injected with Vibrio alginolyticus at 105 cfu shrimp-1 was detected earlier than in those injected with 103 cfu shrimp-1. Shrimp received LvnPKC-dsRNA for 1 days specifically depleted the expression of LvnPKC mRNA in hemocytes compared those of diethylpyrocarbonate water treatment. After that, significantly decreased expressions of lipopolysaccharide - and ß-1,3-glucan-binding protein, prophenoloxidase-activating enzyme, peroxinectin, prophenoloxidase I, and prophenoloxidase II in the prophenoloxidase-activating system; lysozyme and cytosolic manganese superoxide dismutase and mitochondrial manganese superoxide dismutase in the antioxidant system were observed. We therefore concluded that LvnPKC is involved in immune defense of L. vannamei exposed to hypothermal stress or infected with V. alginolyticus.

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.

Silencing tyrosine hydroxylase retards depression of immunocompetence of Litopenaeus vannamei under hypothermal stress.

Tyrosine hydroxylase (TH), the first and rate-limiting step in the synthesis of catecholamines, is required in catecholamine synthesis of the neuroendocrine regulatory network against stress in shrimp. The immunocompetence, catecholamine biosynthesis, and carbohydrate metabolites were evaluated in Litopenaeus vannamei received L. vannamei TH (LvTH) double-stranded (ds)RNA, diethyl pyrocarbonate-water, or non-targeted dsRNA for 3 days then transferred from 28 to 20 or 28 °C. The immunocompetence of LvTH-depleted shrimp held at 28 °C was promoted, and those were downregulated under hypothermal stress and revealed higher level than the other two dsRNA treatments. Meanwhile, the decrease of catecholamine biosynthesis was observed in LvTH-depleted shrimp held at 28 °C, and those were elevated under hypothermal stress and revealed lower levels, compared to two dsRNA treatments. The reduced carbohydrate metabolites was observed in LvTH-depleted shrimp held at 28 °C, and those were upregulated under hypothermal stress and showed lower levels than the other two dsRNA treatments. It was therefore concluded that LvTH-depleted shrimp revealed enhanced immunocompetence and reduced carbohydrate metabolites when exposed to a hypothermal stress condition, and in the meantime, even though catecholamine biosynthesis was downregulated, no significant difference was observed in DA or NE levels.

The transcription of atypical protein kinase C in hemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, during the molt stage and injection of pathogen-associated compounds.

Protein kinase C (PKC), which is involved in cell signaling pathways, comprises a family of serine/threonine kinases ubiquitously present in animals and its members are grouped on the basis of structural and activation characteristics into novel, classical, and atypical PKC forms. In this study, an atypical PKC of Macrobrachium rosenbergii, designated MraPKC, was successfully cloned, and its protein comprised structural domains similar to those of atypical PKC homologues, including the Phox and Bem1 (PB1) domain, a zinc finger phorbol-ester/DAG-type signature, protein kinase signatures, and a cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase C-terminal domain. Phylogenetic analyses revealed a close evolutionary relationship between MraPKC and aPKCs of insects. MraPKC transcripts were detected in all tissues examined through an RT-PCR, with the highest level detected in muscles. A quantitative real-time PCR was used to evaluate MraPKC expression in hemocytes of M. rosenbergii in various molt stages, and in prawn challenged with Vibrio alginolyticus, Lactococcus garvieae, and white spot syndrome virus (WSSV) as well as in prawns injected with pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PG), and polyinosinic:polycytidylic acid (Poly:IC). Results revealed that the expression pattern of MraPKC was distinctly modulated during molting, with significant enhancement in the C stage. MraPKC transcripts significantly increased in hemocytes of prawns infected with L. garvieae at 6-24 h and those injected with PG at 12-24 h. In contrast, significantly decreased expression of MraPKC was observed in hemocytes of prawns injected with V. alginolyticus and LPS for 3 and 12 h, respectively, and a similar phenomenon was observed in hemocytes of those injected with WSSV and Poly:IC for 12 h each. Therefore, MraPKC might play crucial roles in biological processes, and it may mediate the signaling pathway induced by varied pathogens for the potential regulation of host innate defense.

Involvement of dopamine beta-hydroxylase in the neuroendocrine-immune regulatory network of white shrimp, Litopenaeus vannamei.

In shrimp, the biosynthesis of catecholamines, including dopamine and norepinephrine, is required for physiological and immunological responses against stress. Dopamine beta-hydroxylase (DBH), a copper-containing monooxygenase enzyme that plays an important role in catecholamine synthesis of the neuroendocrine regulatory network, was identified in Litopenaeus vannamei. In the present study, the potential role of DBH in the immunocompetence of L. vannamei was further estimated by depleting DBH by pharmaceutical inhibition of disulfiram and a gene silencing technique of L. vannamei DBH-double-stranded (ds)RNA (LvDBH-dsRNA). Immunocompetence was evaluated following the determination of the total hemocyte count, differential hemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, phagocytic activity, and the clearance efficiency as well as the susceptibility against Vibrio alginolyticus infection. At 30-120 min after shrimp had received disulfiram, they exhibited significantly reduced total hemocyte count, phenoloxidase activity of hemocytes in hemolymph, respiratory bursts of hemocytes in hemolymph and per hemocyte, phagocytic activity, clearance efficiency, and survival ratio against V. alginolyticus infection, compared to those injected with saline. In addition, the significantly lower total hemocyte count, phagocytic activity, clearance efficiency, and resistance to V. alginolyticus infection were observed in shrimp that received LvDBH-dsRNA at 3 days post injection compared to those injected with diethyl pyrocarbonate-water or non-targeting gene-dsRNA. The DBH depleted L. vannamei revealed immunosuppression and decreased the survival ratio to V. alginolyticus infection, which indicated that DBH played a crucial role in the neuroendocrine-immune regulatory network.

The hot-water extract of leaves of noni, Morinda citrifolia, promotes the immunocompetence of giant freshwater prawn, Macrobrachium rosenbergii.

The hot-water Morinda citrifolia leaf extract (HMLE) was prepared for in vitro assessment on phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity (PA). Furthermore, the HMLE was administrated in the diet at 0.6, 3, and 6 g (kg diet)-1 for Macrobrachium rosenbergii, and the potential effects on the immunocompetence of prawns were evaluated. PO activity, RBs, and PA in hemocytes incubated with the HMLE at 140, 20, 20, and 140 mg l-1 significantly increased. The immune parameters of the total hemocyte count (THC), differential hemocyte count (DHC), RBs, PO activity, superoxide dismutase (SOD) activity, PA, transglutaminase (TG) activity and hemolymph clotting time were evaluated before and after 1, 3, 5, 7, and 9 weeks of the feeding trial. During 9 weeks of the feeding trial, higher THCs, DHCs, RBs, PO, and TG as well as accelerated clotting times were observed in prawns fed HMLE-containing diets at 0.6 g kg-1. The mRNA expressions of prophenoloxidase, TG, crustin, and lysozyme of prawns fed HMLE-containing diets at 0.6 g kg-1 for 9 weeks of the feeding trial significantly increased. The susceptibility of prawns fed the HMLE at 0.6 g kg-1 to Lactococcus garvieae infection significantly decreased, and the relative survival percentage was 23.1%. We therefore found that HMLE administrated through the diet at 0.6 g kg-1 was capable of enhancing the immunity and resistance against L. garvieae in M. rosenbergii.

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 ß-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 ß-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.

Dopamine beta-hydroxylase participate in the immunoendocrine responses of hypothermal stressed white shrimp, Litopenaeus vannamei.

Dopamine beta-hydroxylase (DBH) plays a critical role in catecholamine (CA) synthesis of neuroendocrine regulatory network, and is suggested to be involved in the immunoendocrine responses of invertebrate against bacterial challenge. DBH has been identified in white shrimp, Litopenaeus vannamei, and further investigation on its potential function was conducted after hypothermal stress, pharmaceutical inhibition and gene silencing in the present study. Cloned DBH L. vannamei (LvDBH), belonging to the Copper type II, ascorbate-dependent monooxygenases, was characterized by a DOMON domain, a Cu2_monooxygen domain and three glycosylation sites, and its expression was abundant in thoracic ganglia and haemocytes determined by quantitative real-time PCR. The effects of hypothermal stress showed that LvDBH expression in thoracic ganglia, haemocytes and hepatopancreas as well as the DBH contents in haemocytes and dopamine (DA) and norepinephrine (NE) levels in haemolymph are obviously up-regulated. L. vannamei receiving disulfiram for 30-120 min revealed the inhibition of DBH and NE contents in haemocytes and haemolymph respectively, but high level of DA in haemolymph was noticed. Besides, a significant decrease of LvDBH expression in thoracic ganglia, haemocytes and hepatopancreas were also observed. Subsequently, LvDBH expression was successfully silenced in thoracic ganglia, haemocytes and hepatopancreas of shrimp that received LvDBH-dsRNA for 3 days, and meanwhile, a decrease of DBH contents in haemocytes accompanied by decreased levels of NE and DA in haemolymph were also observed. These results indicate that LvDBH possesses the functional domains responsible for CAs synthesis, and therefore, inhibiting DBH contents in haemocytes by disulfiram and by LvDBH-dsRNA resulted in the impaired synthesis of NE from DA in haemolymph. These also suggest that the increased release of DA and NE in haemolymph for potential modulation of physiological or immunological responses is the consequence of the upregulated LvDBH expression and DBH contents in L. vannamei exposed to hypothermal stress.

The known two types of transglutaminases regulate immune and stress responses in white shrimp, Litopenaeus vannamei.

Transglutaminases (TGs) play critical roles in blood coagulation, immune responses, and other biochemical functions, which undergo post-translational remodeling such as acetylation, phosphorylation and fatty acylation. Two types of TG have been identified in white shrimp, Litopenaeus vannamei, and further investigation on their potential function was conducted by gene silencing in the present study. Total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, transglutaminase (TG) activity, haemolymph clotting time, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when shrimps were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or TG dsRNAs. In addition, haemolymph glucose and lactate, and haemocytes crustin, lysozyme, crustacean hyperglycemic hormone (CHH), transglutaminaseI (TGI), transglutaminaseII (TGII) and clotting protein (CP) mRNA expression were determined in the dsRNA injected shrimp under hypothermal stress. Results showed that TG activity, phagocytic activity and clearance efficiency were significantly decreased, but THC, hyaline cells (HCs) and haemolymph clotting time were significantly increased in the shrimp which received LvTGI dsRNA and LvTGI + LvTGII dsRNA after 3 days. However, respiratory burst per haemocyte was significantly decreased in only LvTGI + LvTGII silenced shrimp. In hypothermal stress studies, elevation of haemolymph glucose and lactate was observed in all treated groups, and were advanced in LvTGI and LvTGI + LvTGII silenced shrimp following exposure to 22 °C. LvCHH mRNA expression was significantly up-regulated, but crustin and lysozyme mRNA expressions were significantly down-regulated in LvTGI and LvTGI + LvTGII silenced shrimp; moreover, LvTGII was significantly increased, but LvTGI was significantly decreased in LvTGI silenced shrimp following exposure to 28 and 22 °C. Knockdown of LvTGI and LvTGI + LvTGII also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. The same consequences have been confirmed in LvTGII silenced shrimp in our previous study. These results indicate that LvTGI and LvTGII not only reveal a complementary effect in gene expression levels but also play a key function in the immune defence mechanism of shrimp, by regulating the haemolymph coagulation, immune parameters and immune related gene expression, and in the regulation of carbohydrate metabolism.

Roles of dopamine receptors in mediating acute modulation of immunological responses in Macrobrachium rosenbergii.

Dopamine (DA) was found to influence the immunological responses and resistance to pathogen infection in invertebrates. To clarify the possible modulation of DA through dopamine receptors (DAR) against acute environmental stress, the levels of DA, glucose and lactate in the haemolymph of Macrobrachium rosenbergii under hypo- and hyperthermal stresses were measured. The changes in immune parameters such as total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and phagocytic activity (PA) were evaluated in prawns which received DAR antagonists (SCH23390, SCH, D1 antagonist; domperidone, DOM, D2 antagonist; chlorpromazine, CH, D1+2 antagonist) followed by hypo- (15 °C) and hyperthermal (34 °C) stresses. In addition, pharmacological analysis of the effect DA modulation was studied in haemocytes incubated with DA and DAR antagonists. The results revealed a significant increase in haemolymph DA accompanied with upregulated levels of glucose and lactate in prawns exposed to both hypo- and hyperthermal stresses in 2 h. In addition, a significant decrease in RBs per haemocyte was noted in prawns which received DAR antagonists when they exposed to hyperthermal stress for 30 min. In in vitro test, antagonism on RBs, SOD and GPx activity of haemocytes were further evidenced through D1, D1, D1+D2 DARs, respectively, in the meantime, no significant difference in PO activity and PA was observed among the treatment groups. These results suggest that the upregulation of DA, glucose and lactate in haemolymph might be the response to acute thermal stress for the demand of energy, and the DAR occupied by its antagonistic action impart no effect on immunological responses except RBs in vivo even though the modulation mediated through D1 DAR was further evidenced in RBs, SOD and GPx activities in vitro. It is therefore concluded that thermal stress mediate stress responses not only through DAR but also via diverse pathways, and DA might modulate the levels of RBs, SOD and GPx activities mainly through D1 DAR.

A first insight into temperature stress-induced neuroendocrine and immunological changes in giant freshwater prawn, Macrobrachium rosenbergii.

Haemolymph norepinephrine (NE); total haemocyte count (THC); respiratory bursts (RBs); superoxide dismutase (SOD), phenoloxidase (PO), and phagocytic activity; and prophenoloxidase (proPO)-system-related genes (lipopolysaccharide- and ß-1,3-glucan-binding protein: LGBP, proPO, peroxinectin: PE, and α2-macroglobulin: α2-M) in haemocytes of Macrobrachium rosenbergii were investigated after transferring them from 28 °C to 22 °C, 28 °C, and 34 °C respectively. The results revealed that haemolymph NE, hyaline cells (HCs), and PO activity per granulocyte increased from 30 to 120 min of exposure, and however, RBs and phagocytic activity significantly decreased from 30 to 120 min of exposure as well as granular cells (GCs), semigranular cells (SGCs), and SOD activity decreased from 60 to 120 min of exposure for the prawns subjected to temperature stress. The proPO-system-related gene expression markedly increased with 60-120 min of exposure for the prawns transferred from 28 °C to 22 °C and 34 °C, except α2M at 120 min. These results provide a first insight into the effects of temperature stress on haemolymph NE level and immune functions in prawns and suggest that temperature-stress-induced acute modulation in immunity is associated with the release of haemolymph NE in M. rosenbergii.