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.

Dietary administration of mangosteen, Garcinia mangostana, peel extract enhances the growth, and physiological and immunoendocrinological regulation of prawn, Macrobrachiumrosenbergii.

In this study, we investigated the potential immunostimulatory effects of mangosteen (Garcinia mangostana) peel extract on Macrobrachium rosenbergii, specifically in enhancing immunity and resistance against Lactococcus garvieae. We employed a dietary administration approach to assess the impact of different extract preparations from mangosteen peel, namely mangosteen peel powder (MPP), boiled mangosteen peel powder (MPB), and mangosteen peel extract (MPE). Following the administration of mangosteen peel extract, we evaluated growth performance, innate immune parameters, and disease resistance in the prawns. The results revealed a significant increase in total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), as well as phagocytic activity and clearance efficiency against L. garvieae. Based on these findings, we suggest that mangosteen peel extract can be utilized as an immunostimulant for prawns through dietary administration, regulating immune responses and enhancing resistance against pathogens by modulating carbohydrate metabolism.

The immunostimulatory effects of Theobroma cacao L. pod husk extract via injection and dietary administrations on Macrobrachium rosenbergii and its resistance against Lactococcus garvieae.

The extract from Theobroma cacao L, pod husk served as the immunostimulant to enhance the immunity and resistance against Lactococcus garvieae of Macrobrachium rosenbergii. In this study, we employed the injection method and dietary administration method to determine the effect of cocoa pod husk (CPH) on M. rosenbergii. The non-specific immune parameters and disease resistance were evaluated after the prawn injected with 1 µg prawn-1 CPH extract (C1), 3 µg prawn-1 CPH extract (C3), and 5 µg prawn-1 CPH extract (C5) for 1, 3, and 7 days. The results showed a significant increase of total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity and clearance efficiency to L. garvieae. The non-specific immune parameters, physiological parameters, and disease resistance and growth performance were evaluated after the prawn fed with 1 g kg-1 CPH extract diet (CD1), 3 g kg-1 CPH extract diet (CD3) and 5 g kg-1 CPH extract diet (CD5). The results showed a significant increase in all immune parameters and showed a significant decrease in physiological parameters. No significant difference was observed in growth performance of prawn fed with the CPH containing diet. Both injection and dietary method showed a significant increase in disease resistance against to L. garvieae. We therefore recommend that CPH extract can be used as a immunostimulant for prawn by dietary administration to regulate immune responses, and carbohydrate metabolism lead to enhance resistance against pathogen.

Injection administration of mangosteen, Garcinia mangostana, husk extract enhances physiological and immune-neuroendocrinological regulation of Macrobrachiumrosenbergii.

The study aimed to investigate the effects of injecting mangosteen husk hot-water extracts (MHE) on immune and physiological factors in Macrobrachium rosenbergii. Different doses of MHE (10, 20, and 40 µg prawn-1) were injected into the prawns, and various immune and physiological parameters were evaluated. The results revealed that higher doses of MHE (20 and 40 µg prawn-1) led to significant increases in immune parameters, improved phagocytic activity, and clearance efficiency. However, certain parameters, such as phenoloxidase activity per granulocyte, plasma glucose, and lactate levels were decreased after injection. Moreover, prawns injected with MHE and subjected to hypothermal stress exhibited changes in haemolymph dopamine and norepinephrine. Prawns injected with MHE for 7 days showed increased survival rates when challenged with Lactococcus garvieae. The relative survival percentages were 11.8%, 46.6%, and 47.1% for MHE doses of 10, 20, and 40 µg prawn-1 injection, respectively, indicating enhanced resistance to the pathogen. In conclusion, injecting MHE can act as an immunostimulant and physiological and neuroendocrine regulator for prawns, enhancing their resistance to L. garvieae.

Molecular characterization and functional roles for Vibrio alginolyticus resistance of an octopamine/tyramine receptor of the white shrimp, Litopenaeus vannamei.

Octopamine and Tyramine are biogenic amines that have been demonstrated to play an important immunological role in white shrimp, Litopenaeus vannamei. G protein-coupled receptors, known as seven-transmembrane domain receptors, are a variety of neurotransmitter receptors which are sensitive to biogenic amines for initiating the cell signaling pathway. In present study, we cloned and characterized an octopamine/tyramine receptor (LvOA/TA-R) from the hemocytes of L. vannamei, with a 1194 b.p. open reading frame that encodes 398 amino acids. Several bioinformatics analyses indicated that LvOA/TA-R had seven conserved hydrophobic transmembrane domains. The phylogenetic analysis and multiple sequence alignment indicated that LvOA/TA-R was orthologous to the OA/TA receptor of tiger shrimp, P. monodon. LvOA/TA-R was expressed in hemocytes and nervous tissue including circumoesphageal connective tissue and the thoracic and abdominal ganglia. Significant increases in LvOA/TA-R occurred in hemocytes of L. vannamei under Vibrio alginolyticus infection within 30-60 min of infection. Here, we demonstrated that LvOA/TA-R expression is upregulated in response to Vibrio alginolyticus infection and appears to be functionally responsible for the observed immune response. These results suggest that LvOA/TA-R mediates regulation of immunity, which promotes the resistance of L. vannamei to V. alginolyticus.

The effect of banana blossom on growth performance, immune response, disease resistance, and anti-hypothermal stress of Macrobrachium rosenbergii.

Banana (Musa acuminata) blossom contains high nutritional value and bioactive compounds. In this study, Macrobrachium rosenbergii were fed with diets containing banana blossom powder (BBP) at 10 and 20 g kg-1, hot-banana blossom (BBH) extract at 10 and 20 g kg-1, and the basal diet for 56 days. The growth performance, physiological response and immune parameters were evaluated. The results showed that a significantly higher percentage weight gain (PWG) and percentage length gain (PLG) in prawns fed with BBH diet. The feed efficiency (FE) significantly increased in prawns fed BBP. The prawn fed both BBH and BBP diet showed higher survival rate than control group. The prawn fed with BBH showed a significant increase in total haemocyte count (THC) and different haemocyte count (DHC), whereas phenoloxidase (PO) activity and respiratory bursts (RBs) significant increase in prawns fed both BBP and BBH diet. Furthermore, M. rosenbergii fed with both BBP and BBH diets showed significantly higher phagocytic activity and clearance efficiency against Lactococcus garvieae infection. At the end of the 56 days of feeding trial, the susceptibility of prawns to L. garvieae infection and hypothermal (18 °C) stress were evaluated. The results showed that prawns fed BBH diets had a significantly higher survival rate against L. garvieae than those of fed with the basal diet. Anti-hypothermal stress was observed in prawns fed both BBP and BBH diets showing no significant difference in haemolymph glucose in prawns subjected to 18 °C and 28 °C, whereas the norepinephrine level in haemolymph of prawns fed with BBH diets subjected to 18 °C was significantly lower than in prawns subjected to 28 °C. In summary, we recommend addition of hot-banana blossom extract to the diet of M. rosenbergii at 20 g kg-1 to promote growth performance, improve physiological function, enhance immunity, increase anti-hypothermal stress, and to increase resistance against L. gavieae.

Cloning and characterization of a key enzyme in octopaminergic pathway: Tyramine beta-hydroxylase from Litopenaeus vannamei, as expressed during Vibrio alginolytics infection and hypothermal stress.

Tyramine beta-hydroxylase (TBH) is needed for the biosynthesis of the octopamine (OA) from tyramine (TA). Both OA and TA act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. In this study, TBH was identified in white shrimp, Litopenaeus vannamei, and further investigation on its potential function was conducted after inducing hypothermal stress and Vibrio alginolyticus infection. TBH of L. vannamei (LvTBH) was comprised 2178 nucleotide residues and contained an open reading frame encoding 408 amino acids, belonging to the Copper type II, ascorbate-dependent monooxygenases, was characterized by two Cu2_monooxygen domains and five glycosylation sites. LvTBH expression was especially abundant in muscle, and mainly in brain and thoracic ganglia of nervous system, eyestalk tissues, epithelium, and stomach, as determined by quantitative real-time PCR. The effects of hypothermal stress showed significant increases in LvTBH at 15, 30 and 60 min in brain and at 30 min in haemocyte, accompanied by an increase in OA level in haemolymph from 15 to 60 min. Significant increases in LvTBH occurred at 15, 30 and 60 min in haemocyte and at 60 min in brain tissue, and was proportional to the OA level of haemolymph under Vibrio alginolyticus infection from 30 to 60 min. Here, we demonstrated that LvTBH is functionally responsible for biogenic amine synthesis, suggesting that the increased release of OA in haemolymph for potential modulation of physiological and immunological responses is the consequence of the upregulated LvTBH gene expression in L. vannamei exposed to hypothermal stress and Vibrio alginolyticus infection.

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.

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.

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 ß-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.

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.

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.

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.

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.

Dietary administration of sodium alginate ameliorated stress and promoted immune resistance of grouper Epinephelus coioides under cold stress.

Grouper, Epinephelus coioides, fed a diet containing sodium alginate at 0 (control, named C) and 1.0 g kg-1 (named S) at a temperature of 28 °C for 12 days, were then further individually transferred to 28 (two groups named C-28 and S-28) or 20 °C (two groups named C-20 and S-20), and immune parameters and stress indexes were measured at the beginning and after 6, 12, 24 and 48 h of exposure. Examination of immune parameters revealed that the alternative complement activity (ACH50), lysozyme activity, phagocytic activity, superoxide dismutase, and respiratory bursts significantly increased in groupers fed the sodium alginate-containing diet for 12 days, and were higher in the S-28 than those of the C-28 and S-20 groups, which were higher than those of the C-20 group from 6 to 48 h except for ACH50 at 48 h, respiratory bursts at 48 h, and lysozymes at 6 h. For the assessment of stress indicators, cortisol, glucose, and lactate levels of serum significantly decreased in grouper fed the sodium alginate-containing diet for 12 days, and were higher in the C-20 group than those of the C-28 and S-20 groups, which were higher than those of the S-20 group at 6-48 h. In another experiment, grouper fed the test diet for 12 days at a temperature of 28 °C were challenged with Photobacterium damselae subsp. piscicida at a dose of 5 × 103 colony-forming units (cfu) (g fish)-1, and then individually transferred to 28 or 20 °C. The survival rate of challenged fish of the C-28 group was significantly lower than those of challenged fish of the C-20 and S-28 groups, which were significantly lower than that of challenged fish of the S-20 group. All challenged fish of the S-20 group survived. Survival rates over 144 h were 30.0%, 70.0%, and 56.7% for the C-28, C-20, and S-28 groups, respectively. Our results indicated that dietary sodium alginate administration downregulated stress response indicators, enhanced immune responses, and prevented impacts of physiologic stress responses, immunosuppression, and susceptibility to P. damselae subsp. piscicida in grouper subjected to cold stress. Grouper cultured at 28 °C were more susceptible to P. damselae subsp. piscicida infection.

Cloning and characterization of tyrosine hydroxylase (TH) from the pacific white leg shrimp Litopenaeus vannamei, and its expression following pathogen challenge and hypothermal stress.

Tyrosine hydroxylase (TH) belongs to the biopterin-dependent aromatic amino acid hydroxylase enzyme family, and it represents the first and rate-limiting step in the synthesis of catecholamines that are required for physiological and immune process in invertebrates and vertebrates. Cloned Litopenaeus vannamei TH (LvTH), containing a short alpha helix domain, a catalytic core, a regulatory domain, a phosphorylation site and two potential N-linked glycosylation sites as presented in vertebrate and insect THs without acidic region and signal peptide cleavage sites at the amino-terminal, exhibited a similarity of 60.0-61.2% and 45.0-47.0% to that of invertebrate and vertebrate THs, respectively. Further, LvTH expression was abundant in gill and haemocytes determined by quantitative real-time PCR. L. vannamei challenged with Vibrio alginolyticus at 10(5) cfu shrimp(-1) revealed significant increase of LvTH mRNA expression in haemocytes within 30-120 min and in brain within 15-30 min followed with recuperation. In addition, shrimps exposed to hypothermal stress at 18 °C significantly increased LvTH expression in haemocytes and brain within 30-60 and 15-60 min, respectively. The TH activity and haemolymph glucose level (haemocytes-free) significantly increased in pathogen challenged shrimp at 120 min and 60 min, and in hypothermal stressed shrimp at 30-60 and 30 min, respectively. These results affirm that stress response initiates in the brain while haemocytes display later response. Further, the significant elevation of TH activity in haemolymph is likely to confer by TH that released from haemocytes. In conclusion, the cloned LvTH in our current study is a neural TH enzyme appears to be involved in the physiological and immune responses of whiteleg shrimp, L. vannamei suffering stressful stimulation.

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.