Enhancement of immunity and disease resistance in Litopenaeus vannamei through injection of tyramine formulated with polyethylene glycol.

This study investigates the prolonged effect of immune disease resistance in Litopenaeus vannamei through the administration of tyramine (TA) formulated with polyethylene glycol (PEG). Facing the challenges of intensive farming, environmental stress, and global climate changes, innovative approaches to improve shrimp health are essential. The research focuses on the role of biogenic amines in stress response and immune regulation, demonstrating that TA, especially when combined with PEG, significantly prolongs immunity and resistance against Vibrio alginolyticus. The experimental design included administering TA, PEG, and TA-PEG, followed by evaluations of immunity, lactate and glucose levels, and immune-related gene expressions. Results showed notable prolonged effects in total hemocyte count, phenoloxidase activity, and phagocytic activity in the TA-PEG group, indicating enhanced immune activation period. Additionally, the expression of prophenoloxidase system-related genes was significantly upregulated in the TA-PEG group. Furthermore, the TA-PEG group exhibited a significantly higher survival rate in a susceptibility test against V. alginolyticus. The results of this study confirm that the combined use of PEG can effectively extend the immunostimulatory duration of TA.

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.

The acute modulation of norepinephrine on immune responses and genes expressions via adrenergic receptors in the giant freshwater prawn, Macrobrachium rosenbergii.

Norepinephrine (NE), immunocompetent parameters (total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to Lactococcus garvieae), and prophenoloxidase (proPO) system-related genes (lipopolysaccharide- and ß-1,3-glucan-binding protein, LGBP; prophenoloxidase, proPO; peroxinectin, PE; α2-macroglobulin, α2-M) expressions were investigated in Macrobrachium rosenbergii received NE through injection at 50 pmol/prawn after 0, 30, 60, and 120 min. Furthermore, the PO activity, RB, SOD activity, phagocytic activity and proPO system-related genes expressions were determined in haemocytes incubated with cacodylate buffer (CAC), NE, and NE co-treated with various adrenergic receptor (AR) antagonists in vitro. Results showed that NE, THC, granular cells, PO activity, SOD activity, proPO system-related genes expressions, and phagocytic activity and clearance efficiency to L. garvieae increased; PO activity per granulocyte and RB per haemocyte decreased from 30 to 120 min; semigranular cells and RB increased in the initial 30 min, and then decreased at 120 min when the prawns received NE by injection. In vitro studies, all the determined immune parameters and genes expressions were significantly decreased in haemocytes incubated with NE after 30 min. The negative effects of NE were prevented on the PO activity and phagocytic activity by the ß-AR antagonist of metoprolol (Met), on the SOD activity by the ß-AR antagonist of propranolol (Pro), on the RB by the ß-AR antagonist of Met and prazosin (Pra), and on the proPO system-related genes expressions by α-AR antagonist of Pra. These results show that NE modulates prawn haemocytes proPO system-related genes expressions via α1-AR, PO activity and phagocytosis via ß1-AR, respiratory burst via α1-and ß1-ARs, and SOD activity via ß2-AR. It is concluded that NE stimulates the regulation of immunocompetence parameters and proPO system-related genes expressions in an acute response to maintain homeostasis of M. rosenbergii, which is primarily mediated through α1-, ß1-and ß2-ARs.

Norepinephrine regulates prophenoloxidase system-related parameters and gene expressions via α- and ß-adrenergic receptors in Litopenaeus vannamei.

The total (THC) and differential haemocyte counts (DHC), phenoloxidase (PO) activity, and prophenoloxidase (proPO) system-related genes were investigated in haemocytes of Litopenaeus vannamei that received saline, norepinephrine (NE), and NE co-treated with various adrenergic receptor (AR) antagonists both in vivo and in vitro. Results showed that semi-granular and granular cells of shrimp which received NE, NE + phentolamine (Phe), NE + prazosin (Pra), NE + propanolol (Pro) and NE + metoprolol (Met) significantly decreased, while the PO activity of the shrimp received NE + Phe in vivo was significant higher than all the other treatments. PO activities of haemocytes exposed to saline, Pra + NE, and Met + NE were significantly higher than those of haemocytes exposed to NE, Phe + NE, and Pro + NE in vitro. Similar phenomena in lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP), proPO-I, proPO-II, serine proteinases (SP), and peroxinectin (PE) messenger (m)RNA expressions of haemocytes exposed to saline, NE, and NE co-treated with various AR antagonists were observed both in vivo and in vitro. No significant differences were observed for LGBP and proPO-II mRNA expressions between haemocytes treated with saline and Pra + NE, for proPO-I mRNA expression between haemocytes treated with saline and Met + NE; or for SP and PE mRNA expressions among haemocytes treated with saline, Pra + NE, and Met + NE. These results suggest that stress-induced NE may promote the migration of circulating granulocytes to the site of the injection and the existing proPO mRNA translation which had been stored in granulocytes. NE downregulated the LGBP, proPO-I, proPO-II, SP, and PE gene transcription by haemocytes via α1-, ß1-, α1-, α1- and ß1-, and α1- and ß1-ARs, respectively, which subsequently decreased the PO activity by α1- and ß1-ARs in haemocytes of L. vannamei.

Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, and immune status of white shrimp, Litopenaeus vannamei larvae.

In this study, the probiotic, Bacillus subtilis E20, isolated from the human health food, natto, was used for white shrimp, Litopenaeus vannamei, larvae breeding to improve the larval survival rate and development by adding probiotic to the rearing water at (control), 10(8), and 10(9) cfu L(-1) salt water once every 3 days during the 14 days of breeding experiment. Thereafter, stress tolerance and immune status of postlarvae were evaluated. Shrimp larval development was significantly accelerated after adding the probiotic to the larval rearing water at a level of 10(9) cfu L(-1). The survival rate of larvae was significantly higher in the treatment with 10(9) cfu L(-1) compared to the control and the treatment with 10(8) cfu L(-1) after all larvae had metamorphosed to postlarvae. Adding the probiotic to the shrimp larvae rearing water produced a weak inhibition of bacterial growth by an analysis of the total bacterial count and presumptive Vibrio count. For stress tests, no postlarvae died when they were reared in water in which the temperature was decreased from 30 to 2 degrees C at a rate of 0.1 degrees C min(-1). Postlarvae had significantly lower cumulate mortality in the treatments with 10(8) and 10(9) cfu L(-1) compared to the control when they were suddenly exposed to fresh water and 60 per thousand salt water. A significant decrease in the cumulative mortality of postlarvae treated with the probiotic at a level of 10(9) cfu L(-1) was recorded after the sudden transfer to 300 mg L(-1) nitrite-N compared to the control and treatment with 10(8) cfu L(-1). The analysis of immune-related gene expressions showed that the gene expression of prophenoloxidase I, prophenoloxidase II, and lysozyme of larvae were significantly increased after being reared in probiotic-containing water at the levels of 10(8) and 10(9) cfu L(-1). However, no significant difference in serine proteinase or glutathione peroxidase gene expressions was recorded in this study. It is therefore suggested that 10(9) cfu L(-1) of probiotic, B. subtilis E20 adding to rearing water for shrimp larva breeding.

Immune response of white shrimp, Litopenaeus vannamei, after a concurrent infection with white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus.

In the present study, we investigated immunological changes in viral-infected white shrimp, Litopenaeus vannamei. White shrimp were infected with white spot syndrome virus (WSSV) or co-infected with WSSV and infectious hypodermal and hematopoietic necrosis virus (IHHNV) as detected by polymerase chain reaction (PCR). The complete (100%) mortality rate of shrimp was caused by viral infection due to immune parameters being suppressed including decreases in phenoloxidase activity, total hemocyte counts, differential hemocyte counts, and the gene expressions of prophenoloxidase and peroxinectin. In addition, increases in lipopolysaccharide and beta-1,3-glucan-binding protein of hemocytes and the hepatopancreas, and respiratory bursts per cell, and a decrease in superoxide dismutase were found in viral-infected shrimp, which may have been related to the defense against viral infection.

A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.

The effect of Vibrio alginolyticus infection on caspase-3 expression and activity in white shrimp Litopenaeus vannamei.

Complementary (c)DNA encoding cysteine aspartate protease-3 (caspase-3) messenger (m)RNA of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the caspase sequences of Penaeus merguiensis, Spodoptera frugiperda, Spodoptera littoralis, and Penaeus monodon. The 1212-bp cDNA contained an open reading frame (ORF) of 951bp, a 56-bp 5'-untranslated region (UTR), and a 205-bp 3'-UTR containing the poly(A) tail. The molecular mass of the deduced amino acid (aa) sequence (316aa) was 28.7kDa with an estimated pI of 6.46. It contains two hits in the caspase family, including a p20 domain profile and p10 domain profile. From aa residues 183 to 194, there was a single pattern hit for QACRG, a caspase family cysteine active site. Caspase-3 mRNA was widely distributed in all tissues; especially in haemocytes and lymphoid organ, but was weakly expressed in muscles, ganglia, spermaries, and ovaries. Shrimp injected with Vibrio alginolyticus induced transcription of caspase-3 mRNA and increased caspase-3 activity which contributed to the occurrence of apoptosis and DNA fragmentation in haemocytes.

Immune responses and gene expression in white shrimp, Litopenaeus vannamei, induced by Lactobacillus plantarum.

The total haemocyte counts, phenoloxidase (PO) activity, respiratory bursts, superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to Vibrio alginolyticus, as well as prophenoloxidase (proPO), lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP), serine protein (SP), and peroxinectin (PE) mRNA transcription of L. vannamei, and its susceptibility to V. alginolyticus when the shrimp were fed diets containing Lactobacillus plantarum at 0 (control), 10(7), and 10(10) cfu (kg diet) (-1) for 48 and 168 h were evaluated. The results indicated that PO activity, SOD activity, clearance efficiency to V. alginolyticus, proPO and PE mRNA transcription, and the survival rate after challenge with V. alginolyticus all significantly increased, but the total haemocyte counts significantly decreased in shrimp fed a diet containing Lac. plantarum at 10(10) cfu (kg diet) (-1) for 168 h. However, no significant differences in phagocytosis, LGBP, or SP mRNA expression of shrimp were observed among the different treatments. It was concluded that administration of Lac. plantarum in the diet at 10(10) cfu (kg diet) (-1) induced immune modulation and enhanced the immune ability of L. vannamei, and increased its resistance to V. alginolyticus infection.

Molecular cloning and characterisation of prophenoloxidase cDNA from haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, and its transcription in relation with the moult stage.

Expression of prophenoloxidase (proPO) cDNA was determined from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus, kuruma shrimp Marsupenaeus japonicus, and white shrimp Litopenaeus vannamei. The proPO of M. rosenbergii was constitutively expressed. The 2,547-bp cDNA contained an open reading frame (ORF) of 2,013 bp, a 96-bp 5'-untranslated region, and a 438-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (671 aa) was 76.7 kDa with an estimated pI of 7.05. It contained putative copper-binding sites, a complement-like motif (GCGWPRHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in giant freshwater prawn proPO. Comparison of amino acid sequences showed that prawn proPO is similar to the proPO of penaeid, crayfish and lobster. Prawn proPO was only synthesised in haemocytes. The proPO transcript was significantly increased in the A stage and achieved the highest level in the B stage, and then declined sharply in the C stage and reached the lowest level in the D(2)/D(3) stage.

Molecular cloning and characterisation of prophenoloxidase from haemocytes of the white shrimp, Litopenaeus vannamei.

cDNA encoding prophenoloxidase (proPO) of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus (accession no.: AF521949) and kuruma shrimp Marsupenaeus japonicus (accession no.: AB0733223). proPO of L. vannamei was constitutively expressed. The 2471-bp cDNA contained an open reading frame (ORF) of 2058 bp, a 96-bp 5'-untranslated region, and a 317-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (686 amino acids) was 78.1 kDa with an estimated pI of 6.02. It contained putative copper binding sites, a complement-like motif (GCGWPQHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in white shrimp proPO. Comparison of amino acid sequences showed that white shrimp proPO is more closely related to the proPO of another penaeid than to that of a freshwater crayfish. White shrimp proPO mRNA was synthesized in haemocytes and not in the hepatopancreas or muscle. The activation responses of the proPO of the white shrimp to an exogenous protease (trypsin), a detergent (sodium dodecyl sulphate), and algal and microbial cell wall components (laminarin, sodium alginate, zymosan, and lipopolysaccharide), and its susceptibility to protease inhibitors in vitro resemble the proPO activation system of other crustaceans. These facts suggest that the proPO system in haemocytes of the white shrimp Litopenaeus vannamei serves an important function in non-self recognition and host immune reactions.

The peroxinectin of white shrimp Litopenaeus vannamei is synthesised in the semi-granular and granular cells, and its transcription is up-regulated with Vibrio alginolyticus infection.

Peroxinectin mRNA expression in the different types of haemocytes of white shrimp Litopenaeus vannamei was studied by in situ hybridisation using digoxigenin-UTP-labelled riboprobes and reverse transcription-polymerase chain reaction (RT-PCR). Granular cells (GC) and a mixture of semi-granular cells (SGC) and hyaline cells (HC) were separated by 70% Percoll gradient centrifugation. Peroxinectin was synthesised in both GC and the mixture of SGC-HC. An in situ hybridisation assay indicated that peroxinectin mRNA expression occurred in GC and SGC, but not in HC. Peroxinectin transcript up-regulated significantly, whereas haemocyte count decreased significantly at 6, 12 and 24 h post Vibrio alginolyticus-injection with slower restoration as compared to that of saline-injected shrimp. The RT-PCR assay indicated that peroxinectin exists extensively in several species of decapod crustaceans including L. vannamei, freshwater prawn Macrobrachium rosenbergii, common caridina Caridina pseudodenticulata, stone crab Thalamita crenata and mud crab Scylla serrata suggesting that this protein plays an important role in defence against pathogens.

Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing.

A gram-negative, rod-shaped bacterium identified as Vibrio alginolyticus was isolated from diseased Litopenaeus vannamei (also called Penaeus vannamei) in Taiwanese culture ponds. The diseased shrimp displayed poor growth, anorexia, inactivity, reddish pleural borders of antennae, uropods and telson, opaque and whitish musculature, and mortality. In histological preparations, melanized hemocytic granulomas were observed in the connective tissue around hemal sinuses together with hemocytic aggregation in necrotic musculature. Six isolates of Vibrio were collected from diseased shrimp at 3 farms, and these were evaluated for characteristics including morphology, physiology, biochemistry and sensitivity to antibiotics. The results indicated that the isolates belonged to a single species that grew in 1 to 8% NaCl, at 10 to 40 degrees C and on TCBS (thiosulfatecitrate-bile sucrose) agar, and that gave positive catalase, O/F (Oxidation/Fermentation), lysine decarboxylase, gelatinase and cytochrome-oxidase tests. Identification of CH003 (1 of 6 isolates) was confirmed by PCR assay for V. alginolyticus (expected amplicon 1486 bp). The 16S rDNA sequence (GenBank accession number AY373027) gave 99.9% sequence identity to V. alginolyticus (GenBank accession number X74690). The calculated 96 h LD50 dose of the isolated strain was 3.0 x 10(5) colony forming units (CFU) shrimp(-1) (6.6 x 10(4) CFU g(-1)).