Changes in the histological organization and spheroid formation in lymphoid organ of Penaeus monodon infected with yellow head virus

The changes in the histological and three dimensional organizations of lymphoid organ and characteristics of lymphoid cells after chronic infection with yellow head virus (YHV) in Penaeus monodon were investigated. The vascular cast of infected lymphoid organ showed less branching and dramatically shortened terminal capillaries that formed the lymphoid tubules, and only large stumps of these lymphoid tubules remained. This might occur because the terminal ends of the tubules were damaged from YHV infection, as stromal cells and hemocytes in the LT wall were infected and formed foci that could give rise to lymphoid spheroids that broke away from the original lymphoid tubules. Histologically, there was a decrease of PAS-stained connective tissue materials in lymphoid spheroids as well as a decrease of stromal cells as marked by anti-vimentin antibody. This indicated that stromal cells together with type 1 fibers and associated extracellular matrix degenerated in lymphoid spheroids, while type 2 or reticular fibers proliferated and encapsulated the spheroids.     

Histological and three dimensional organizations of lymphoid tubules in normal lymphoid organ of Penaeus monodon

The normal lymphoid organ of Penaeus monodon (which tested negative for WSSV and YHV) was composed of two parts: lymphoid tubules and interstitial spaces, which were permeated with haemal sinuses filled with large numbers of haemocytes. There were three permanent types of cells present in the wall of lymphoid tubules: endothelial, stromal and capsular cells. Haemocytes penetrated the endothelium of the lymphoid tubule's wall to reside among the fixed cells. The outermost layer of the lymphoid tubule was covered by a network of fibers embedded in a PAS-positive extracellular matrix, which corresponded to a basket-like network that covered all the lymphoid tubules as visualized by a scanning electron microscope (SEM). Argyrophilic reticular fibers surrounded haemal sinuses and lymphoid tubules. Together they formed the scaffold that supported the lymphoid tubule. Using vascular cast and SEM, the three dimensional structure of the subgastric artery that supplies each lobe of the lymphoid organ was reconstructed. This artery branched into highly convoluted and blind-ending terminal capillaries, each forming the lumen of a lymphoid tubule around which haemocytes and other cells aggregated to form a cuff-like wall. Stromal cells which form part of the tubular scaffold were immunostained for vimentin. Examination of the whole-mounted lymphoid organ, immunostained for vimentin, by confocal microscopy exhibited the highly branching and convoluted lymphoid tubules matching the pattern of the vascular cast observed in SEM.     

The roles of haemocytes and the lymphoid organ in the clearance of injected Vibrio bacteria in Penaeus monodon shrimp

In order to study the reaction of Penaeus monodon haemocytes, live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled. Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that large numbers of haemocytes encapsulated the bacteria at the site of injection. A rapid decrease of live circulating bacteria was detected in the haemolymph. Bacterial clearance in the haemolymph was induced by humoral factors, as observed by agglutinated bacteria, and followed by uptake in different places in the body. Bacteria mainly accumulated in the lymphoid organ (LO), where they, or their degradation products, could be detected for at least 7 days after injection. The LO consists of folded tubules with a central haemal lumen and a wall, layered with cells. The haemolymph, including the antigens, seemed to migrate from the central tubular lumen through the wall, where the bacteria are arrested and their degradation is started. Electron microscopy of the LO revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It is proposed that haemocytes settle in the tubule walls before they phagocytose. Immunostaining suggests that many of the haemocytes degranulate in the LO, producing a layer of fibrous material in the outer tubule wall. These findings might contribute to the reduced haemocyte concentration in the haemolymph of diseased animals or following injection of foreign material. It is proposed that the LO is a filter for virtually all foreign material encountered in the haemolymph. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessel in most decapod crustaceans that do not possess a LO. The experimental shrimp appeared to contain many LO spheroids, where bacterial antigens were finally observed as well. It is proposed that the spheroids have a degradation function for both bacterial and viral material, and that their presence is primarily related to the history of the infectious burden of the shrimp.     

Proteomic analysis of altered proteins in lymphoid organ of yellow head virus infected Penaeus monodon

A comparative proteomic analysis was employed to identify altered proteins in the yellow head virus (YHV) infected lymphoid organ (LO) of Penaeus monodon. At 24 h post-infection, the infected shrimps showed obvious signs of infection, while the control shrimps remained healthy. Two-dimensional electrophoresis of proteins extracted from the LO revealed significant alterations in abundance of several proteins in the infected group. Protein identification by MALDI-TOF MS and nanoLC-ESI-MS/MS revealed significant increase of transglutaminase, protein disulfide isomerase, ATP synthase beta subunit, V-ATPase subunit A, and hemocyanin fragments. A significant decrease was also identified for Rab GDP-dissociation inhibitor, 6-phosphogluconate dehydrogenase, actin, fast tropomyosin isoform, and hemolymph clottable protein. Some of these altered proteins were further investigated at the mRNA level using real-time RT-PCR, which confirmed the proteomic data. Identification of these altered proteins in the YHV-infected shrimps may provide novel insights into the molecular responses of P. monodon to YHV infection.     

Antiserum to the gp116 glycoprotein of yellow head virus neutralizes infectivity in primary lymphoid organ cells of Penaeus monodon

Yellow head virus (YHV) is an invertebrate nidovirus that has caused mass mortality of cultured Penaeus monodon in Asia. In this study, we investigated whether mouse polyclonal antiserum raised against the YHV gp116 or gp64 structural glycoproteins could neutralize YHV infectivity as determined using an in vitro quantal assay in primary cultures of lymphoid organ cells. Anti-gp116 antiserum showed virus-neutralizing activity whereas anti-gp64 antiserum failed to inhibit infection. The results suggest that gpl16 antiserum blocks binding of virions to cellular receptors to facilitate YHV entry into lymphoid organ cells.     

Monoclonal antibodies specific to haemocytes of black tiger prawn Penaeus monodon

Monoclonal antibodies specific to haemocytes of Penaeus monodon were generated from a mouse immunized with a mixture of SDS-treated and formalin-fixed haemocytes. Hybridoma clones were selected by immunohistochemistry against fixed haemocytes, heart, lymphoid organ, and haemopoietic tissue, and Western blot against haemocyte extract and haemolymph. Sixteen monoclonal antibodies specific to haemocytes were obtained and could be divided into six groups according to their binding capacities to various haemocyte proteins in Western blot analyses, 102, 43, approximately 20, 61, 175 and approximately 230 kDa, and their differences in recognition of haemocyte sub-populations. The first group of antibodies strongly recognized a small subset of semi-granulocytes (SG) and hyalinocytes (H) but occasionally stained lightly a very small population of granulocytes (G). The antibodies also bound to a group of cells in haemopoietic tissue as well as cells located at the inner layers of the tubules in the lymphoid organ but not in the spheroid. The second group of antibodies strongly bound to a large sub-population of G and SG with coarse granules but did not bind to most of the H. This group of antibodies also cross-reacted with cells in the outer layer of the tubules in the lymphoid organ. The third group of antibodies recognized all G and only a small portion of SG. The fourth, fifth and sixth groups bound to sub-populations of G, SG and H in similar proportions. None of the antibodies showed any cross-reactivity to other components in haemolymph. The common antigens recognized by the first and the second groups of antibodies in the haemopoietic tissue and the lymphoid organ may reflect relationships among these organs in the development of the sub-populations of G and SG. Haemopoietic tissue may be the site for haemocyte production and the lymphoid organ may be the site for further differentiation of at least two different lines of haemocytes.     

Allatostatins of the tiger prawn,Penaeus monodon (Crustacea: Penaeidea)

More than 40 peptides belonging to the -Y/FXFGL-NH(2) allatostatin superfamily have been isolated and identified from the central nervous system (CNS) of the tiger prawn, Penaeus monodon (Crustacea: Penaeidea). The peptides can be arranged in seven sub-groups according to the variable post-tyrosyl residue represented by Ala, Gly, Ser, Thr, Asn, Asp, and Glu. Two of the residues (Thr and Glu) have not been observed in this position previously in either insects or crustaceans. Also reported for the first time for allatostatins, two of the peptides are N-terminally blocked by a pyroglutamic acid residue. The yields of certain peptides with similar amino acid sequences to each other were, in some instances, very different. As an example, the yield of ANQYTFGL-NH(2) was 2pmol, compared with ASQYTFGL-NH(2), with a yield of 156 pmol. There are several possibilities to account for this. If, as in all species so far investigated, there is a single allatostatin gene in P. monodon, then it would appear that different sub-populations have contributed mutant forms of particular peptides to the extract. Another, less likely possibility is that this species has more than one allatostatin gene, producing a variable array of peptides albeit in different molar ratios. Several peptides were present apparently as a result of the loss of one or more residues at the N-terminus of a larger form, either due to N-terminal degradation or specific post-translational processing. The number of peptides identified exceeds that for any other insect or crustacean species previously investigated. None is identical to any of the 60-70 insect allatostatins so far identified, and only three are common to other crustaceans. Immunohistochemical study of the CNS of P. monodon, with the same antisera as used to monitor the purification, confirms the widespread nature and complexity of allatostatinergic neural pathways in arthropods. Thus, all neuromeres of the brain, and all except one of the ventral cord ganglia, possess allatostatin neurons and extensive areas of allatostatin-innervated neuropile. In addition to the cytological evidence that the allatostatins act as neurotransmitters, associated with tissues as varied as eyes and legs, their presence in neurohemal areas such as the sinus gland and the perineural sheath of the thoracic ganglia suggests a neuroendocrine function. As well as posing a challenge to physiologists assigning specific functions to the allatostatins, their extensive intra-species multiplicity, linked to their inter-species variability, also presents a complex problem to geneticists and evolutionists.     

Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon

P.-C. LIU, K.-K. LEE AND S.-N. CHEN. 1996. The pathogenicity of six Vibrio harveyi strains in tiger prawn, Penaeus monodon , was studied, using both live bacteria and extracellular products (ECP). The organisms originally isolated from diseased penaeids were more virulent using both live bacteria and ECP (LD₅₀, 4.87–8.65 times 10⁴colony-forming units (cfu) and 1.20–1.51 μg protein g^-1body weight) than the two reference strains originally isolated from either sea water (ATCC 25919; LD₅₀, 3.18 times 10⁶cfu and 2.70 μg protein g^-1body weight) or diseased Talorchestia sp. (ATCC 14126, 0.418 times 10⁶cfu and 2.34 μg protein g^-1body weight). Each strain was reisolated from the haemolymph and the hepatopancreas of moribund prawns following each bacterial challenge. Both the live bacteria and the ECPs of the penaeid isolates exhibited stronger proteolytic (caseinase), phospholipase and haemolytic activities than those of the reference strains. These results indicate that there are differences between penaeid and non-penaeid isolates of V. harveyi in pathogenicity and reveal that proteases, phospholipases, haemolysins or exotoxins might play leading roles in the pathogenicity of V. harveyi in the tiger prawn, Penaeus monodon .     

High expression of a novel leucine-rich repeat protein in hemocytes and the lymphoid organ of the black tiger shrimp Penaeus monodon

A novel leucine-rich repeat (LRR) cDNA has been cloned from hemocytes of the black tiger shrimp Penaeus monodon by 5' rapid amplification of cDNA ends. The full-length of P. monodon LRR (PmLRR) consisted of 2604 bp with a 1686-bp open reading frame, encoding 561 amino acids. The deduced protein contained a high proportion of leucine residues (17%) and had significant homology to LRR-containing proteins from bacteria to humans. Sixteen tandem LRR motifs of 23-24 amino acids in length occurred in the primary sequence. The computed 3D structure revealed a horseshoe shape consisting of alternately repeated strand and helical domains. Such structures are generally considered to mediate protein - protein interactions and to our knowledge, this is the first report of an LRR protein from a crustacean. PmLRR expression was tissue-specific (i.e. highest in hemocytes, intestine and lymphoid organ) suggesting that it may play some roles in shrimp defense against pathogens. A preliminary test suggested that PmLRR may be down-regulated after viral injection.     

Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon

FMRFamide-like immunoreactivity (FLI) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using a combination of three anti-FMRFamide-like peptide (FLPs) monoclonal antibodies. Approximately 3000 small neuronal cell bodies in the lamina ganglionalis; 100 medium to large size at the ganglion between the medulla interna and the medulla terminalis; and 250 medium size around the medulla terminalis were stained intensely. The neuronal processes in neuropils of the medulla externa, medulla interna, medulla terminalis, sinus gland and some nerve fibers in the optic nerve were also recognized. The small cell bodies, approximately 1500 cells, anterior to the medulla externa were stained inconsistently and the neuronal processes were not observed from these cells. Isolation of FLPs from 9000 eyestalks was performed using methanol/acetic/water (90:1:9) extraction. After the extract was partially purified using C18 cartridges, it was further purified by five to seven steps of RP-HPLC using three kinds of columns: C18; C8; and cyano, and three solvent systems: acetonitrile/trifluoro acetic acid; aceonitrile/heptafluoro butyric acid; and acetonitrile/triethyl ammonium acetate. Dot-ELISA using the combination of the same antibodies was used to monitor FLPs in the fractions during purification processes. Seven new sequences of FLPs were identified which can be divided into four subgroups according to the primary structure of the C-terminus: (1) GDRNFLRFamide; (2) AYSNLNYLRFamide; (3) AQPSMRLRFamide, SQPSMRLRFamide, SMPSLRLRFamide and DGRTPALRLRFamide; and (4) GYRKPPFNGSIFamide. These data indicate the high complexity of this peptide family in which multiple forms are usually exist.     

Immunolocalization of allatostatin-like neuropeptides and their putative receptor in eyestalks of the tiger prawn, Penaeus monodon

Allatostatin (AST)-like immunoreactivity (IR) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using four anti-AST antibodies. Depending on the antisera, AST-like immunoreactivity was detected in neuronal bodies of the lamina ganglionalis, cell bodies anterior to the medulla externa and cell bodies on the anterior and posterior of the medulla terminalis. Neuronal processes in neuropiles of the medulla externa, medulla terminalis, sinus gland and nerve fibers in the optic nerve were also recognized. No IR in cell bodies or in nerve fibers was found in the medulla interna. Strong AST-like immunoreactivity was found in hundreds of cells of the X organ. The localization of AST-like peptides suggests that they function as neurotransmitters and/or neuromodulators. Antiserum to the Drosophila AST receptor (Dar-2) recognized a single protein in P. monodon eyestalk protein extracts that was identical in size to that found in Drosophila protein extracts. Using this antiserum the putative P. monodon AST receptor was localized to the sinus gland in both juvenile and adult eyestalks. To our knowledge this is the first demonstration of a neuropeptide receptor localized to the crustacean sinus gland. This suggests that ASTs may function directly on the sinus gland as a neuromodulator. In juvenile eyestalks, the putative AST receptor was also localized to neuronal X organ cells of the medulla terminalis in males but not in females. The significance of this sex-specific receptor localization is unclear but emphasizes that ASTs function within the nervous system of the eyestalk.     

A double WAP domain-containing protein PmDWD from the black tiger shrimp Penaeus monodon is involved in the controlling of proteinase activities in lymphoid organ

A homolog of mammalian secretory leucocyte proteinase inhibitor or SLPI known as a double WAP domain (DWD) protein has been found in penaeid shrimp and believed to play an important role in innate immune system of the shrimp. The PmDWD identified from the Penaeus monodon EST database was investigated for its expression under pathogen infection. Infections by Vibrio harveyi and white spot syndrome virus (WSSV) up-regulated the expression of the PmDWD, which was peaked at about 24 h post infection and, then, subsided to more or less normal level. The PmDWD was expressed in various tissues of normal, 24-h WSSV-injected and leg-amputated shrimp, predominantly in the hemocytes. The expression was dramatically increased in lymphoid organ upon WSSV infection and leg amputation. The recombinant PmDWD (rPmDWD) was not active against the commercial proteinases: trypsin, chymotrypsin, elastase and subtilisin while its mutant rPmDWD_F70R was active against the subtilisin. By using agar diffusion assay, the rPmDWD inhibited the crude proteinases from lymphoid organs of leg-amputated and WSSV-infected shrimp. It inhibited the crude proteinases from Bacillus subtilis as well. Unlike the mammalian SLPIs, the rPmDWD had no antimicrobial activity against various bacteria.     

Passive immunization of the tiger prawn, Penaeus monodon, using rabbit antisera to Vibrio harveyi

Passive immunization, toxicity neutralization and the persistence of passive protection in the tiger prawn ( Penaeus monodon ) were investigated using rabbit antisera to the formalinized extracellular products (ECP) (RαECP) and/or formalinized bacterial cells (RαBC) of luminescent Vibrio harveyi strain 820514 originally isolated from diseased tiger prawns. Rabbit antiserum to bovine serum albumin (RαBSA) or phosphate-buffered saline (PBS, pH 7·2) both served as controls. The toxicity of ECP to prawns was neutralized by pre-incubation with RαECP. Passive immunization by pre-injection of RαBC or RαECP into prawns 3 d in advance protected against a lethal dose challenge of bacteria. To determine the persistence of passive protection by rabbit antiserum in tiger prawns, the RαBC, RαECP, RαBSA or PBS were injected into prawns. At 10, 17 or 24 d post-immunization, groups of prawns were given a lethal dose challenge of bacteria. The prawns in the two control groups were all killed within the first 2 d following challenge at all three challenge dates, Pre-injection with RαBC and RαECP provided total protection for 10 and 17 d, respectively, with all treated prawns surviving for at least 2 weeks post-challenge. This is the first study using mammalian antisera to investigate toxicity neutralization, passive immunization and persistence of passive protection by rabbit antisera in prawns. The results could be useful in future studies on virulence mechanisms and disease control of vibriosis in cultured prawns.     

Sulfakinin neuropeptides in a crustacean. Isolation, identification andtissue localization in the tiger prawn Penaeus monodon.

The sulfakinin (SK) family of neuropeptides are characterized by a C-terminal octapeptide sequence that begins with two acidic residues (most commonly DD), and ends with YGHMRF-NH2, usually with the tyrosyl residue sulfated. So far, sulfakinins have only been identified in insects and the present study was initiated to investigate if the family is more widely distributed within the arthropods. Purification of an extract of the central nervous system of the giant tiger prawn Penaeus monodon has revealed three novel members of the sulfakinin peptide family. One of the peptides, Pem SKI, has the sequence 相似文献   

Ovarian function in the giant tiger prawn (Penaeus monodon) as determined by in vitro bioassay.

Ovary tissue fragments of the giant tiger prawn Penaeus monodon were incubated in vitro with L-methionine[35S] plus L-cysteine[35S] as a metabolic labeling reagent. The labeled cytoplasmic and secreted proteins synthesized in vitro during incubations under various conditions were subjected to SDS polyacrylamide electrophoresis and visualized by autoradiography. Vitellogenin (Vg) was immunologically identified and shown to be actively synthesized and released into the incubation medium. The synthesis and release of Vg into the incubation medium was optimized and shown to be linear over a 16-h period. Comparisons between different ovarian regions and different stages of development revealed that the level of Vg synthesis and accumulation in the incubation media was variable depending on stage of development and region within the ovary. Coincubation of ovarian fragments with sinus gland extracts showed a dose-related inhibition of total protein and Vg synthesis. The in vitro ovarian bioassay is suitable for examining the effect of hormonal inputs of P. monodon.