Apoptosis of tiger shrimp (Penaeus monodon) haemocytes induced by Escherichia coli lipopolysaccharide

This study was aimed at investigating the toxicity mechanism of lipopolysaccharide (LPS) on Penaeus monodon haemocytes at a cellular level. Reactive oxygen species (ROS) production, nitric oxide (NO) production, non-specific esterase activity, cytoplasmic free-Ca^2 + (CF-Ca^2 +) concentration, DNA damaged cell ratio and apoptotic cell ratio of in vitro LPS-treated haemocytes were measured by flow cytometry. Two concentrations of Escherichia coli LPS (5 and 10 μg mL^? 1) were used. Results showed that ROS production, NO production and CF-Ca^2 + concentration were significantly induced in the LPS-treated haemocytes. Ratio of DNA damaged cell and apoptotic cell increased caused by LPS, while esterase activity increased at the initial 60 min and dropped later. The initial increase in esterase activity suggested that LPS activated the release of esterase, and the later decrease might result from apoptosis. These results indicated that LPS would induce oxidative stress on shrimp haemocytes, and cause Ca^2 + release, DNA damage and subsequently cell apoptosis. This process of ROS/RNS-induced Ca^2 +-mediated apoptosis might be one of the toxicity mechanisms of LPS on shrimp haemocytes.     

Dopamine depresses immunity in the tiger shrimp Penaeus monodon

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Photobacterium damsela were measured when tiger shrimp Penaeus monodon (13.5+/-1.5 g) were individually injected with saline or dopamine at 10(-8), 10(-7), or 10(-6)mol shrimp(-1). Results showed that a transient period of immunosuppression occurred between 2 and 8h after injection of dopamine for all immune parameters except circulating haemocytes, and all immune parameters had returned to control values within 8-16 h after receiving dopamine. The injection of dopamine also significantly increased the mortality of P. monodon challenged with the pathogen Pho. damsela. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility of P. monodon to Pho. damsela.     

Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon)

An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 x 10(5). Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the clotting system and the prophenoloxidase-activating system, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.     

High-density linkage maps and sex-linked markers for the black tiger shrimp (Penaeus monodon)

We report on the construction of sex-specific high-density linkage maps and identification of sex-linked markers for the black tiger shrimp (Penaeus monodon). Overall, we identified 44 male and 43 female linkage groups (2n = 88) from the analysis of 2,306 AFLP markers segregating in three full-sib families, covering 2,378 and 2,362 cM, respectively. Twenty-one putatively homologous linkage groups, including the sex-linkage groups, were identified between the female and male linkage maps. Six sex-linked AFLP marker alleles were inherited from female parents in the three families, suggesting that the P. monodon adopts a WZ-ZZ sex-determining system. Two sex-linked AFLP markers, one of which we converted into an allele-specific assay, confirmed their association with sex in a panel of 52 genetically unrelated animals.     

Tissue-specific expression and regulation of the haemolymph clottable protein of tiger shrimp (Penaeus monodon)

The clottable protein (CP) involved in Penaeus monodon haemolymph coagulation has previously been characterized and cloned. Polyclonal antibodies against purified CP were also prepared from rabbit serum. By Western blot analyses, we showed occurrence of CP in the shrimp central nervous system, gill, and lymphoid organ. Results of RT-PCR further indicated that the central nervous system, gill, and lymphoid organ transcribed more CP, heart and hepatopancreas transcribed less, while the haemocytes and the muscle did not. We further analyzed the CP distribution within shrimp lymphoid organ by immunohistochemical method, CP was found to localise in stromal cells of lymphoid organ rather than in the developing haemocytes. In addition, concentrations and regulation of the plasma CP under normal and artificially traumatic conditions were studied with rocket immunoelectrophoresis. The average plasma CP concentration in normal intermolt shrimps was elevated from 3 mg ml(-1) to above 12 mg ml(-1) after successive blood-withdrawing for a week. The production and secretion of CP apparently were increased more than 4 folds to compensate its loss. Our result also suggested that the shrimp sinus gland endocrine system is not directly required for the expression and up-regulation of CP.     

Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon)

We cloned the complementary deoxyribonucleic acid (cDNA) of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon). It was 2207 bp long and included a 1959-bp coding region, a 40-bp flanking region at the 5' end, and a 208-bp flanking region at the 3' end. The deduced, 652-amino acid sequence had a molecular mass of 71 481 Da and an estimated isoelectric point (pI) of 5.2. Based on phylogenetic analysis, the gene is clustered with the hsc70 proteins of invertebrates and vertebrates. In native gel electrophoresis, recombinant P. monodon hsc70 expressed in an Escherichia coli system is tightly associated with carboxymethylated alpha-lactalbumin (CMLA), which indicates that hsc70 probably functions as a chaperone. In an in vitro adenosine triphosphatase assay, recombinant hsc70 hydrolyzed adenosine triphosphate to adenosine-5'-diphosphate and increased hydrolysis activity by binding to unfolded peptide, CMLA. In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp. The expression of hsc70 messenger ribonucleic acid (mRNA) in hemocytes increased 2- to 3-fold at the first hour after shrimp experienced heat shock and 0.5-hour recovery. Hsc70 mRNA decreased gradually to the background level. Cloning and characterizing the P. monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins with the stress or immune responses of shrimp.     

Development of gene transfer technology for black tiger shrimp, Penaeus monodon

An effective foreign gene transfer method for shrimp would have several potential uses in the shrimp culture industry, such as in preventing infectious diseases. We evaluated two gene transfer methods and used black tiger shrimp, Penaeus monodon, as a model target species. For a promoter, we used the 1,592-bp promoter region of the EF-1alpha gene, a house-keeping gene, of kuruma shrimp Marsupenaeus japonicus. The promoter region was linked to either the gene for green fluorescence protein (GFP) or the gene for chloramphenicol acetyl transferase (CAT). The fusion genes were designated pJEF-GFP and pJEF-CAT, respectively. The pJEF-GFP gene was introduced into fertilized eggs of black tiger shrimp by microinjection and particle gun bombardment. The survival rate of the microinjected eggs was 17.6%, and 1.0% of the treated embryos were found to be GFP-positive. However, the GFP-positive embryos were damaged and embryogenesis did not progress. The survival rate of the particle-bombarded eggs was 60.6%, and 0.42% of the treated embryos were found to be GFP-positive. Ubiquitous GFP expression was observed from 8 hr post-fertilization and these embryos developed and hatched normally. The pJEF-CAT gene was introduced into fertilized eggs of black tiger shrimp using the optimized conditions of the particle gun bombardment. CAT activity was observed from 1 to 7 days post-fertilization, with the highest activities being observed at 5 and 7 days post-hatching.     

The carotenoids of wild and blue disease affected farmed tiger shrimp (Penaeus monodon, Fabricus)

1. The main carotenoids in wild Penaeus monodon exoskeleton were astaxanthin di- and mono-esters, astaxanthin, and beta-carotene. 2. Wild P. monodon exoskeleton contained on average 26.3 ppm total carotenoid; normally pigmented farmed shrimp had a similar concentration (25.3 ppm). 3. Exoskeletons of farmed "blue" P. monodon (i.e. blue-coloured, as opposed to the normally red-blue/black banded shrimp) contained significantly less total carotenoid (4.3-7 ppm). The only major carotenoid being astaxanthin. 4. Commercially available diets contained only trace quantities of canthaxanthin. 5. Nutritional deficiency with respect to carotenoids is suggested as the cause of blue disease in farmed P. monodon.     

Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.     

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.     

Alteration of egg activation by protease inhibitors in the black tiger shrimp,Penaeus monodon

In penaeoid shrimp, contact of spawned eggs with seawater induces egg activation. However, little is known about the factors that influence egg activation in Penaeus monodon. Therefore, the main objective of the present study was to determine whether shrimp-produced proteases that are released in seawater are essential for egg activation. Female shrimp were allowed to spawn in artificial seawater containing protease inhibitors. It was shown that 4-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF) and soybean trypsin inhibitor (SBTI) inhibited egg activation. High doses of APMSF and SBTI induced only 1–2% complete egg activation. Moreover, when the APMSF- and SBTI- treated eggs were subsequently washed, egg activation did not resume. In contrast, other protease inhibitors, pepstatin A, E-64, and ethylene glycol tetraacetic acid, did not inhibit egg activation, as evident by approximately 98% complete activation. Our results suggest that serine proteases, which are most likely trypsin-like proteases, released in seawater may be involved in egg activation of P. monodon.     

Induction of the acrosome reaction in black tiger shrimp (Penaeus monodon) requires sperm trypsin-like enzyme activity

Trypsin-like enzymes in egg water (EW), a natural acrosome reaction (AR) inducer, are known for their importance in shrimp AR. In this report, we describe a unique phenomenon of the AR of black tiger shrimp (Penaeus monodon) sperm. It was completed within 45-60 sec and comprised only the acrosomal exocytosis and depolymerization of the sperm head anterior spike. We used peptidyl fluorogenic substrates to show the presence of trypsin-like enzymes in P. monodon EW and sperm, but minimal activities of chymotrypsin-like enzymes. In sperm, these trypsin-like enzymes existed both on the sperm surface and in the acrosome. The acrosomal enzyme was revealed as a 45-kDa band by fluorogenic substrate in-gel zymography. Although EW possessed high trypsin-like enzyme activities, they were not essential for the AR induction; EW pretreated with an irreversible trypsin inhibitor, or heat-inactivated EW (HI-EW), to abolish the trypsin-like activities could still induce the AR. The HI-EW-induced AR was inhibited by the presence of a membrane impermeant soybean trypsin inhibitor (SBTI) in the sperm suspension, indicating the significance of sperm-borne trypsin-like enzymes (on the surface and/or in the acrosome) in this AR process. However, pretreatment of sperm with SBTI followed by its removal from the suspension still allowed the AR to occur within 5 min of sperm exposure to HI-EW. Since trypsin-like activity of the SBTI-pretreated sperm surface at 5 min after SBTI removal was at the minimal level, our results suggest the importance of the acrosomal trypsin-like enzyme in the AR process.     

Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand

In 2001-2002 throughout Thailand, black tiger shrimp Penaeus monodon farmers reported very unusual retarded growth. We have called this problem monodon slow growth syndrome (MSGS). Based on decreased national production, estimated losses due to this phenomenon were in the range of 13 000 million baht (approximately 300 million US dollars) in 2002. Since rearing practices had not changed, it was considered possible that the MSGS problem may have arisen from a new or existing pathogen. To examine this possibility, cultivated shrimp were sampled from 32 commercial rearing ponds that reported abnormally slow growth from eastern, central and southern regions of Thailand. Shrimp were randomly sampled from each pond and grouped into normal and small shrimp. Normal shrimp were defined as those with body weights (BW) of 24 g or more while small shrimp were defined as those that weighed 16.8 g or less. Pleopods were used for detection of monodon baculovirus (MBV), heptopancreatic parvovirus (HPV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) using specific polymerase chain reaction (PCR) assays. In addition, some shrimp were processed for normal histopathology and transmission electron microscopy (TEM). Most of the shrimp specimens were infected by at least 1 of these viruses but many had dual or multiple infections. Prevalence of HPV and combined HPV/MBV infections in the small shrimp was significantly higher than in the normal shrimp. In addition to the viruses, a new microsporidian species, gregarines and bacteria were also observed but were not significantly associated with the MSGS problem. Some of the small shrimp gave negative results for all these pathogens by PCR and histology and no new and unique histopathology was recognized in any of the samples. The findings suggested that HPV infection was a contributing factor but not the overriding factor responsible for MSGS. It is possible that MSGS is caused by an unknown pathogen or by some other presently unknown, non-pathogenic factor.     

Up-regulation of ribophorin I after yellow head virus (YHV) challenge in black tiger shrimp Penaeus monodon

This work constitutes the second report from a continuing investigation of shrimp genes that may be involved in apoptosis associated death resulting from yellow head virus (YHV) infection. Here, we describe from the black tiger shrimp Penaeus monodon, a ribophorin I-like gene that is probably a subunit of the oligosaccharyltransferase complex (OST), a key enzyme in N-linked glycosylation that occurs in the endoplasmic reticulum. The OST complex also contains DAD1 (defender against apoptotic death 1) that has been reported to control apoptosis and that we have previously reported from P. monodon. The full length ribophorin I of P. monodon comprised 2157 bp with the ORF of 1806 bp corresponding to 601 deduced amino acids and three putative N-linked glycosylation sites. Analysis revealed hydrophobic properties implying that it could be a membrane protein. Tissue distribution analysis using real-time RT-PCR with SYBR Green revealed that ribophorin I was endogenously expressed in all examined tissues of normal shrimp. However, unlike DAD1 that was down-regulated after YHV challenge, ribophorin I expression was up-regulated and remained high until the moribund stage.