Photobacterium damselae ssp. damselae associated with diseased black tiger shrimp Penaeus monodon Fabricius in India

AIM: To characterize and identify Photobacterium damselae ssp. damselae present in black gill diseased Penaeus monodon collected from east coast of India. METHODS AND RESULTS: Photobacterium damselae ssp. damselae was isolated from hepatopancreas, muscles and gills by using the thiosulfate citrate bile salts sucrose agar supplemented with 1.5% NaCl (TCBS-1) medium. A total of 32 Ph. damselae ssp. damselae isolates were studied together with two reference strains. The biochemical tests and analysis of ureC and 16S rRNA genes confirmed the phenotypic characterization of the isolates as Ph damselae ssp. damselae. Experimental infection studies revealed that the LD50 values of P. monodon and P. indicus ranged from 2x10(3) to 5x10(5) CFU per shrimp and from 4x10(2) to 2x10(4) CFU per shrimp, respectively. CONCLUSIONS: Photobacterium damselae ssp. damselae was found in the internal organs of P. monodon and it showed pathogenic to shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the Ph. damselae ssp. damselae present in the black gill diseased P. monodon in India and therefore might serve as a basis for future studies and diagnosis purpose to shrimp culturists.     

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.     

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.     

Identification and characterization of syntenin binding protein in the black tiger shrimp Penaeus monodon

Shrimp exhibit a diverse response to viral infection that is manifested in drastic up- and down-regulations of a variety of genes. In our previous work, we identified syntenin of the shrimp Penaeus monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection, its message being greatly upregulated in the acute phase of the infection. In order to further explore the link between Pm-syntenin and viral infection, we performed a yeast two-hybrid screening of a P. monodon cDNA library, using Pm-syntenin as bait. One of the molecules that specifically interacted with Pm-syntenin was the receptor-binding domain of alpha-2-macroglobulin (alpha2M). A GST pull-down assay showed that GST-alpha2M, but not GST alone, was capable of co-precipitating syntenin. Another GST pull-down assay showed that GST-syntenin, but not GST alone, was capable of co-precipitating alpha2M. In addition, mutant analyses showed that the N-terminal 131 amino acids of syntenin were both necessary and sufficient to bind the C-terminus receptor-binding domain of alpha2M. Furthermore, WSSV-infected Pm showed a significant upregulation of the alpha2M message, suggesting that both syntenin and its protein partner alpha2M are upregulated in the acute phase of a WSSV infection. Taken together with a previous report showing the co-localization of alpha2M and syntenin in the exosome of a dendritic cell line, it is likely that syntenin, through its interaction with alpha2M, plays an important role in the immune defense mechanisms of viral infections of shrimps.     

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.     

Clearing mechanisms of Vibrio vulnificus biotype I in the black tiger shrimp Penaeus monodon

Vibrio species' infections are a common sequelae to environmental stress or other disease processes in shrimp, but the mechanism by which the shrimp eliminate the bacteria is poorly understood. In this study, the penetration, fate and the clearing of V. vulnificus were investigated in Penaeus monodon. A bacterial disease isolate from a shrimp farm was identified as V. vulnificus biotype I. Polyclonal antiserum was raised in rabbits against the bacterium and the specificity was verified by ELISA and immunoblot against a range of Vibrio spp. and other gram-negative bacteria. The bacteria were then administered to P. monodon juveniles by injection, immersion and oral intubation. An indirect immunoperoxidase technique was employed in a time course study to follow the bacteria and bacterial antigens in the tissue of the shrimp. Bacteria were cleared by a common route, regardless of the method of administration. Observations in immersion challenge were similar to a combination of those for oral and injection challenges. With immersion, bacteria entered the shrimp through damaged cuticle or via insertion points of cuticular setae. Shortly after entry, whole bacterial cells were observed in the haemolymph and connective tissue. They were either phagocytosed by haemocytes, or broken down outside host cells. Haemocytes containing bacterial cells or antigens (HCB) were observed in the connective tissue and haemolymph. HCB accumulated around the hepatopancreas, midgut, midgut-caecum, gills, heart and lymphoid organ. Free bacterial antigens also accumulated in the heart and lymphoid organ. Bacteria entering through the mouth by oral intubation or immersion were broken down so that only soluble or very fine particles entered the hepatopancreas. Bacterial antigens passed through the hepatopancreas into the haemolymph. Antigens were initially observed in the haemolymph sinuses and subsequently accumulated in the heart and lymphoid organ. Bacterial antigens were released from the shrimp, initially through the gills and subsequently through hepatopancreatic B-cells, branchial podocytes and sub-cuticular podocytes.     

Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon.

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study.     

Bacteriological study of shrimp, Penaeus monodon Fabricius, hatcheries in India

Shrimp hatcheries often face problems of mortality caused by diseases. To understand the bacteriological status of shrimp, Penaeus monodon Fabricius, hatcheries in India, a study of hatchery water at different points was conducted in several hatcheries located along the east and west coast of India. The species composition of the bacterial flora was also determined. The total plate counts of raw sea water on tryptic soya agar ranged from 10² to 10⁴ ml^–1, whereas it ranged from 10⁴ to 10⁶ ml^–1 in larval tanks. In the larval tanks, the proportion of Vibrio species ranged from 50% to 73%, as compared to 31% in raw sea water. A mixed bacterial flora was observed in hatchery water; however, in the larval tanks, the flora in the larvae was predominantly made up of Vibrio species. A few of the tested Vibrio isolates were non-virulent to shrimp larvae under experimental conditions. Over 90% of the strains were resistant to amoxycillin, ampicillin, cephalexin, cephazolin, cloxacillin and sulphafurazole. Most strains showed sensitivity to tetracycline, chloramphenicol, and quinolones such as norfloxacin and ciprofloxacin.     

Molecular cloning and tissue distribution of the Toll receptor in the black tiger shrimp, Penaeus monodon

The black tiger shrimp (Penaeus monodon) is economically important in many parts of the world, including Thailand. Shrimp immunity is similar to that of other invertebrate organisms; it consists of an innate immunity system. Toll or Toll-like receptors (TLRs) play an essential role in recognizing the cleaved form of the cytokine Sp?tzle, which is processed by a series of proteolytic cascades activated by secreted recognition molecules. We isolated a full-length Toll receptor from P. monodon. The cloned full-length sequence of the PmToll cDNA consists of 4144 nucleotides, containing a 5'-UTR with 366 nucleotides, a 3'-terminal UTR with 985 nucleotides, with a classical polyadenylation signal sequence AATAAA, a poly A-tail with 27 nucleotides, and an open reading frame coding for 931 amino acids. The deduced amino acid sequence of PmToll is a typical type I membrane domain protein, characteristic of TLR functional domains. It includes a putative signal peptide, an extracellular domain consisting of leucine-rich repeats, flanked by cysteine-rich motifs, a single-pass transmembrane portion, and a cytoplasmic TLR domain. PmToll was expressed in all tissues tested, including gill, hemocytes, heart, hepatopancreas, lymphoid organs, muscle, nerve, pleopod, stomach, testis, and ovary. The deduced amino acid of PmToll is closely related to that of other shrimp Tolls, especially FcToll. Further studies elucidating the mechanism of action of Tolls will be of benefit for understanding the defense mechanisms of this economically important aquatic species.     

Hepatopancreatic nuclease of black tiger shrimp Penaeus monodon unlikely to be involved in viral triggered apoptosis

Nucleases are phosphodiesterases that hydrolyze DNA and/or RNA. In a search for shrimp nucleases involved in apoptosis, we discovered a nuclease from hepatopancreatic cDNA of the black tiger shrimp Penaeus monodon. The full-length nuclease gene was amplified and revealed to contain 1668bp corresponding to 381 deduced amino acid residues in the mature enzyme. Sequence analysis indicated 83% nucleic acid identity and 89% amino acid identity to a nuclease from the Kuruma shrimp Penaeus japonicus (also called Marsupenaeus japonicus). Comparative analysis of sequences, conserved motifs and phylogenetic trees indicated that P. monodon nuclease (PMN) belonged to the family of DNA/RNA non-specific endonucleases (DRNSN). RT-PCR analysis using primers specific for PMN mRNA with seven different shrimp tissues revealed that expression in normal shrimp was restricted to the hepatopancreas. Semiquantitative RT-PCR analysis of PMN using hepatopancreatic mRNA from normal shrimp and from shrimp challenged with white spot syndrome virus (WSSV) indicated significant up-regulation of PMN in the hepatopancreas (P<0.05) at the early stage of viral infection but a return to baseline levels as gross signs of disease developed. At the same time, expression was always confined to the hepatopancreas and never seen in other tissues, including those reported to be prime targets for WSSV and subject to increased levels of apoptosis after infection. The results suggested that PMN is probably a digestive enzyme that is unlikely to be involved in hallmark DNA digestion associated with apoptosis.     

Immunomodulation by dietary beta-1, 3-glucan in the brooders of the black tiger shrimp Penaeus monodon

The present study evaluated the effectiveness of beta-1,3-glucan derived from Schizophyllum commune in enhancing shrimp survival as well as haemocyte phagocytosis and superoxide anion production in brooder Penaeus monodon. Pond-reared P. monodon adults (135 +/- 25 g) stocked in outdoor or indoor tanks were fed either a test diet containing beta-1,3-glucan (2.0 g kg(-1) or a glucan-free control diet for 40 days. Their survival was compared. The brooders reared in indoor tanks were analysed at days 0, 1, 3, 6, 12, 24, 30 and 40 for their haemocyte phagocytic activity and superoxide anion production. The results showed that regardless of indoor or outdoor rearing the survival rate of shrimp fed the glucan diet was significantly higher (P<0.001) than that of the control group. The brooders showed enhanced haemocyte phagocytic activity, cell adhesion and superoxide anion production when glucan was administered in their diets. The immunostimulatory enhancement peaked at day 24 after starting the dietary exposure and subsequently decreased to the pre-feeding level at the end of the 40 days feeding trial.     

Changes in integument histology and protein expression related to the molting cycle of the black tiger shrimp, Penaeus monodon

We investigated changes in the histology and protein expression in the epidermis and sub-epidermis of the black tiger shrimp (Penaeus monondon) during the molting cycle. The epidermis consists of a cell layer located beneath the cuticle, while the sub-epidermis is mainly composed of sub-epidermal cells and tegumental glands. During the molting cycle, the epidermal cells increase in cell height and number, and the sub-epidermis increases in its storage of carbohydrate, protein, mucus, and other unidentified substances at the time of the active period of cuticular regeneration. At the early premolt (stage D0), the epidermal cells are tidily organized, but short. Storage of carbohydrate and protein in the sub-epidermis is not observed. During the rest of the premolt (D1-4 stages) and the early postmolt A stage, epidermal cell height and sub-epidermal deposition are increased, and reached a maximum during the D4 to A stages. The period of late postmolt stages B-C3 is the time for a decrease in epidermal cell height and sub-epidermal depositions. Lastly at intermolt stage C4, the epidermal cells become short, and untidily organized. Sub-epidermal deposition is not observed. Protein expression in the epidermis and sub-epidermis was observed by SDS-PAGE. This revealed that the profile of a protein band with a molecular mass of 57 kDa corresponded with the profile observed by histochemistry. All results point to the conclusion that both the epidermis and sub-epidermis play major roles in cuticular regeneration. It may also reflect the level of metabolic activity of the integument during the molting cycle. In addition, for the first time, this work provides direct evidence of the epidermal and sub-epidermal changes that occur during the molting cycle of the black tiger shrimp.     

Enhanced growth and resistance to Vibrio challenge in pond-reared black tiger shrimp Penaeus monodon fed a Bacillus probiotic

The bacterial probiont Bacillus S11 (BS11) was used as a supplement in feed (PF) for black tiger shrimp Penaeus monodon in 2 earthen pond field-trials carried out for 100 d during 2 different seasons in Thailand. Growth and survival were compared with those of shrimp receiving an unsupplemented feed (UF). In the hot and cool seasons, respectively, shrimp fed PF grew significantly larger and had significantly higher survival than shrimp fed UF (p < 0.05). Projected yields on an annual basis (two 100 d crops) were 49% greater with PF-fed shrimp. In 8 d challenge tests using the luminescent bacterium Vibrio harveyi 1526, shrimp fed UF all died within 6 d while survival for shrimp fed PF was 5 and 9%.     

Host associated mixed probiotic bacteria induced digestive enzymes in the gut of tiger shrimp Penaeus monodon

The shrimp Penaeus monodon was used for the isolation of digestive enzyme producing host-associated probiotic bacteria. Gut was isolated from a healthy animal completely and morphologically different bacterial isolates were screened for the production of hydrolytic enzymes, such as, protease, amylase, lipase and cellulases. Based on their ability to produce enzymes, the potent probiotic bacteria were identified as Bacillus subtilis and B. licheniformis and these two were used for the preparation of probiotic diet for experimental trials. Probiotic diet was prepared by mixing the shrimp feed with 2 g probiotic/100 g artificial diet (F1), 4 g/100 g (F2), 6 g/100 g (F3), 8 g/100 g (F4) and 10 g/100 g (F5). Juvenile shrimp was fed with probiotic and control diet for a period of 7 weeks at 5 and 8% body weight for the first 3 and 7 weeks, respectively. After seven weeks, whole gut was dissected out and protease activity was estimated as 145 ± 12.3 U/g in control animal and increased as 710 ± 15.2 U/ g in F5 feed groups. Amylase activity was 139 ± 10.4 U/g in control and increased as 209 ± 13. 3 U/g in F5 group. Cellulase activities were 171 ± 9.3 in F5 groups and the control group showed only 102 ± 12.4 U/g. Lipase activity was 78 ± 3 U/g in F1 groups and it increased as 85 ± 5 U/g in F3 groups. These findings indicate the potential of host-associated bacteria to enhance the production of enzymes in the gut of juvenile 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.     

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.