[Show abstract][Hide abstract] ABSTRACT: Monodon baculovirus (MBV) causes slow growth of the shrimp being due to its infection in the hepatopancreas, the organ producing digestive enzymes and for nutrient storage. It has long been documented that the virus is transmitted from broodstock to offspring via contamination in rearing water by feces of the infected broodstock that contained sloughing-off damaged hepatopancreatic cells containing MBV. As a management practice, the washing of eggs and nauplii with disinfectant has been recommended to eliminate MBV that are attached to their surface and thus prevent the infection at the later development stage of the shrimp. However, we detected the MBV infection in postlarvae and juveniles of the black tiger shrimp Penaeus monodon, even after our repeated attempts to eliminate the virus by washing the eggs and nauplii with povidone iodine as disinfectant. Therefore we used MBV-infected broodstock to identify the cause of this problem by tracing the presence of MBV in hepatopancreas and ovary of the broodstock, eggs, nauplii and postlarvae, using histology with haematoxylin-eosin staining, polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) specific for polyhedrin, a protein produced by MBV. We found all the ovaries collected from the broodstock, which were detected MBV-positive in the hepatopancreas by histology, PCR and ISH, were also PCR-positive. By ISH, positive signals were detected in the cell membrane, cytoplasm and nuclear membrane of the oocytes. The eggs and nauplii from the MBV-positive broodstock were also positive by PCR, in both “wash” and “no-wash” specimens. By ISH, positive signals were detected in ooplasm and subcuticular region of nauplii, as well as inside its body. Using IHC, positive signals were detected inside the body and appendage of the nauplius. Taken all these together, it is most likely that MBV could be vertically transmitted through trans-ovarian route. Hence, simply washing the eggs and nauplii with disinfectant may not be an adequate procedure to eliminate the MBV infection in offspring from MBV-infected broodstock.
[Show abstract][Hide abstract] ABSTRACT: Vitellogenesis in crustaceans is a process by which a major yolk protein, vitellin (Vn) is proteolytically produced from vitellogenin (Vg) and deposited into developing oocytes. Vitellogenesis is regulated by a gonad-inhibiting hormone (GIH) produced in the eyestalk ganglia. In this study, Vg and GIH mRNA expression and their physiological concentrations at the protein level were examined during ovarian maturation in domesticated broodstock of Penaeus monodon. GIH mRNA was expressed at the highest level in the eyestalk ganglia of the shrimp with immature ovary while the GIH peptide was actively released into the hemolymph. The release of GIH dropped dramatically at stage I of the ovary onwards conforming to its negative regulatory function on Vg synthesis. Vg mRNA expression study confirmed that Vg was synthesized in both the ovary and the hepatopancreas of P. monodon. The expression of Vg increased as ovarian maturation progressed similarly to that demonstrated in the wild broodstock. Vg protein was found in the hemolymph since stage I of ovarian maturation suggesting a rapid release of Vg into the hemolymph before deposition into oocytes as shown by a significant increase of Vn in the ovary at the following stage. Unlike previous studies in wild P. monodon broodstock, Vn was localized to follicle cells of late perinucleolar oocytes and to both follicle cells and ooplasm of the vitellogenic oocytes of domesticated broodstock. We speculate that the incorporation of Vn from follicle cells to the oocytes occurred more slowly in domesticated shrimp; this may account for the retarded reproductive maturation of the domesticated broodstock comparing with the wild broodstock. Our study thus provides insights on vitellogenesis in domesticated P. monodon that will be useful for improvement of their reproductive maturation.
[Show abstract][Hide abstract] ABSTRACT: Sand polychaetes (Perinereis nuntia) have been used as a live feed to boost reproductive maturation before mating in hatchery farms in Thailand. However, no study has evaluated the effect of polychaetes on performance and physiology of the sperm of the domesticated black tiger shrimp (Penaues monodon). Therefore, this study aims to study the effects of sand polychaetes on growth, survival, reproductive performance and sperm physiology and morphology of domesticated male broodstock P. monodon. After feeding with either polychaetes or commercial pellets for four weeks, growth and survival rates of polychaete-fed group were significantly higher than those of pellet-fed group. Spermatophore weight and total sperm counts of the polychaete-fed group were significantly higher than those of the pellet-fed group at Weeks 3 and 4, whereas % abnormal sperm cells and % acrosome reaction of polychaete-fed group were significantly lower and higher than those of pellet-fed group only at Week 4, respectively. Physiology of spermatophores in the polychaete-fed group was white and opaque while that of the pellet-fed group was pigmented. Morphological changes of sperm revealed less abnormal sperms (misshaped half, misshaped head, and misshaped tails) in the polychaete-fed group. Nutritional analysis revealed that polychaetes have significantly higher total protein, fat contents and essential fatty acids (arachidonic and eicosapentaenoic acids) but significantly lower fiber content. Moreover, histological analysis of hepatopancreas revealed more vacuoles, which are storages for glycogen and lipids in the polychaete-fed group than in the pellet-fed group. Therefore, this study provides an evidence that polychaetes provide benefits to growth, survival and sperm performance for boosting reproductive maturation in male domesticated broodstock Penaeus monodon.
[Show abstract][Hide abstract] ABSTRACT: We exploited Artemia as a double-stranded (ds)RNA-delivery system to combat viral diseases in shrimp. First, the transformed Escherichia coli (E.coli) expressing red fluorescent protein (RFP) was tested in the Artemia enrichment process. RFP signals detectable in the gut of Artemia under confocal microscope were evident for the successful encapsulation. Second, the Artemia enrichment process was performed using E.coli producing Laem-Singh virus (LSNV)-specific dsRNA, which has been previously shown to inhibit the viral infection in the black tiger shrimp Penaeus monodon by intramuscular injection and oral administration. The enriched Artemia nauplii were confirmed to contain dsRNA-LSNV by RT-PCR, and were subjected to the feeding test with P.monodon postlarvae. Quantitative RT-PCR indicated that a number of LSNV copies in most of the treated shrimp were, at least, 1000-fold lower than the untreated controls. During 11-17 weeks after feeding, average body weight of the treated group was markedly increased relative to the control group. A smaller differential growth rate of the treated group as compared to the control was also noticed. These results suggested that feeding shrimp with the dsRNA-enriched Artemia can eliminate LSNV infection, which is the cause of retarded growth in P.monodon. The present study reveals for the first time the therapeutic effect of dsRNA-enriched Artemia for shrimp disease control.
Antiviral research 08/2013; 100(1). DOI:10.1016/j.antiviral.2013.08.005 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Marine invertebrates respond to fluctuations in their environment throughout development and growth. Homeostatic adaptation is mediated in part through the regulation of Na+‐K+ ATPase enzymes and members of this class of enzymes are localized to the antennal gland of many crustaceans. In this study, we investigated the morphological and biochemical changes of the antennal glands in eight families of cross‐breed post‐larval shrimp subjected to hypotonic (10 ppt) and isotonic (20 ppt) conditions. The greatest changes in the antennal glands were detected in the hypotonic condition with families two and five cross‐breed shrimp exhibiting the highest tolerance to this diluted salinity. The changes detected in hypotonic tolerant shrimp included enlargement of coelomosac and labyrinth spaces, the presence of many dense cytoplasmic globules in podocytes as well as highly wavy apical architecture of labyrinth cells. Several alterations were also observed at the subcellular level such as widening of the intercellular spaces between podocytes, organelle destruction, and decreased basal membrane in‐folding in labyrinth cells. In addition, shrimp exposed to hypotonic conditions displayed decreased expression of both α and β subunits of Na+‐K+ ATPase and decreased enzymatic activity. The changes observed in animals upon exposure to hypotonic stress are discussed.
Aquaculture Research 06/2013; 44(7). DOI:10.1111/are.12162 · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral inhibition by double-stranded (ds)RNA is a potential therapeutic approach for controlling shrimp viral diseases. Here, we describe the successful oral application of dsRNA targeting Laem-Singh Virus (LSNV) to diminish monodon slow growth syndrome (MSGS) in Thai Penaeus monodon. Shrimp feed formulated with bacterially expressed LSNV-dsRNA was given to shrimp for 9 weeks. RT-PCR results revealed that all control shrimp were LSNV-positive at the end of experiment, while the shrimp that received dsRNA-feed exhibited 20-60% LSNV reduction. The average body weight of treated shrimp (number of shrimp=100) was significantly higher than that of the control group. Such increase is likely due to the elimination of MSGS caused by LSNV, as size variation of the treated group is much lower than that in the control group. This study demonstrates for the first time that feed with LSNV-specific dsRNA promotes the overall growth of P. monodon and relieves MSGS condition in LSNV-infected shrimp. The work reaffirms the potential of dsRNA application for controlling viral disease in shrimp farming.
Journal of Biotechnology 02/2013; 164(4). DOI:10.1016/j.jbiotec.2013.01.028 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7x1.1microm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10nm) and an electron-dense exospore (2nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.