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The hepatopancreas of the shrimp is the target organ of several pathogens. Diagnosis of diseases of the hepatopancreas usually required an isolation of the organ and the process cannot avoid sacrificing the shrimp. To save life of the animal, this study was aimed at taking hepatopancreatic tissue from the shrimp by biopsy process. Thirty-two sexually mature adults of the black tiger shrimp Penaeus monodon were used as the shrimp model and small pieces of their hepatopancreatic tissue were taken by needle biopsy. Specific position of the needle penetration was suggested to avoid damage to the heart and the gills. From the lateral view of the carapace of the shrimp, this position was at one-third from the rostral end of a line drawn from the last dorsal spine of the rostrum to the posterior margin of the carapace. From the top view, this point was located at a medial quarter of a line drawn from the midline of the carapace toward the lateral extremity. The needle was inserted vertically at a depth of 1 cm into the hepatopancreas, and small pieces of the tissue were quickly withdrawn into the syringe. After the operation, the shrimp were released into rearing tanks and observed for any mortality or morbidity for two months, during which all the shrimp survived and remained apparently healthy. The hepatopancreatic tissues taken from the biopsy were processed for polymerase chain (PCR) reaction method, fresh smear and histology. The PCR revealed the presence of Penaeus monodon densovirus (PmoDNV) in 40% of the samples. The fresh smear and histology revealed good quality of cell morphology and histological features, as well as PmoDNV inclusions in the PCR-positive samples. This study suggests that needle biopsy of hepatopancreas to detect pathogens, or to evaluate health status, of this organ in shrimp is possible, at least in P. monodon.

World production of farmed shrimp is focused on a few species. At present, the Pacific whiteleg shrimp Litopenaeus (Penaeus) vannamei tops the list, followed by the black tiger shrimp Penaeus monodon and a few others. The former attains the market size between 15 and 25 g within three months in culture, while P. monodon requires at least four months to reach the marketable size of ~25 g and larger. It is therefore desirable to produce stocks of P. monodon with a fast-growth-rate trait, either through selective breeding or via other modes of scientific invention. Among others, chromosome manipulation of P. monodon that confers three sets of chromosomes (3n) to the shrimp, a condition called triploidy and a feat that was achieved through a non-GMO technique, could be the answer. Thermal chromosome set manipulation for triploid induction is considered as a safe and environmentally friendly technique to produce sterile offspring for genetic protection, and prevent genetic pollution from aquaculture stocks in the wild. The black tiger shrimp can be induced to yield a high percentage of triploid offspring with abnormal reproductive histology. Two studies applying different induction methods briefly at the very early stage of embryo formation—one from Australia (chemical shock) and another from Thailand (cold shock) have reported successful results. The triploid shrimps produced from chemical shock method had reduced growth rate, while that from cold shock displayed higher growth rate compared to shrimp with the natural double set of chromosomes. Gender distribution of the triploid P. monodon induced by cold shock was skewed towards females, the larger size of the two sexes and hence is favored more in aquaculture. Because of the favorable results of the cold shock method, it is currently pursued to produce triploid P. monodon for commercial purposes. The production is accomplished through an automatic cold-shock induction system, consisting of spawning detection and cold shock treatment sections. This triploid induction project is an extension of the ongoing selective breeding program of a specific pathogen -free P. monodon that has already been commercially launched, and is part of an initiative to promote sustainable genetic stock improvement protocols for this shrimp species.

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 hematoxylin–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.

Molt-inhibiting hormone (MIH) is commonly localized in the X-organ sinus gland complex (XOSG) of the crustacean. The present study aimed to elucidate the expression of MIH at the subcellular and cellular levels in the eyestalks and integument of juvenile Penaeus monodon during the molting cycle. Gene expression of Pem-MIH1 in the optic lobes showed a single PCR product at 172 bp, and was restricted only to the eyestalk XOSG but not detected in pleopods, cephalothorax integument, muscle, hepatopancreas or retina. Quantitative analysis of gene expression in the eyestalks demonstrated no significant alteration of Pem-MIH1 mRNA in XOSG during the molt cycle. Immunohistochemistry using antibody against recombinant molt-inhibiting hormone-like peptide (anti-MIH-like) revealed variable staining intensities of individual MTXOs but were most persistently intense in SGs. During the molt cycle, the mean numbers of XO immunoreactive cells were slightly oscillated but not statistically different. The quantitative immunohistochemistry measured from XOSG illustrated minimal fluctuations of the values obtained suggesting the periodical synthesis and release of MIH from XOSG system during the molt cycle. The positive reaction was also detected in the tegumental glands located at the retina and the integument of walking leg, base of eyestalk and cephalothorax of the shrimp. The large variations of immunostaining and amount of reactive tegumental glands in the eyestalk were noted throughout the molt cycle. The numbers of the MIH-like immunoreactive glands obtained from the retina as well as the integument base did not significantly change during the molt cycle but tend to increase during postmolt to intermolt and decrease during premolt. These results suggest that the MIH-like peptides in the tegumental glands are probably released mostly during the late premolt. Our findings thus propose the novel storage site of MIH besides SG of the eyestalk including a new target tissue, an epidermal cell, the role of which may link to the complicated molting regulation and/or the dark-adaptation of shrimp.

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.

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 11-17weeks 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.

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.

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.

This study seeks to determine the best method for preventing the second body (PBII) extrusion as a means to induce triploidy in the black tiger shrimp Penaeus monodon . Chemical (cytochalasine-B and 6-dimethylaminopurine) and temperature (heat and cold) shocks were applied to newly fertilized eggs. Cold shock that was administered at 8 °C, for 10 min at 8 min post-spawning, was the best method for PBII triploidy induction, as evidenced by the highest percentage of forming three pronuclei in syngamy. Therefore, cold shock induction was employed to further explore the advantage of triploid over diploid shrimp. After hatching of the cold-shocked eggs, the larvae were allowed to reach juvenile and adult levels of development. The number and amount of chromosomes were determined in juveniles and adult stages, using Fluorescence Activating Cell Sorting methods, by which the shrimp were divided into diploid and triploid groups. At day 150 in culture, the average body weight of the triploid females (35.2 ± 3.2 g) and triploid males (31.5 ± 3.5 g) was significantly higher ( P P. monodon , and its feasibility for commercial production.

Monodon Slow-Growth Syndrome (MSGS), a pathological condition in the black tiger shrimp Penaeus monodon, is associated with Laem–Singh virus (LSNV) infection. Infected shrimp grow slowly when the virus invades the part of the retina called the zona fasciculata. Since molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH) are synthesized in the optic lobe and are related to molting activity and growth of the shrimp, the purpose of this study is therefore to determine the levels of these hormones and related parameters in MSGS shrimp. P. monodon juveniles were sampled from normal and MSGS ponds and were divided into small-negative, large-negative, small-positive and large-positive groups, depending on the size of the shrimp and whether they were LSNV-negative or LSNV-positive. Individual shrimp were measured for duration of each molt stage and molt interval. Levels of MIH1 and CHH1 transcripts were determined from the optic lobe, using real-time reverse transcriptase–polymerase chain reaction. The results revealed that the small-positive and the small-negative shrimp did not differ in durations of molt stages or molt intervals, as well as on the levels of MIH1 transcript in the optic lobe, MIH1 peptide in the optic lobe and hemolymph, or ecdysteroids in the hemolymph. Differences in molting activities and related transcript and hormones were observed between the small- and large-sized shrimp, but not in the status of LSNV infection. While levels of CHH1 transcript in the optic lobe of both small-negative and small-positive shrimp did not differ, levels of CHH1 peptide, as well glucose, in the hemolymph of the small-positive shrimp were significantly lower than those of the small-negative group. Levels of glycogen in the hepatopancreas of the small-positive shrimp were also significantly higher than that of the small-negative ones. The results suggest that growth retardation in MSGS shrimp is related to the suppression of the release of CHH1 peptide by LSNV invasion in the zona fasciculata, consequently causing decreased hepatopancreatic glycogenolysis and persistent hypoglycemia, resulting in growth stunting.

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)-labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti-FITC-conjugated gold-lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar-associated components of the HE. This revealed that both Con A and WGA-binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold-labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N-acetyl-β-d-glucosamine (sugar affinity to WGA).

Vibriosis is a common bacterial disease that can cause high mortality and morbidity in farmed shrimp. Since compounds from seaweed have been reported to have anti-bacterial and immunostimulant activity, this study was conducted to determine whether solvent extracts from the red seaweed Gracilaria fisheri might be a possible alternative for prevention and treatment of shrimp vibriosis caused by Vibrio harveyi. Seaweed extracts prepared using ethanol, methanol, chloroform and hexane were evaluated for anti-V. harveyi activity by the disc-diffusion method. The ethanol, methanol and chloroform extracts showed activity against a virulent strain of V. harveyi with potency (minimal inhibitory concentrations in the range of 90-190 μg ml(-1)) equivalent to the antibiotic norfloxacin. The ethanol extract was not toxic to the brine shrimp Artemia salina when it was fed to them for enrichment prior to their use, in turn, as feed for postlarvae of Penaeus monodon. Postlarvae fed with these enriched Artemia gave significantly lower mortality than control postlarvae after challenge with V. harveyi. In addition, P. monodon juveniles injected with the ethanol extract showed a significant increase in the total number of haemocytes and an increased proportion of semi-granulocytes and granulocytes when compared to control shrimp. The activities of phenoloxidase and superoxide dismutase were also increased, with an accompanying increase in superoxide anion production. When these juvenile shrimp were challenged with V. harveyi, mortality was markedly reduced compared to that of control shrimp. The results indicated that ethanol extracts of G. fisheri had immunostimulant and antimicrobial activity that could protect P. monodon against V. harveyi.

A recent study showed Thalassiosira weissflogii to be a diatom containing suitable nutrition for larviculture of the black tiger shrimp, Penaeus monodon. Accurate and practical identification of this diatom species is therefore important for commercial hatcheries. The purpose of this study was to establish a DNA-based method of identification to supplement morphological examination, avoiding confusion with other Thalassiosira sp. Primers, 18SF/28SR1, specific for ribosomal DNA genes (3′-end of 18S rDNA through 5′-end of 28S rDNA, covering two internal transcribed spacers), were employed as a first-step polymerase chain reaction, followed by a second nested amplification using specifically designed primers, ITS1-F-D/ITS1-R-D. The nested-PCR result revealed specificity in the detection, distinguishing T. weissflogii from T. pseudonana, Cyclotella meneghiniana, and Chaetoceros sp., and the PCR fragment of the amplified region had a sequence that was 99% identical to the T. weissflogii sequence held by GenBank.

The purpose of this study was to compare the performance of two species of diatoms, Thalassiosira weissflogii and Chaetoceros gracilis, in the larviculture of the black tiger shrimp Penaeus monodon. Shrimp larvae were fed with either C. gracilis, T. weissflogii, or a combination of the two species of diatoms. The larvae fed solely with T. weissflogii or a combination of the two types of diatom had significantly higher survival rates and faster metamorphosis than those fed solely with C. gracilis. The numbers of diatom cells consumed by larvae during 3-h periods were determined, revealing that larvae consumed significantly higher numbers of C. gracilis than T. weissflogii. However, when the protein, total fatty acid, and polyunsaturated fatty acid content of the two species of diatom are compared, significantly higher amounts of each are found in T. weissflogii. Converting the number of diatom cells consumed into equivalent protein, total fatty acid, eicosopentaenoic acid, and decosahexaenoic acid reveals that larvae fed with T. weissflogii received significantly higher amounts of all the nutrients, compared to those consuming C. gracilis. The results showed an advantage of feeding T. weissflogii to C. gracilis in enhancing survival and metamorphosis in P. monodon larvae.

The optic lobe of the female-shrimp eyestalk was selected as the target tissue for suppressive subtractive hybridization (SSH) because it is a place for synthesis of many hormones and peptides/enzymes involved in molting and growth. SSH was performed to screen genes differentially expressed in the optic lobe between large female (LF; body weight > 90 percentile of weight distribution curve) and small female (SF; body weight E- value − 4 ) indicating little available molecular information on the optic lobe. Among the EST with relatively high homology to GenBank records, 5 candidate ESTs had homology to records for genes involved in cell differentiation/proliferation, cell cycle and hormone processing. These included genes resembling cyclophilin, cyclophilin A, fibrillarin, SPARC and PC2. These candidate ESTs were selected to confirm the reproducibility of the SSH data using semi-quantitative RT-PCR normalized with EF-1α. Pearson's correlation analysis confirmed that the index of relative cyclophilin, SPARC and fibrillarin-like expression was negatively correlated with body weight ( p 2 ) based on the index of relative cyclophilin-like expression in the optic lobe of female P. monodon .

Black tiger shrimps (Penaeus monodon) are able to survive and can be reared under various salinities, possibly by the cellular adaptation of their excretory system, particularly the antennal gland, which is known to regulate body fluid in crustaceans. We have investigated the morphological and biochemical alterations of the antennal glands in shrimp reared in 7, 15, or 30 ppt seawater. Drastic changes occur in animals reared under 7 ppt conditions. Ultrastructural studies of the antennal gland in shrimps reared in 7 ppt seawater have revealed that podocytic cells in the coelomosacs ramify with more cytoplasmic processes forming the filtration slits, and that the tubular labyrinth cells possess more mitochondria in their basal striation and a wider tubular lumen than those found in the other groups. Many apical cytoplasmic blebs from labyrinth cells have also been seen in the lumen of the labyrinths under 7 ppt conditions, a feature that is not as prominent under the other conditions. The expression and activity of the Na(+)/K(+)-ATPase in the antennal gland are also correlated with the surrounding environment: the lower the salinity, the higher the expression and activity of the enzyme. Immunohistochemistry results have demonstrated the highest staining intensity in the labyrinth cells of shrimps reared under 7 ppt conditions. Our findings thus suggest that one of the adaptation mechanisms of this shrimp to the surrounding salinity is the regulation of Na(+)/K(+)-ATPase expression in the antennal gland, in conjunction with subcellular changes in its excretory cells.

This study aimed to investigate morphological changes of developing oocytes during oogenesis in the black tiger shrimp Penaeus monodon as well as elucidate the cellular pathway for the formation of cortical rods, the egg jelly precursor. The differentiating female germ cells in the mature ovary were divided into four stages (Oc 1–4 ) based on the changes in ultrastructural characteristics. The prominent features in the developing oocytes (Oc 1 Oc 2 Oc 3 ) were the abundance of ribosomes and dilated rough endoplasmic reticulum (RER) in Oc 1 and Oc 2 and the presence of yolk granules and lipid droplets in Oc 3 all indicating active synthesis of protein and lipid components. The main characteristic of the mature oocyte (Oc 4 ) was the presence of cortical rods (CRs) which were composed of the tightly packed structural units each resembling a bottle-brush. Immunostaining of cortical rod proteins which were part of the structural units indicated that they were first synthesized in the RER–Golgi complex of Oc 2 and transported into the extracellular crypts of the mature oocyte (Oc 4 ) where they were assembled into CRs.

The aim of this study was to determine whether magnesium (Mg2+) in seawater is required for egg activation of the black tiger shrimp Penaeus monodon and whether manipulation of Mg2+ levels can be used to delay the process and thereby synchronize egg activation. Female P. monodon broodstock were allowed to spawn in artificial seawater containing Mg2+ at varying levels with respect to the normal (100%) level: 100%, 50%, 20% and 0%. Egg activation occurred normally at 100% Mg2+, incompletely at 50% and 20% Mg2+ levels and did not occur at all with 0% Mg2+. The fertilization rate with 100% Mg2+ was observed to be 83%, but fertilization failed to take place in all the other groups. The fertilization rate was restored from 0% to 76% following the 20% Mg2+ level treatment when Mg2+ levels returned to normal (100%) as soon as spawning was completed. This study suggests that the level of Mg2+ in seawater plays a vital role in P. monodon egg activation, and that commencement of this process could be delayed by manipulation of the Mg2+ level during and immediately after spawning.

Gregarines are parasitic protozoa that occasionally parasitize the gut lumen of penaeid shrimp and other crustaceans. Here we describe the morphology of gregarine trophozoites found in the black tiger shrimp Penaeus monodon using light microscopy (LM) and scanning (SEM) and transmission (TEM) electron microscopy. Using LM with fresh preparations and with paraffin sectioning followed by hematoxylin-eosin staining, several trophozoites were discovered in the foregut and midgut, and gametocysts were found in the hindgut. Trophozoites existed both as solitary individuals and in association and exhibited either caudofrontal or lateral patterns. They were cylindrical in shape, 60 to 300 microm long by 10 to 60 microm wide, and consisted of 2 parts: a small anterior portion known as a protomerite, which was separated by a septum from a larger posterior portion known as a deutomerite. Under SEM, both the protomerite and deutomerite were found to be entirely covered with longitudinal, parallel pellicular folds 0.1 microm thick. Under TEM, the deutomerite was seen to contain a nucleus with a single eccentric nucleolus and several areas of peripherally located chromatin. The cytoplasm of both the protomerite and deutomerite contained abundant vesicles and granules of different sizes. The pellicle consisted of a double layer of electron-dense membranes separated by an electron-lucent area 30 nm wide and containing a microfilament. A few microfilaments were also observed in the cytoplasm underneath the pellicle, possibly serving as locomotive apparatus for the parasite. Based on its morphology, this gregarine appears similar to those of the genus Nematopsis.

Monodon slow-growth syndrome (MSGS) is a condition of Penaeus monodon cultivation ponds characterized by abnormally slow growth and coefficients of size variation greater than 35%. Shrimp from these ponds often show the presence Laem-Singh virus (LSNV) in the lymphoid organ, heart and other tissues. We compared various tissues of abnormally small and normal size (large) P. monodon from 3 MSGS ponds and from 2 reference ponds with normal growth (control ponds) both histologically and for the presence of LSNV. Small and large shrimp from the MSGS ponds were positive for LSNV by reverse transcriptase polymerase chain reaction (RT-PCR) tests while shrimp from one normal-growth pond were positive and from another negative. The LSNV infections were confirmed by transmission electron microscopy (TEM) and in situ hybridization (ISH). The only striking difference between the small and large LSNV-positive shrimp was retinopathy exclusively in the small shrimp from the MSGS ponds. Retinopathy comprised abnormally enlarged haemolymphatic vessels, haemocytic infiltration and rupture of the membrane that separated the fasciculated zone from the overlying row of retinular cells. By TEM and ISH, LSNV was detected in the fasciculated zone and in onion bodies of the organ of Bellonci in the small shrimp from the MSGS ponds, but not in those tissues and cells of the large shrimp from the MSGS pond or from the normal-growth ponds, whether LSNV-positive or not. The results suggested that retinopathy associated with LSNV may be linked causally to stunting of P. monodon in MSGS ponds.

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 )s perm. 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 45kDa 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.

One of the important peptide hormones that control reproduction in crustaceans is gonad-inhibiting hormone (GIH). GIH is known to modulate gonad maturation by inhibiting synthesis of vitellogenin (Vg), the precursor of yolk proteins. In this study, a cDNA encoding a GIH (Pem-GIH) from the eyestalk of Penaeus monodon was cloned using RT-PCR and RACE techniques. Pem-GIH cDNA is 861 bp in size with a single ORF of 288 bp. The deduced Pem-GIH consists of a 17-residue signal peptide and a mature peptide region of 79 amino acids with features typical of type II peptide hormones from the CHH family. Pem-GIH transcript was detected in eyestalk, brain, thoracic and abdominal nerve cords of adult P. monodon. The gonad-inhibiting activity of Pem-GIH was investigated using the RNA interference technique. Double-stranded RNA, corresponding to the mature Pem-GIH sequence, can trigger a decrease in Pem-GIH transcript levels both in eyestalk ganglia and abdominal nerve cord explant culture and in female P. monodon broodstock. The conspicuous increase in Vg transcript level in the ovary of GIH-knockdown shrimp suggests a negative influence for Pem-GIH on Vg gene expression, and thus implies its role as a gonad-inhibiting hormone. This is the first report to demonstrate the use of double-stranded RNA to elucidate the function of GIH in P. monodon.

Owing to the problem of male infertility in the domesticated shrimp Penaeus monodon, this study was conducted to reveal the morphological events of an acrosome reaction (AR) of sperm of this highly valuable species. The AR observed in an in vitro incubation of sperm with egg water (EW) and that during actual spawning was compared. Under transmission and scanning electron microscopy, sperm taken from the female thelycum was composed of a posterior main body, a central cap and an anterior single spike. Upon contact with EW, the sperm underwent two phases of AR: acrosomal exocytosis and spherical mass formation. The former was composed of a degeneration of the spike, swelling of the cap region and rupture of the acrosomal pouch. The latter began with polymerization of materials within the subacrosomal region and ended with re-configuration of the subacrosomal region into an electron-dense spherical mass. The AR of the sperm observed during spawning revealed similar morphological events, with degeneration of the spike upon contact with the vitelline envelope and formation of the spherical mass while penetrating into jelly material produced by protruding cortical rods. The results suggest the presence of AR inducers derived from the vitelline envelope and cortical rods of the egg. This study forms the basis for an evaluation of infertility regarding to AR in the domesticated P. monodon male.

Serotonin (5-hydroxytryptamine, 5HT) has been reported to induce ovarian maturation and spawning in the crayfish Procambarus clarkii and white Pacific shrimp Litopenaeus vannamei . The aim of this study was to explore the role of exogenous 5HT on the reproductive performance of the black tiger shrimp Penaeus monodon . 5HT solution was injected into domesticated P. monodon broodstock at 50 μg/g body weight and ovarian maturation and spawning were recorded. The presence of 5HT in the ovary and oviduct of P. monodon was also studied by immunohistochemistry and its levels in the ovary by enzyme link immunoabsorbance assay (ELISA). The 5HT-injected P. monodon developed ovarian maturation and spawning rate at the level comparable to that of unilateral eyestalk-ablated shrimp. Hatching rate and the amount of nauplii produced per spawner were also significantly higher in the 5HT-injected shrimp, compared to the eyestalk-ablated shrimp. 5HT-positive reactions were found in the follicular cells of pre-vitellogenic oocytes, in the cytoplasm of early vitellogenic oocytes and on the cell membrane and cytoplasm of late vitellogenic oocytes. 5HT in the ovary was present at 3.53 ± 0.26 ng/mg protein level in previtellogenic stage and increased to 17.03 ± 0.57 ng/mg protein level in the mature stage of the ovary. The results suggest a significant role of 5HT, possibly directly on the ovary and oviduct, on the reproductive function of female P. monodon .

A new, apparently innocuous virus was found while investigating the cause of monodon slow growth syndrome (MSGS) in cultured black tiger shrimp (Penaeus monodon). It was identified via plasmid vector clones of E. coli containing randomly amplified cDNA fragments produced from total nucleic acid extracts of hemolymph from MSGS shrimp. Of 421 clones, 30 that failed to give positive dot blot hybridization with a digoxigenin (DIG)-labeled shrimp DNA probe were sequenced and compared to GenBank records. Of these, 22 corresponded to known shrimp DNA records. Of eight that did not, one (20A) showed significant deduced amino acid sequence similarity to RNA-dependent RNA polymerases (RdRp) of the viruses in the family Luteoviridae and alignment revealed commonly conserved amino acids including a GDD motif believed to be at the enzyme active site. However, phylogenetic analysis showed that the virus sequence did not cluster with the Luteoviridae or other known RNA virus sequences. Thus, in accordance with frequent practice, it was named according to the area where it was first collected as Laem-Singh virus (LSNV). In situ hybridization with a DIG-labeled 20A insert revealed strong cytoplasmic staining confined to the lymphoid organ (LO), the heart and hepatopancreatic connective tissue in both normal and MSGS shrimp. RT-PCR assays based on the 20A clone sequence also gave positive results with both normal and MSGS shrimp. Transmission electron microscopy (TEM) of LO tissue revealed viral-like particles of approximately 27 nm diameter (within the Luteoviridae size range) in locations that matched those of positive in situ hybridization reactions in parallel samples. Although not directly associated with MSGS in Penaeus monodon, the presence or effect of this virus with other crustacean species is presently unknown.

The problem of growth retardation in cultured black tiger shrimp, Penaeus monodon was observed at one commercial shrimp farm in East Africa in mid 2004. The mean body weight of the shrimp at the sixth month in affected ponds was only 19 ± 4 g, which was 30% less than the expected size of normal shrimp grown within the same period at this farm. A preliminary examination for pathogenic bacteria, parasites and viruses revealed mild bacterial infections in a few specimens but all were negative for 7 known pathogenic shrimp viruses, either by assays using polymerase chain reaction (PCR) technology or by histological examination. A subsequent examination of a second lot of samples prepared for transmission electron microscopy (TEM) revealed the presence of previously undescribed lesions in the lymphoid organ and gills. In semithin sections stained with toluidine blue, these were apparent as large cytoplasmic inclusions that were not visible in normal tissue sections stained with hematoxylin and eosin. By TEM, the cytoplasmic inclusions contained large numbers of non-enveloped, icosahedral virions of approximately 25 nm diameter. Given the unique nature of the lesions and the negative results with molecular tests for known shrimp viruses, it is possible that the particles seen constitute a new viral pathogen of shrimp. The retarded growth problem in East Africa resembles a similar problem called monodon slow growth syndrome (MSGS) that has been problematic with P. monodon in Thailand since 2001. Whether the slow growth in Africa and Thailand are due to the same or related pathogens or some other factor(s) remains to be explored.

The aims of this study were to determine the effects of Vibrio bacterin, with or without carboxymethyl β-1,3-glucans (CMBG), on the protection of black tiger shrimp Penaeus monodon against V. harveyi infection, and to determine the mechanisms underlying the protection. The study was done with two groups of shrimp, one in short-term treatments in concrete tanks and the other in long-term treatment in commercial ponds. Healthy juvenile black tiger shrimp were provided with formalin-killed V. harveyi (bacterin) and CMBG, which were top-dressed on commercial pellets, for 10 d. They were then challenged with virulent V. harveyi and the relative percent survival (RPS) was determined. The shrimp hemolymph was also studied to determine hemocyte counts and phagocytic, bactericidal, and phenoloxidase activities. In the commercial ponds, the shrimp were given commercial pellets top-dressed with the two substances for 2 months. The shrimp were sampled and challenged with virulent V. harveyi and the RPS deter...

This report describes morphological changes in the eggs in the black tiger shrimp Penaeus monodon upon contact with seawater, the process known as egg activation. Eggs from wild P. monodon broodstock were collected at 15-s intervals post-spawning during the first 15 min, and at 15-min intervals thereafter for 2 h. The samples were fixed and processed for light, scanning and transmission electron microscopy. As soon as the egg was released into seawater, the cortical rods began to emerge from the crypts on the periphery of the egg, and elevated the thin investment coat that covered the surface of the egg. Sperm in the first phase of the acrosome reaction were observed on both the egg and the surface of the investment coat. The rods protruded from the surface and were completely expelled out within 45 s. I0mmediately after complete extrusion, the cortical rods began to break up and formed the jelly layer around the egg. By this time, the interaction between the sperm at the second phase of the acrosome reaction and egg began. The hatching envelope had started formation at 1-min post-spawning, and was completed within 13–15-min post-spawning. The first and second polar bodies extruded from the egg at 3–5- and 10–15-min post-spawning, respectively. It was apparent that after the hatching envelop had formed, additional sperm could not enter the egg. This study suggests that the critical period for the egg–sperm interaction in P. monodon is within 45-s post-spawning.

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.

Records of PCR test results for white spot syndrome virus (WSSV) were reviewed in Thailand from 1998 to 2000 for wild Penaeus monodon broodstock purchased by hatcheries and for post-larvae (PL) brought by farmers to diagnostic laboratories for testing. Samples for PCR comprised DNA extracts from the last pleopod dissected from broodstock females after the first spawning and DNA extracts from whole, homogenized PL. There was a consistent pattern of fluctuation in percentage of WSSV-positive broodstock and PL. In broodstock, the fluctuation pattern was similar each year, with a low percentage (0 to 6 %) from January to May and a higher percentage (6 to 18%) for the rest of the year, with a peak from September to November. The fluctuation pattern for PL was similar but offset with peaks and troughs occurring approximately 2 mo after those for the broodstock. The peak percentages of broodstock-positive samples were roughly constant from year to year, but those for PL decreased progressively in magnitude from 1998 to 2000. Examination of a small number of hatcheries in 2000 revealed that the percentage of WSSV-positive PL samples was significantly lower for hatcheries that routinely discarded WSSV-PCR-positive wild broodstock when compared to hatcheries that did not.

A rapid and sensitive PCR-ELISA has been developed for detection of hepatopancreatic parvovirus (HPV) in Penaeus monodon . The specific primer set amplified 156 bp fragment and could detect as a little as 0·01 fg of purified HPV DNA which equivalent to three viral particles. No cross-reactivity was observed when nucleic acid templates from white spot syndrome virus, yellow-head virus, monodon baculovirus and shrimp were tested. The crude DNA simple prepared from hepatopancreas can be used as DNA template and provide a favorable result. Using this technique for detection of HPV infection in 87 carrier shrimps revealed the higher sensitivity and efficiency of detection when compared to histological examination and conventional PCR. Sixty-two percent infection was detected by PCR-ELISA from samples with HPV negative diagnosed by histological examination. Therefore, this sensitive and specific method is promisingly useful for early detection of HPV infection in broodstock, carriers and for ex situ application where large numbers of samples can be analyzed simultaneously.

Hepatopancreatic parvovirus (HPV) infects the hepatopancreas in penaeid shrimp and retards their growth. The DNA sequence of HPV from Thai shrimp Penaeus monodon (HPVmon) differs from HPV of Penaeus chinensis (HPVchin) by approximately 30%. In spite of this difference, commercial PCR primers (DiagXotics) developed from HPVchin to yield a 350 bp PCR product do give a 732 bp prod- uct with HPVmon DNA template. On the other hand, the sen- sitivity of HPVmon detection with these primers and with hybridization probes designed for HPVchin is significantly lower than it is with HPVchin. To improve sensitivity for HPV- mon detection, we used the sequence of the 732 bp HPVmon PCR amplicon described above to develop specific PCR primers (H441F and H441R) and hybridization probe. The primers could detect as little as 1 fg of purified HPVmon DNA while the 441 bp digoxygenin-labeled PCR product gave strong, specific reactions with in situ hybridization and with hybridization blots. In contrast, negative results were obtained using DNA from all other pathogens tested and from DNA of P. monodon. Supernatant solution from boiled, fresh shrimp fecal and postlarval samples homogenized in 0.025% NaOH/0.0125% SDS could be used to detect as little as 0.1 pg HPVmon DNA by the PCR reaction. By dot blot hybridization, a visible signal was obtained with purified HPVmon DNA at 0.01 pg, but detection in spiked feces and postlarval samples was only 1 and 0.1 pg, respectively.

Hepatopancreatic parvovirus (HPV) causes disease in several species of penaeid shrimp. Heavy infections may result in poor growth and reduced production for shrimp farmers. From one southern Thai shrimp pond with a high prevalence of HPV infection, 790 shrimp were sampled randomly and the hepatopancreas (HP) removed. Most HP were preserved in liquid nitrogen. However, every 10th HP (79 total) was divided into 2 parts appropriately fixed for examination by transmission electron microscopy (TEM) and light microscopy. Based on light microscopy, the prevalence of HPV infection in the pond was approximately 30% and its presence was confirmed by TEM of parallel samples. The virus was subsequently purified from hepatopancreatic homogenates of the samples preserved in liquid nitrogen. Negative staining of the purified viral preparation revealed unenveloped, icosahedral viral particles 22 to 24 nm in diameter. Agarose gel electrophoresis of nucleic acid extracts revealed the presence of 2 fragments, one very intense (5.8 kb) and the other weak (4.2 kb). The larger fragment was degraded by DNase I and S1 nuclease, indicating single-stranded DNA (ssDNA) characteristic of the viral family Parvoviridae. The smaller fragment was degraded by DNase I but not by S1 nuclease, indicating that it comprised double-stranded DNA. A genomic DNA library of the 5.8 kb ssDNA was constructed in pUC18 and a clone containing a 659 bp fragment specific and sensitive for HPV was selected for sequencing. Based on this sequence, an HPV-specific primer set was designed to yield a 156 bp amplicon by polymerase chain reaction (PCR) amplification. The expected 156 bp amplicon was obtained only in the presence of HPV DNA template (at as little as 1 fg purified DNA) and not with nucleic acid templates extracted from healthy shrimp tissue or other shrimp pathogens. It is hoped that this PCR assay will be useful to shrimp aquaculturists for early detection and screening of shrimp larvae, parental broodstock or other possible carriers of HPV in the shrimp cultivation system.

PmNOBII was first described from experimentally infected shrimp, but contemporary reports showed that white-spot virus infections in several penaeid shrimp species exhibited similar gross signs and histopathology. Using laboratory infected specimens of Penaeus monodon , DNA of the non-occluded baculovirus PmNOBII was extracted and digested with BamHI and EcoRI. Resulting DNA fragments were ligated with Bluescribe vector using T4 ligase and competent cells of Escherischia coli JM 107 were transformed. Two recombinant clones that gave negative hybridization with P. monodon DNA but positive hybridization with PmNOBII DNA were selected. Inserted DNA fragments of 0.9 kbp and 4.2 kbp were obtained from these clones after plasmid digestion with BamHI and EcoRI. These fragments were subsequently labeled with digoxygenin for visualization and tested using the in situ DNA hybridization technique with tissues from PmNOBII infected and non-infected laboratory shrimp. For viral infected nuclei identified by H and E staining in parallel samples, the 4.2 kbp fragment gave a stronger DNA hybridization signal than did the 0.9 kbp fragment. The 4.2 kbp fragment was then used for in situ DNA hybridization tests with commercially or experimentally cultivated shrimp specimens showing gross signs and histopathology characteristic of white-spot virus infection. Field signs of the disease included general reddish coloration, white granules of 1–2 mm under the cuticle and rapid mortality. Normal histology (H and E) revealed Cowdry-A type nuclear inclusions that developed to produce basophilic hypertrophied nuclei typical of PmNOBII, and transmission electron microscopy revealed characteristic rod shaped virions. All these specimens gave positive hybridization results, and included cultivated shrimp specimens of Penaeus chinensis, P. indicus, P. japonicus, P. merguiensis, P. monodon and P. vannamei obtained from various countries in Asia between August 1993 and January 1995. The data indicate that PmNOBII, or closely related variants, are currently responsible for a widespread epizootic in the Asian shrimp farming industry.

Objective: To examine the structure of the oviduct of the shrimp Penaeus monodon. Methods: The oviducts of P. monodon with three different major groups of ovarian development (Group (Gr.) 1: Stages I & V; Gr. 2: Stages II & III; and Gr. 3: Stage IV) were examined by light, transmission electron, and scanning electron microscopies, respectively. Results: The epithelium of the oviduct in Gr. 1 was composed of tall simple columnar cells with their basal nuclei located on the basement membrane and its thick collagen fibers. In Gr. 2, the oviduct seemed to produce some substances and their epithelial cells became transitional with centrally located nuclei and formed some vacuoles. Obviously, the epithelial cells in Gr. 3 (at Stage IV) were disorganized, disrupted, and shed accumulated spherical secretory substances including some cellular contents into the lumen. Conclusions: The structural changes of the P. monodon oviduct were related to ovarian maturation stages (Grs. 1–3). Prior to spawning, only the oviduct epithelium at ovary Stage IV produced and secreted a number of spherical secretion substances into the lumen. These substances may act as the oviductal lubricants to facilitate the spawning process.

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 0.7 × 1.1 μm 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 (10 nm) and an electron-dense exospore (2 nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848 bp 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.

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.

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is widespread in cultured Penaeus monodon and P. vannamei in Thailand. It causes runt-deformity syndrome that is characterized by physical abnormalities and stunted growth in P. vannamei, but causes no apparent disease in P. monodon. In both species, the virus may produce Cowdry Type A inclusions in tissues of ectodermal and mesodermal origin, but these are common in P. vannamei and rare in P. monodon. The virus can be more easily detected in both species by IHHNV-specific PCR primers. By in situ hybridization (ISH) using specific IHHNV probes, fixed phagocytes associated with myocardial cells tended to show strong positive reactions in both shrimp species. Ovarian and neural tissue (neurons in the nerve ganglia and glial cells in the nerve cord) were ISH positive for IHHNV only in P. vannamei. By transmission electron microscopy, necrotic cells were found in the gills of IHHNV-infected P. vannamei, while paracrystalline arrays of virions and apoptotic cells rather than necrotic cells were found in the lymphoid organ of IHHNV-infected P. monodon. Thus, it is possible that apoptosis in P. monodon contributes to the absence of clinical disease from IHHNV. These findings reveal different responses to IHHNV infection by the 2 shrimp species. A curious feature of IHHNV infection in P. monodon was inconsistency in the comparative viral load amongst tissues of different specimens, as detected by both ISH and real-time PCR. This inconsistency in apparent tissue preference and the reasons for different cellular responses between the 2 shrimp species remain unexplained.

This research is aimed at developing an automatic spawning detection system for the black tiger shrimp, Penaeus monodon. This system is composed of instruments which are attached to the inner wall of the spawning tank that contains light sensing circuits which change light intensity into electrical signals. The detection unit is composed of a polyvinyl chloride (PVC) pipe containing a light emitting diode (LED) bulb that serves as a light source transmitter, and a light-dependent resistor (LDR) that serves as a light receiver which transforms light intensity into electrical resistance. Placed between the LED bulb and the LDR is a 135 μm nylon mesh screen that is stretched horizontally inside the pipe to filter out shrimp eggs. Changing signals from the LDR turns on (or off) a 220 V bulb. This device detects spawning activity as early as 22 s after the onset of spawning. There is a correlation that exists between the number of spawned eggs and the time of the onset of spawning, with earliest detection occurring with the highest number of eggs.

This study focused on apoptosis in various tissues of the black tiger shrimp Penaeus monodon following white spot syndrome virus (WSSV) injection. The study included: (1) light microscopy (LM) and transmission electron microscopy (TEM) of various tissues; (2) fluorescent LM of nuclear DNA by staining with 4, 6-diamidine-2-phenyl indole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labelling (TUNEL) techniques; and (3) determination of caspase-3 activity. Juvenile P. monodon were injected with WSSV, and several tissues of ectodermal and mesodermal origin were studied at different intervals after injection. The total haemocyte count had decreased to one-tenth of its original level 60 h after WSSV injection. By LM, extensive destruction by WSSV was observed in the stomach epithelium, gills, hematopoietic tissue, hemocytes and the heart, but the most severely affected tissue was the subcuticular epithelium. TEM revealed that at 6 h post-injection (p.i.) the chromatin of infected nuclei was marginated, and by 24 h p.i. the nuclei were filled with enveloped and non-enveloped WSSV virions. At later stages of the infection, the nucleus extruded WSSV particles. Chromatin margination and nuclear condensation and fragmentation (i.e. signs of apoptosis) were observed as early as 6 h p.i. in all affected tissues, but occurred in cells without WSSV virions rather than in cells with virions. The occurrence of apoptosis was supported by data obtained using TUNEL and by DAPI-staining and progressed from 6 to 60 h p.i. In addition, caspase-3 activity in WSSV-infected shrimp was about 6-fold higher than that in uninfected shrimp. The data strongly suggests that apoptosis occurs following WSSV infection in P. monodon, but the extent to which it contributes to shrimp mortality requires further investigation.

Histological, cytochemical and ultrastructural changes in giant black tiger shrimp Penaeus monodon were investigated at various time intervals after injection with yellow head virus (YHV). Hemocytes, lymphoid organs (LO) and gills were the main focus of the study. After injection with YHV, onset of mortality varied from 36 h onward. By normal hematoxylin and eosin staining, the 3 tissues showed clear and increasing prevalence of nuclear condensation, pyknosis and karyorrhexis from approximately 36 h post-injection (p.i.) until death, although pathology was evident in the LO as early as 12 h p.i. in some shrimp. By nuclear DNA staining with 4',6-diamidino-2-phenylindole (DAPI) and by specific labeling of 3'-OH ends of nuclear DNA using a technique called terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL), cells of the 3 tissues showed evidence of chromatin condensation and DNA fragmentation, respectively. Both are generally considered to be characteristic of apoptosis. In addition to TUNEL labeling, evidence for DNA fragmentation was supported by the appearance of approximately 200 base pair DNA ladders at approximately 48 h p.i. in hemocytes of YHV-infected but not uninfected shrimp. Transmission electron microscopy (TEM) of LO tissue revealed features of apoptosis in tissues of YHV-infected shrimp only. These included marginated, condensed and fragmented chromatin without concurrent cytoplasmic damage. Histological, cytochemical, ultrastructural and biochemical data were consistent with the hypothesis that widespread and progressive apoptosis occurred in susceptible shrimp infected with YHV. Although no specific tests were carried out to determine whether this purported apoptosis was the cause of mortality, moribund shrimp had extensive deterioration of vital tissues such as the hemolymph, gills, heart and LO, suggesting that many essential bodily functions had been severely compromised. This probably resulted in the gross signs of lethargy and weakness seen, and it is reasonable to suggest that further, progressive deterioration could have led to the collapse of vital functions followed by death.

Commercial, intensive, earthen shrimp ponds (188) in southern Thailand were stocked with postlarvae (PL) of Penaeus monodon that had tested positive or negative for white-spot syndrome virus (WSSV) infection by polymerase chain reaction (PCR) assay. All the PL were grossly healthy. At 2 wk intervals after stocking, shrimp from each pond were examined for gross WSSV lesions and tested for WSSV by PCR. Shrimp from all the ponds stocked with WSSV-PCR-positive PL (Group 0, n = 43) eventually showed gross signs of white-spot disease (WSD) at an average of 40 d after stocking. Of the remaining ponds stocked with WSSV-PCR-negative PL (n = 145), some remained WSSV-PCR-negative throughout the study (Group 5, n = 52), while others (93) became WSSV-PCR-positive after stocking, during the first month (Group 1, n = 23), second month (Group 2, n = 40), third month (Group 3, n = 24), or fourth month (Group 4, n = 6). Crop failure was defined as a pond drain or forced harvest before 14 wk or 98 d of cultivation. For Group 0 the proportion of ponds failing was 0.953, while it was only 0.019 for Group 5. Thus, the relative risk of failure for Group 0 was approximately 50 times that of Group 5. The relative risk of failure for Group 0 was also 3 times that for ponds stocked with WSSV-PCR-negative PL. Obviously, not all WSSV outbreaks resulted in crop failure. Of the 93 ponds stocked with PCR-negative PL that later yielded WSSV-PCR-positive shrimp, 53% reached successful harvest. The study showed that PCR screening of PL and rejection of WSSV-positive batches before stocking could greatly improve the chances of a successful harvest.

White spot syndrome virus (WSSV) of the black tiger prawn Penaeus monodon is a recently discovered baculo-like virus disease which is currently the cause of very serious and widespread losses in the shrimp industry in Thailand and elsewhere in Asia. Three suspected crab carriers of this virus commonly found in shrimp-rearing areas were investigated. These were Sesarma sp., Scylla serrata and Uca pugilator. All these crabs could be infected with WSSV by injection and they sustained heavy viral infections for up to 45 d (confirmed by normal histology, specific in situ DNA hybridization and PCR amplification) without visible signs of disease or mortality. All of them also transferred the disease to P. monodon via water while physically separated in aquarium cohabitation tests. Transfer of the virus to the shrimp was monitored using in situ DNA hybridization and PCR assay at 12 h intervals after cohabitation began. With U. pugilator, WSSV could be detected in the shrimp cohabitants after 24 h using PCR amplification and after 60 h using in situ hybridization. With S. serrata, the shrimp were positive for WSSV after 36 h using PCR and after 60 h using DNA in situ hybridization. With Sesarma sp. they were positive after 48 h using PCR and 72 h using in situ hybridization. These laboratory studies demonstrated that crab carriers of WSSV may pose a real threat to cultivated shrimp. However, the studies were carried out in containers with a small volume and with relatively clean sea water as compared to shrimp cultivation ponds. Pond-based studies are now needed to determine whether factors such as pond volume, pond water quality and shrimp and crab behavior can influence the rate and success of transfer.

The activities of the enzymes N-acetyltransferase (NAT) and hydroxyin-dole-O-methyltransferase (HIOMT) and the hormone melatonin were studied in the optic lobe of the subadult giant tiger shrimp Penaeus monodon. Compared with the level in other species, a relatively high level of NAT activity that was temperature- and pH-dependent were observed. The NAT enzyme had a relatively high maximum velocity (Vmax, 100 pmol/hr/μg protein) and low Michaelis constant (Km, 22 μM), when tryptamine is used as substrate. In contrast to the high level of NAT activity, HIOMT activity and melatonin levels were low in the optic lobe of the giant tiger shrimp. Sex differences in the levels of NAT activity and melatonin, which are observed in a freshwater species Macrobrachium rosenber-gii, were not noticeable in the saltwater species P. monodon, at least not when they were in their subadult stage.