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Seawater (SW)-acclimated Nile tilapia, Oreochromis niloticus, reared under a salinity 30 environment had lower growth and survival than the freshwater (FW)-acclimated fish. It was hypothesized that cells of the SWacclimated fish had not been able to synthesize an adequate level of a compatible osmolyte, myo-inositol (MI), in adjusting to the salinity 30 environment. In this study, MI supplements, at 250, 500, and 750 mg/kg pellets, were provided to the fish through top-dressing. After the 30-day feeding trial, the following parameters were determined: final body weights; survival; biomass increase; feed conversion ratio (FCR); plasma osmolality and ions; and two transcripts in the gills mips250 and mipa1 encoding enzymes responsible for MI biosynthesis. The SW-acclimated O. niloticus receiving 500-mg MI supplement had significantly higher survival, biomass increase, and lower FCR than those of the SW-acclimated fish receiving no supplement. At 500-mg MI supplemental level, the increasing values of plasma osmolality and Na+ observed in SW-acclimated fish were significantly attenuated. The transcript mipa1, but not mips250, was markedly up-regulated in the SW-acclimated O. niloticus, compared with that of the FW-acclimated fish. Again, MI at 500-mg supplement attenuated the upregulation significantly. This study suggests that MI supplement at the optimum level enhanced the performance of SW-acclimated O. niloticus, and through yet unknown mechanisms, attenuated some of their physiological responses to the osmotic stress. [ABSTRACT FROM AUTHOR]

Fishmeal is the main source of protein in the shrimp feed industry and is normally derived from trash fish. As such, the production of fishmeal has an adverse effect on the marine environment by taking away small and juvenile fish, leading to depletion of marine species. There is a need for alternative sources of protein which will substitute fishmeal in the aquaculture industry. This study evaluated the components and nutritional efficacy of bioflocs, which were used to substitute fishmeal protein. The effect of bioflocs diets on growth performance, survival rate, and immune response in shrimp compared to normal fishmeal feed were determined. Bioflocs were harvested from the shrimp ponds (C:N ratio >12:1) at Shrimp Village, Chaiya district, Surat Thani, Thailand. The total protein in bioflocs was about 48% and the total lipid was about 5% (dried weight) and the percentages of essential amino acids (EAA) and fatty acids (EFA) in bioflocs were similar to those of fishmeal feed. Shrimp fed with the different dietary bioflocs feed regimens [% to replace fishmeal; 0% (B0), 25% (B25), 50% (B50), 75% (B75), and 100% (B100)] for 42 days revealed that all growth parameters were almost similar to those of the control shrimp (shrimp fed with normal fishmeal, B0) including final body weight, weight gain, specific growth rate, and feed conversion ratio. Remarkably, the survival rates, the levels of immune parameters, and expression of immune genes (proPO-I, PEN-4 and dicer) were significantly higher in bioflocs fed shrimp, especially in B25 and B50 shrimp. Moreover, B25 and B50 bioflocs fed shrimp showed notably increased survival rates following Vibrio parahaemolyticus (V. parahaemolyticus) infection. In conclusion, the present study demonstrates that shrimp survival and immunity are enhanced by biofiocs substituted fishmeal. Significantly, the bioflocs diets activated the immune response to prevent V. parahaemolyticus infection. • Bioflocs was produced using molasses as a carbon source at the C:N ratio >12:1. • Bioflocs powder had equivalent values of essential amino acids and fatty acids to fishmeal. • Bioflocs substitution fishmeal enhanced shrimp growth and immune activities. • Bioflocs feed had a capacity to control V. parahaemolyticus infection in shrimp. • Bioflocs powder revealed a potential to replace fishmeal protein in shrimp feed pellets. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Abstract: 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 10min at 8min 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±3.2g) and triploid males (31.5±3.5g) was significantly higher (P <0.0001) than that of their diploid counterparts (24.5±0.5g for females and 23.1±3.8g for males), having a ratio of 2 females:1 male for triploid shrimp, and 2 females:3 males for diploid shrimp. These results reveal the advantages of growing triploid over diploid P. monodon, and its feasibility for commercial production. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP), the fungus-related micro-organism that infects the hepatopancreas of marine shrimp, is believed to cause growth retardation of the infected shrimp. As the hepatopancreatic cells are composed of different cell types that support the digestive system and innate defense, malfunction of the infected cells could lead to growth retardation and weakness. This study aimed to determine of the percentage of each hepatopancreatic cell type infected by EHP and its infectious levels in the normal and growth-retarded Pacific whiteleg shrimp Litopenaeus vannamei. Histological examination revealed that all the cell types examined were infected with EHP, with significantly higher (p<0.01) percentage on the M- and Fcells, compared to other cell types. The R-, B- and F-cells of the growth-retarded shrimp were infected at significantly lower (p<0.05) percentage than the same cell types of the normal shrimp. By PCR, positive reactions of EHP infection were detected in all shrimp samples, both in the normal and growth-retarded shrimp, but only at the nested PCR level. Also, the relative density of the EHP load in the hepatopancreas of the two shrimp groups did not differ statistically. These results suggest that growth retardation in L. vannamei currently exists may not be due to EHP infection. [ABSTRACT FROM AUTHOR]

Abstract: 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 135μm 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 220V bulb. This device detects spawning activity as early as 22s 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. [Copyright &y& Elsevier]

Hepatopancreatic microsporidiosis, the shrimp disease caused by Enterocytozoon hepatopenaei (EHP) infection, has been widely spread in farmed Litopenaeus vannamei in Thailand. The symptoms include decreased feeding, muscle waste, slow growth, and chronic mortality. Since 5-aminolaevulinic acid (5-ALA), a non-protein amino acid, has been known to enhance ATP production in the cell through the increased formation of hemoproteins in the electron transport complex of mitochondria, it was hypothesized that providing 5-ALA to EHP-infected shrimp might reduce the mortality caused by the infection. Naturally EHP-infected L. vannamei were randomly sampled from a commercial shrimp pond that showed signs of hepatopancreatic microsporidiosis. The shrimp samples were reared under 30-ppt seawater in 40-L plastic tanks, divided into four groups; each receiving commercial pellets supplemented with different doses of 5-ALA: 0 (control), 15, 30, and 60 ppm. After 21 days of the experiment, it was found that the shrimp receiving 5-ALA at 60 ppm survived at a rate significantly (p < 0.05) higher than that of other groups, and had a significant increase in biomass, compared with that of the control group. Histology of the hepatopancreas of the 60-ppm group revealed significantly (p < 0.01) less percentage of atrophic tubules and larger areas of the vacuoles of the B-cells, suggesting improved hepatopancreatic functions. The levels of the EHP load in the hepatopancreas as revealed by semi-quantitative PCR were increasing in a dose-related manner of 5-ALA supplement. Histology of the hepatopancreas also revealed the presence of EHP. The ATP levels in the hepatopancreas of the 30- and 60-ppm groups were significantly (p < 0.01) higher than that of the control group. Further tests are required in commercial farms to find out if the 5-ALA supplement is beneficial to tackle the problem of hepatopancreatic microsporidiosis at the industrial level. [Display omitted] • Supplementation of 5-ALA in feed to EHP-infected L. vannamei reduced the mortality and increased the biomass of the affected shrimp. • The supplementation decreased the percentage of atrophic hepatopancreatic tubules and increased the areas of the B-cell vacuoles. • The supplement increased ATP levels in the shrimp hepatopancreas. • However, the supplement also increased the EHP load in the hepatopancreas. [ABSTRACT FROM AUTHOR]

Background: Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp. Results: A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3' and 5' RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival. Conclusions: Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD. [ABSTRACT FROM AUTHOR]

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 ( Pmo DNV) in 40% of the samples. The fresh smear and histology revealed good quality of cell morphology and histological features, as well as Pmo DNV 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 . [ABSTRACT FROM AUTHOR]

Co-culture of Nile tilapia, Oreochromis niloticus , with marine shrimp has been an increasing farm practice as evidence has suggested that the co-culture is one of a few ways to mitigate shrimp diseases, especially the acute hepatopancreatic necrosis disease that has affected shrimp culture industry worldwide. Since Nile tilapia are basically freshwater species, having them in co-culture with marine shrimp therefore requires a certain degree of salinity adaptation, in which the fish are readily capable to. The present study was aimed at studying certain hemato-immunological changes of the fish reared under elevated salinity, as well as their susceptibility to pathogens. Nile tilapia (180 g initial BW) were reared under gradually increasing salinity within one month from 0 to 30 ppt at 3 ppt/3 d and maintained for another month in 30-ppt salinity. At the end of the 60-day experimental period, 50% of the fish survived with mortality occurred mainly during the second month when the salinity was at 30 ppt. Their growth rate was retarded to 60% of that of the control fish maintained under freshwater (0 ppt). The fish reared under elevated salinity also had significantly lower hematocrit, higher total white blood cell counts, higher absolute numbers of lymphocytes and thrombocytes and higher serum lysozyme activity, compared to those reared under freshwater. Presence of three representative pathogens: infectious spleen and kidney necrosis virus (ISKNV), Francisella noatunensis and Streptococcus agalactiae ; were determined in the fish reared under freshwater and in those reared under elevated salinity, using polymerase chain reaction (PCR) method. Of the three pathogens, only ISKNV was detected in 1/10 of the freshwater and 6/10 of the elevated-salinity fish. In both groups, the virus was present in the gills, brain, liver, gonads, skin, kidney and spleen, with moderately and severely positive reaction observed in the kidney and spleen. The detection of ISKNV was confirmed by in situ hybridization of the gonads and kidney, using loop-mediated DNA amplification with digoxigenin, which clearly showed dark-brown stain of the positive signals in the PCR-positive samples. These results suggest that a substantial number of O. niltoicus could survive elevated salinity up to full-strength seawater provided that the salinity had been gradually elevated. And under that situation, changes in hemato-immunological functions of the fish did occur, which probably caused lethal proliferation of pre-existing ISKNV that otherwise would remain dormant. [ABSTRACT FROM AUTHOR]

Background: Persistent infection of Penaeus stylirostris densovirus (PstDNV) (also called IHHNV) and its noninfectious inserts in the black tiger shrimp, Penaeus monodon (P. monodon) genome are commonly found without apparent disease. Here, we introduced the method of multiplex PCR in order to differentiate shrimp with viral inserts from ones with the infectious virus. The method allowed us to study the effect of pre-infection of IHHNV, in comparison to IHHNV inserts, on WSSV resistance in P. monodon. Results: A multiplex PCR system was developed to amplify the entire IHHNV genome, ensuring the accurate diagnosis. Field samples containing IHHNV DNA templates as low as 20 pg or equivalent 150 viral copies can be detected by this method. By challenging the two groups of diagnosed shrimp with WSSV, we found that shrimp with IHHNV infection and those with viral inserts responded to WSSV differently. Considering cumulative mortality, average time to death of shrimp in IHHNV-infected group (day 14) was significantly delayed relative to that (day 10) of IHHNV-inserted group. Real-time PCR analysis of WSSV copy number indicated the lower amount of WSSV in the IHHNV-infected group than the virus-inserted group. The ratio of IHHNV: WSSV copy number in all determined IHHNV-infected samples ranged from approximately 4 to 300-fold. Conclusion: The multiplex PCR assay developed herein proved optimal for convenient differentiation of shrimp specimens with real IHHNV infection and those with insert types. Diagnosed shrimp were also found to exhibit different WSSV tolerance. After exposed to WSSV, the naturally pre-infected IHHNV P. monodon were less susceptible to WSSV and, consequently, survived longer than the IHHNV-inserted shrimp. [ABSTRACT FROM AUTHOR]