Molecular cloning,characterization and expression analysis of the fatty acid‐binding protein (MnFABP), involved in dietary lipid sources response in oriental river prawn,Macrobrachium nipponense

A fatty acid‐binding protein (FABP) gene designated as MnFABP10 was cloned and characterized from the freshwater prawn Macrobrachium nipponense. The full‐length cDNA of MnFABP10 was 646 bp encoding a 130 amino acid. Real‐time quantitative RT‐PCR showed that the MnFABP10 gene was expressed in various tissues with the highest expression in the hepatopancreas. The MnFABP10 mRNA levels in the hepatopancreas and ovary of M. nipponense were dependent on the stages of ovarian development. Western blot results revealed a single immunoreactive band with an estimated molecular mass of approximate 14 kDa in the developmental ovary. Then, M. nipponense with an initial body weight of 0.090 ± 0.0010 g were fed with four isonitrogenous and isocaloric diets with different oils, that is, beef tallow (BT), soybean oil (SO), pollack fish oil (FO) and a mixture of fish oil and soybean oil (FO/SO 2 : 1 w/w) for 52 days. The mRNA levels of MnFABP10 in the hepatopancreas were influenced by different lipid sources, with a peak expression observed in prawns fed SO. This study suggests that MnFABP10 may have a putative function in ovary maturation, and its mRNA expression in the hepatopancreas can be regulated by the source of dietary lipids in M. nipponense.     

An evaluation of increasing linolenic acid level in the diet of Macrobrachium nipponense: Lipid deposition,fatty acid composition and expression of lipid metabolism‐related genes

To determine the effects of linolenic acid (LNA, 18:3n‐3) in oriental river prawn (Macrobrachium nipponense), an 8‐week feeding experiment was conducted using six isonitrogenous and isoenergetic semi‐purified diets containing 0.07 g/kg (control), 7.3 g/kg, 16.6 g/kg, 20.2 g/kg, 27.3 g/kg and 36.3 g/kg LNA. The hepatopancreas lipid content decreased significantly when dietary LNA content was >20.2 g/kg. Fatty acid analysis revealed that the percentage of 18:3n‐3 in the hepatopancreas significantly increased with increasing dietary LNA levels, while 20:5n‐3, 22:5n‐3 and 22:6n‐3 levels in the hepatopancreas decreased in a curvilinear manner as dietary LNA increased. Additionally, qRT‐PCR results revealed that hepatopancreas mRNA expression of acetyl‐CoA carboxylase (ACC) decreased with increasing dietary LNA, while the greatest carnitine palmitoyl transferase‐1(CPT1) mRNA expression was observed in the 2.73 g/kg and 36.3 g/kg groups. Furthermore, hepatopancreas mRNA expression of acyl‐CoA delta‐9 desaturase (SCD) and fatty acyl elongase 6(elovl6) was downregulated when prawns fed the diets containing >20.2 g/kg LNA. These results indicate that dietary 18:3n‐3 could decrease lipid deposition through increased fatty acid β‐oxidation and modulated fatty acid synthesis, and alter fatty acid composition by regulating fatty acyl elongase and fatty acyl desaturase mRNA expression in the M. nipponense.     

Molecular cloning of heat shock protein 60 from Marsupenaeus japonicus and its expression profiles at early developmental stages and response to heat stress

Water temperature is an essential environmental factor in aquaculture that affects many aspects of organism, and a rise in water temperature stresses aquatic animals. HSP60 is a major component of the chaperone system and plays an important role in stress response. In this study, the full‐length cDNA sequence of HSP60 from Marsupenaeus japonicus was cloned for the first time, and the tissue distributions and expression profiles of MjHSP60 at the early developmental stages of M. japonicus and under heat stress were verified by qRT‐PCR. The full‐length cDNA sequence of MjHSP60 was 2,303 bp with the deduced amino acid (AA) sequence of 579 AA. MjHSP60 was expressed in all eight tested tissues of M. japonicus and was mainly expressed in the hepatopancreas under normal condition. MjHSP60 was expressed in all of the early developmental stages examined from the fertilized egg to the post‐larval stage, and the expression level of MjHSP60 peaked at zoea II and reached a trough at the transition periods between each two stages (N and M1) and showed a relatively stable expression level at the post‐larval stage. The expression level of MjHSP60 was significantly up‐regulated in the gills, muscle, heart, hepatopancreas and stomach at 3 hr post‐heat‐stress and showed varied sensitivity to heat stress. The expression profile of MjHSP60 in the hepatopancreas and gill showed a time‐dependent manner post‐heat‐stress and peaked at 3 hr post‐heat‐stress. The present study provided a basis for further studies for elucidating the function of MjHSP60 in the heat stress response and the early developmental stages of M. japonicus.     

Production of specific dsRNA against white spot syndrome virus in the yeast Yarrowia lipolytica

White spot syndrome virus (WSSV) is the most aggressive disease affecting cultured shrimp. One possibility to tackle it is by means of RNA interference (RNAi) induced by the presence of double‐stranded RNA (dsRNA). Normally, dsRNA is a product of the cellular machinery to gene regulation, but it can be produced synthetically and introduced into specific tissues or cells and thereby induce RNAi. Although in vitro production of dsRNA is possible, this is high cost. An alternative is to produce dsRNA in vivo using biological systems such as bacteria or yeasts. In this regard, Yarrowia lipolytica offers distinctive advantages for dsRNA production. The objective was to develop a Y. lipolytica strain able to produce dsRNA‐specific against WSSV and to evaluate its antiviral activity in the white leg shrimp Litopenaeus vannamei. From the 0.4 and 0.6 Kb fragments of the ORF89 gene, a dsRNA‐ORF89‐producing construct was built in the plasmid pJC410; the resulting construct (pARY410) was used to transform Y. lipolytica to drive the specific expression of dsRNA‐ORF89. Yeast colonies positive to the WSSV‐ORF89 gene were selected. The expression of dsRNA‐ORF89 and RNAse III was measured being detected at 32 and 48 hr. Subsequently, the antiviral activity of dsRNA‐ORF89 was tested in a WSSV challenge bioassay. The results showed survival in dsRNA‐ORF89 shrimp (25%) compared to control organisms treated with total RNA from the yeast P01‐AS harvested at 32 hr. In conclusion, Y. lipolytica is a convenient host to produce and deliver dsRNA‐ORF89 able to protect WSSV‐challenged shrimp.     

Immune responses of whiteleg shrimp,Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA‐VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune‐related genes were examined to estimate the effect of dsRNA‐VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA‐VP28 treatment, whereas MDA content did not change significantly. Among the ten immune‐related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA‐VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA‐VP28 stimulation indicate that these immune‐related genes were involved in dsRNA‐VP28‐induced innate immunity in shrimp.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

Immunostimulatory effect of mangrove‐derived marine yeasts in Penaeus monodon

This study attempted to assess the efficacy of mangrove‐derived marine yeasts as a source of immuno‐stimulant in the tiger shrimp, Penaeus monodon. The shrimp was fed on the diets in supplementation with yeast biomass at 10% for a period of 15 days and then challenged with the pathogenic Vibrio cholerae. The animals were assessed for immune responses in terms of total haemocyte counts, phenol oxidase and endobiotics. Among the yeasts tested, Rhodotorula minuta was found to have high immunostimulatory effect in the shrimps especially after challenge with the pathogenic vibrio.     

Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP‐infected whiteleg shrimp,Litopenaeus vannamei (Boone, 1931), in India

Stunted growth in pond‐reared Litopenaeus vannamei was observed in different farms located in Tamil Nadu and Andhra Pradesh, India. No mortality was associated with stunted growth. PCR assay on these samples revealed the presence of Enterocytozoon hepatopenaei (EHP) in stunted shrimp. Tissue distribution of EHP in naturally and experimentally infected shrimp was studied by PCR and histology. Histological examination revealed the presence of EHP in hepatopancreas and gut, but not in other organs. The PCR assay revealed the presence of EHP in all the organs tested in both naturally and experimentally infected shrimp. Healthy shrimp were challenged with E. hepatopenaei by intramuscular injection and oral route, and no mortality was observed in both routes after 30 days post‐challenge. Different developmental stages of the microsporidian parasite were observed in the hepatopancreatic epithelial cells. Biochemical parameters such as total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase were measured in the haemolymph of naturally and experimentally EHP‐infected shrimp. All biochemical parameters mentioned were found to be significantly higher in EHP‐infected shrimp when compared to normal shrimp. This is the first report relating AST and ALT levels to EHP infection in naturally and experimentally infected shrimp.     

Identification of RAPD‐SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp,Penaeus monodon Fabricius

White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10³ fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.     

Suitable dietary chitosan improves the growth performance,survival and immune function of tiger shrimp,Penaeus monodon

Different levels of dietary chitosan on growth performance, survival and stress tolerance to air exposure was studied in tiger shrimp, Penaeus monodon. Shrimp (mean initial wet weight about 1.16 g) were fed with six different diets (C0, C0.05, C0.1, C0.2, C0.3 and C0.4) containing six level of chitosan (0%, 0.05%, 0.1%, 0.2%, 0.3% and 0.4% respectively) in triplicate for 60 days. Growth performance [final body wet weight (FBW); weight gain (WG); biomass gain (BG)] of shrimp fed chitosan‐containing diets were higher (P < 0.05) than that of shrimp fed the basal diet, shrimp fed C0.1 diet showed the highest value of growth performance. Survival of shrimp in C0.1 and C0.2 diet groups were higher (P < 0.05) than that of shrimp in C0, C0.05 and C0.4 diet groups but without statistical difference (P > 0.05) in shrimp fed C0.3 diet group. Whole body and muscle lipid contents decreased with increasing dietary chitosan levels. Plasma total cholesterol and triglyceride contents of shrimp fed C0 diet was significantly higher (P < 0.05) than that of shrimp fed chitosan‐containing diets. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities of shrimp fed C0 diet were higher than those of shrimp fed chitosan‐containing diets. Digestive gland malondialdehyde (MDA) and carbonyl protein contents of shrimp fed chitosan‐containing diets were lower (P < 0.05) than that of shrimp fed C0 diet. Total haemocyte count of shrimp fed C0 diet was lower (P < 0.05) than that of shrimp fed chitosan‐containing diets. On the contrary, the haemolymph clotting time of shrimp fed C0 diet was higher (P < 0.05) than that of shrimp fed chitosan‐containing diets. In conclusion, all results suggested that dietary intake containing 0.1% and 0.2% chitosan enhanced the growth of shrimp, whereas a higher level than 0.3% and 0.4% decreased growth of shrimp. Second‐degree polynomial regression analysis of WG and BG indicated that the optimum supplement of dietary chitosan level should be 0.19–0.21%.     

Silencing Pacific white shrimp Litopenaeus vannamei LvRab7 reduces mortality in brooders challenged with white spot syndrome virus

White spot syndrome virus (WSSV) is a major threat for farmed shrimp worldwide. RNA interference (RNAi) is the most recent tool against viral diseases. Rab7 silencing effectively inhibited virus infections in juvenile shrimp, but the antiviral effect in brooders remains unknown. This study found a homologue Penaeus monodon Rab7 gene in Litopenaeus vannamei brooders from Mexico. Sequence identity was >99% to a Thai LvRab7 sequence and >94% to Rab7 sequences from P. monodon or Marsupenaeus japonicus. Animals treated with a partial (494 bp) or a complete (618 bp) LvRab7 dsRNA sequences and challenged 48 h post treatment (hpt) with a high WSSV dose showed 80–88% mortality respectively. Shrimp treated with 4 or 20 μg LvRab7 dsRNA and challenged with a WSSV high dose had 80% mortality each, but it was reduced to 33% and 40%, respectively, with a low dose. Efficacy of dsRNA to reduce shrimp mortality was dependent on virus dose used regardless of dsRNA concentration. A significant reduction in LvRab7 mRNA levels was observed at 120 hpt. In conclusion, silencing LvRab7 in brooders showed a mild antiviral effect against a WSSV challenge at 48 hpt.     

A global view of hepatopancreas and intestinal reveals the potential influencing mechanism of aflatoxin B1 on nutrition and metabolism in Litopenaeus vannamei

The present study was to determine the time‐dependent alterations in growth performance, histomorphology, digestive enzymes activities and the genes expression related to target of rapamycin (TOR) signalling pathway involved in hepatopancreas and intestine of Litopenaeus vannamei at 3, 6, 12, 18, 24 and 30 days after Aflatoxin B1 (AFB1) challenge. The results manifested that AFB1 could induce a significant decrease in growth performance and visible variations of the hepatopancreas and intestine structures in shrimp. Meanwhile, the digestive enzymes activities and genes expression level were significantly lower in the experimental group than in the control group and showed a time‐dependent decrease (p < 0.05). The expression levels of tor and s6k were significantly higher in the experimental group than in the control group from days 6 to 24 and showed a tendency to increase first and then decrease as a whole (p < 0.05). Meanwhile, we analysed candidate pathways involved in hepatopancreas and intestine of shrimp in metabolic response to AFB1. The mainly disturbed pathways related to metabolism in hepatopancreas were involved in pyrimidine, glycosylphosphatidylinositol, glycosaminoglycan, vitamin and amino acid biosynthesis and metabolism, while the mainly disturbed metabolic pathways in intestine were glycosaminoglycan, vitamin and amino acid biosynthesis and metabolism.     

Molecular characterization of transformer‐2 gene in the mud crab Scylla paramamosain and its putative role in sexual development

Transformer‐2 (Tra‐2) is an mRNA splicing factor that acts in concert with Tra to regulate the female sexual development in Drosophila melanogaster. In decapods, as no Tra homologues have been identified, the role of Tra‐2 in sexual development is more often discussed accordingly. In the present study, the full‐length cDNA of Tra‐2 of the mud crab Scylla paramamosain (SpTra‐2) was cloned and characterized. The deduced amino acid sequence of SpTra‐2 contained an RNA‐recognition motif (RRM) flanking by two RS‐rich regions, which are the hallmarks of the Tra‐2 proteins. The SpTra‐2 mRNA was highly abundant in the hepatopancreas, muscles and androgenic glands of the male crabs, implying a potential role in male sexual development. The hypothesis was supported by the RNAi experiments, finding that the injection of SpTra‐2 dsRNA in vivo led to a decreased expression of SpIAG, a key gene in crustacean male sexual differentiation. The relationship between SpTra‐2 and SpIAG was further affirmed by eyestalk ablation (ESA) experiments, which caused SpTra‐2 expression earlier than SpIAG expression in both androgenic glands and hepatopancreas. In addition, treatment with SpTra‐2 dsRNA also reduced the mRNA level of SpSxl, but had no effects on SpDmrt expression, suggesting the sex determination system in S. paramamosain is different from that in D. melanogaster.     

Molecular and functional characterization of Raptor in mTOR pathway from Litopenaeus vannamei

Raptor, a member of the target of rapamycin complex 1 (TORC1), participates in the formation of complex proteins related to the mechanistic target of rapamycin (mTOR) signalling. In this study, a 5,020 bp cDNA of Raptor with an open reading frame (ORF) of 3,804 bp encoding for 1,267 amino acids was cloned from Litopenaeus vannamei. The protein contains three conserved domains: Raptor N, HEAT and WD40 domains. The expression of Raptor gene was detected by qRT‐PCR in different tissues of L. vannamei, including hepatopancreas, intestinal, stomach, eyestalk, gill and muscle. The mRNA expression profiles of Raptor in muscle were also analysed under suppression or stimulation of mTOR signalling pathway. The level of Raptor mRNA significantly increased either at 0.5–6 hr after an injection of rapamycin (RAPA) or after 3 days starvation. Leucine or arginine alleviated the up‐regulation of Raptor gene expression caused by RAPA or starvation. The Raptor gene was successfully suppressed using RNA interference (RNAi) technology, and the gene expression and the protein phosphorylation level of 4EBP1 and S6K were significantly decreased. The results of the study suggested that the expression of Raptor was sensitive to the immunology status of L. vannamei and participated in nutritional metabolism.     

Analysis of viral load between different tissues and rate of progression of white spot syndrome virus (WSSV) in Penaeus monodon

At present the most common and most devastating disease of shrimp is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly by different species of crustaceans carrying the virus. After experimental injection of Penaeus monodon with a known copy number of WSSV in the abdominal muscle, the rate of viral progression in different tissues at 12, 24, 36 and 48 hpi (hours post infection) was assessed using quantitative real‐time PCR. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi, the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable reduction in the rate of viral replication in haemocytes compared with other tissues. The viral load in haemocytes, though increased again beyond 36 hpi, never surpassed the load in the other tissues. The real‐time PCR assay with its high sensitivity and wide dynamic range make it ideal for detecting low‐level WSSV infections that can occur in apparently healthy P. monodon.     

Evaluation of some haemolymph biochemical properties and F‐cell prevalence in hepatopancreas of white leg shrimp (Litopenaeus vanammei) after fed diets containing apple cider vinegar and propionic acid

The present study aimed to evaluate the effect of dietary ACV (apple cider vinegar) and PA (propionic acid) supplementation on some biochemical properties of haemolymph and fibrillar cell (F‐cell) number in the hepatopancreas of shrimp. Five different diets supplemented with 0%, 1%, 2%, 4% ACV and 0.5% PA diets were fed to Litopenaeus vanammei. Some biochemical parameters of haemolymph (cortisol, alkaline phosphatase, albumin, urea, uric acid, creatinine, phosphorus, total hemocyte count) and prevalence of F‐cell in hepatopancreas were examined after 60 days feeding trial. The albumin concentration and total hemocyte count in 2% and 4% ACV and PA groups were significantly higher than those of other groups (p < .05). Moreover, the highest phosphorus concentration was detected in shrimp fed 4% ACV and PA experimental diets (p < .05). No significant differences in haemolymph cortisol, urea, uric acid, creatinine, alkaline phosphatase and tubule area were observed among the experimental groups (p > .05). 2% ACV group showed more F‐cell number in hepatopancreas than other groups while the reduction in F‐cell number was observed in the PA group. Based on these observations, the tested ACV and PA diets remarkably exhibited positive impacts on nutritional value and they may be a potential for adding to shrimp diet.     

Bacterial community composition and distribution in different segments of the gastrointestinal tract of wild‐caught adult Penaeus monodon

Bacterial community associated with the gastrointestinal (GI) tract of aquaculture animals can play important roles in health, nutrition and disease. Compared with the GI tract of aquatic vertebrates such as fish, crustacean GI tract has unique structures and surfaces in different segments that may contribute to differences in the bacterial communities. This study examined the bacterial composition and distribution in different segments along the GI tract and in digesta of wild‐caught adult Penaeus monodon using Automated Ribosomal Intergenic Spacer Analysis (ARISA), real‐time quantitative PCR and clone libraries of 16S rRNA genes. Thirty‐nine bacterial species in four phyla including Proteobacteria (α, β, ε, γ), Firmicutes, Bacteroidetes and Actinobacteria were represented in the GI tract of adult P. monodon. Proteobacteria comprised over 80% abundance of the bacterial community in most segments of the GI tract, except the middle intestine that was dominated by Firmicutes (~50% abundance). The results also showed that bacterial communities showed significant differences along the GI tract segments, particularly the hindgut (p < .001) with Vibrio and Ferrimonas as dominant genera. The knowledge about the distribution of bacteria could be useful in understanding interaction of commensal bacteria and pathogens in different segments, and its potential influence on the effectiveness of probiotic bacteria in the GI tract of shrimp.     

Growth performance,haematological parameters,antioxidant status and salinity stress tolerance of juvenile Pacific white shrimp (Litopenaeus vannamei) fed different levels of dietary myo‐inositol

A 10‐week growth trial was run to evaluate effects of myo‐inositol (MI) on growth performance, haematological parameters, antioxidative capacity and salinity stress tolerance of Litopenaeus vannamei. Six practical diets supplemented with graded levels of MI (designated as MI0, MI600, MI1200, MI2400, MI 3600 and MI4800 for 448.8, 974.2, 1568.0, 2810.6, 3835.5 and 4893.6 mg/kg diet, respectively) were fed to six replicate groups of L. vannamei (mean initial body weight 0.63 ± 0.00 g). The results showed that significant increment of growth performance was observed in shrimp fed MI600 diet than those fed MI1200 diet. Lipid concentration in whole body of the shrimp fed MI600 diet was significantly increased. Shrimp fed MI0 diet had lower total protein (TP) as compared to shrimp fed the MI‐supplemented diets (except MI4800 diet). In general, lower activities of antioxidant enzymes and higher malondialdehyde (MDA) content in haemolymph and hepatopancreas were recorded in shrimp fed MI0 diet, compared to those fed the MI‐supplemented diets. Reduced survival after 7‐h salinity stress was present in shrimp fed MI0 diet as compared to those fed MI4800 diet. Dietary MI requirement for glutathione peroxidase activity of L. vannamei was 2705 mg/kg diet.     

Spawning of female Black tiger shrimp (Penaeus monodon) is not impacted by muscle injection of dsRNA targeted to gill‐associated virus

The ability of domesticated Penaeus monodon, Black Tiger shrimp, to spawn following tail‐muscle injection of dsRNA was examined. Ablated domesticated female broodstock infected subclinically with gill‐associated virus (GAV) were injected with saline or a cocktail of five‐dsRNAs targeting different regions in the GAV ORF1a/1b gene. To track changes in GAV infection loads, TaqMan real‐time PCR was used to quantify mean viral RNA amounts in each of three pleopod clips collected at the time of injection (Day 0) and either immediately after a female spawned or on Day 11 when the trial was terminated. Over the trial, 4 of 19 (21%) saline‐injected shrimp spawned and 12 of 25 (48%) dsRNA‐injected shrimp spawned, with one spawning twice. Egg numbers varied from 25 600 to 459 800 for the saline‐injected shrimp and from 4900 to 213 900 for the dsRNA‐injected shrimp. Of these, one of the four egg batches hatched from saline‐injected shrimp and 9 of the 13 egg batches hatched from dsRNA‐injected shrimp. While variable, egg numbers and hatch rates recorded were typical of those obtained from domesticated broodstock at the commercial hatchery and particularly among females previously spawned. Mean GAV RNA amounts detected in pleopod samples increased in five of the eight saline‐injected shrimp tested by 1.6–227.4‐fold and decreased in 12 of the 15 ds‐RNA‐injected shrimp tested by ?1.1 to ?45.1‐fold. The study demonstrated that tail‐muscle injection of GAV‐specific dsRNA does not adversely impact the ability of P. monodon to spawn.     

Serotonin stimulates ovarian maturation and spawning in the black tiger shrimp Penaeus monodon

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.