Effects of adding sucrose on Penaeus monodon (Fabricius, 1798) growth performance and water quality in a biofloc system

A 60‐day indoor growth trial was conducted to study the effects of biofloc on the growth performance of a Penaeus monodon (Fabricius, 1798), water quality and biological indicators including biofloc volume, chlorophyll‐a, heterotrophic bacteria and Bacillus quantity. Two concentrations of sucrose (0 and 75%) were added daily to the P. monodon culture systems (2.94 ± 0.11 g), which were conducted indoors in fibre‐glass tanks (500 L). Results showed that the final body weight and weight gain of the adding 75% sucrose group were significantly higher (P < 0.05) than that of the control, as well as significantly (P < 0.05) improved specific growth rates and survival rates, and reduced feed coefficient. Adding 75% sucrose promoted heterotrophic bacteria, Bacillus and phytoplankton reproduction, and significantly (P < 0.05) reduced the concentration of ammonia‐N (NH₄‐N), nitrite‐N (NO₂‐N) and nitrate‐N (NO₃‐N). The changes of water quality indicators in the two groups showed the similar trend at the end of the experiment, and the ammonia‐N, nitrite‐N, nitrate‐N and phosphate‐P concentrations in the 75% sucrose group were significantly (P < 0.05) lower than those of the control group, Chlorophyll‐a concentrations peaked at 389.12 μg/L in the biofloc sucrose group at 18:00 h, and heterotrophic bacteria peaked 8 h after sucrose was added. The addition of sucrose also reduced the pH of the water. Our research showed that adding sucrose promoted biofloc formation and shortened the formation time; increased the number of heterotrophic bacteria and algae which might play a role in improving water quality by assimilating ammonia‐N and other harmful substances in the water; supplemented food for P monodon growth; and reduced the feed coefficient.     

Apparent digestibility coefficients of fungal fermented plant proteins in two different penaeid shrimps—A comparative study

Using the 70:30 replacement method and chromium as an inert marker, the digestibility of four fermented oilseed meals/cakes (soybean meal (FSBM), groundnut oil cake (FGNC), rapeseed meal (FRSM), and sunflower oil cake (FSFC)) were determined in Penaeus monodon and Penaeus indicus. Apparent dry matter digestibility (ADMD) of fermented ingredients was ranked as FSBM > FGNC > FRSM > FSFC. The critical variations in apparent protein digestibility (APD) in P. monodon (0.816%) and P. indicus (0.608%) were lower than the ADMD. Appaprent amino acid digestibility (AAD) was >90% in FSBM for both the species and was lower for other ingredients. Protein had a higher digestibility than total amino acids and was in the range of 0.69%–2.71% in P. monodon and 0.32%–2.75% in P. indicus. A correlation between the ADC of total amino acids and protein was found to be r = 0.8229 in P. indicus and r = 0.7447 in P. monodon. Data were further subjected to two way analysis of variance for assessing the digestibility variations between the species. It was observed that P. indicus had higher values of ADMD than P. monodon in FSBM (2.97%) and FRSM (1.22%) and the reverse was true in FGNC and FSFC. The APD was high in P. indicus for FSBM, FGNC and FRSM but not for FSFC. However, significant variations could be noticed in AAD between the species.     

Cloning,characterization, expression analysis and RNAi of Retinoblastoma‐like gene from black tiger shrimp (Penaeus monodon)

Retinoblastoma (Rb) is a multifunctional regulator involved in several key cellular processes, such as cell cycle control, cell differentiation, tumorigenesis and senescence. In this study, an Rb‐like gene, PmRBL, was cloned from black tiger shrimp (Penaeus monodon). The full‐length cDNA sequence of PmRBL is 4,069 bp with an open reading frame of 3,243 bp, which encodes 1,080 amino acids. Quantitative real‐time PCR (qRT‐PCR) analysis indicated that PmRBL was highly expressed in the gills, hepatopancreas and ovaries of P. monodon. The highest PmRBL expression levels were observed in stage III of the ovarian development in P. monodon. RNA interference experiments were conducted to examine the expression profiles of PmRBL, PmCDK2 and PmCyclin E. The knockdown of PmRBL in the ovary and hepatopancreas by dsRNA‐RBL was successful. After dsRNA‐RBL was injected into the shrimp, the relative expression levels of PmCDK2 and PmCyclin E were upregulated at 12–72 hr in the ovaries and hepatopancreas. The localization and level of PmRBL expression in the ovary and hepatopancreas were investigated through in situ hybridization, which revealed consistent results with those of qRT‐PCR. Therefore, PmRBL, PmCDK2 and PmCyclin E may be involved in vitellogenin synthesis and ovarian maturation in P. monodon.     

Apparent digestibility coefficient of poultry by‐product meal (PBM) in diets of Penaeus monodon (Fabricius) and Litopenaeus vannamei (Boone), and replacement of fishmeal with PBM in diets of P. monodon

Nutrient apparent digestibility coefficients (ADCs) of pet food grade poultry by‐product meal (PBM) were determined for black tiger prawn, Penaeus monodon and Pacific white shrimp, Litopenaeus vannamei by the indirect method (reference diet and test diet at 7:3 ratio). Subsequently, an 8‐week growth trial was conducted to evaluate the effects of substitution of fishmeal (FM) with PBM in diets of P. monodon (initial weight = 0.21 ± 0.01 g). In the growth trial, six isonitrogenous and isoenergetic diets PBM0, PBM25, PBM50, PBM75, PBM100 and PBMA100, containing a gradient of PBM 0, 88.7, 177.4, 266, 354.7 and 354 g kg^?1 to replace 0, 92.5, 185, 277.5, 370 and 370 g kg^?1 FM were fed to four replicate groups respectively. The diet PBMA100 was supplemented with DL‐Met to be similar to PBM0. The results showed that both P. monodon and L. vannamei had relatively high ADC of crude protein (77.6% and 84.2% respectively) and gross energy (72.8% and 84.0% respectively) for PBM. Litopenaeus vannamei showed significantly higher digestion ability for PBM than P. monodon (P < 0.05). In growth trial, no significant difference in growth performance was observed among shrimp fed the experimental diets. DL‐Met supplementation did not improve the growth of P. monodon. PBM is a suitable protein ingredient for P. monodon feeds and can be used up to 354.7 g kg^?1 to totally replaced FM.     

EDCs‐induced glucocorticoid receptor related genes expression of the river pufferfish,Takifugu obscurus

Endocrine disrupting chemicals (EDCs) are of substantial concern when contaminating waters. We studied gene expression of larvae from river pufferfish, Takifugu obscurus exposed to EDCs. We cloned the full‐ or partial‐length cDNA sequence of genes related to the cortisol pathway, such as the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), heat shock protein (HSP) 70 and 90, phosphoenolpyruvate carboxykinase (PEPCK) and the FK506‐binding protein 4 (FKBP52). The tissue and time‐dependent expression of mRNA was studied by real‐time PCR in T. obscurus exposed to two representative EDCs (bisphenol‐A and 4‐tert‐octylphenol). HSP70, HSP90, MR and FKBP52 mRNA expression level was much higher than GR and PEPCK expression. GR mRNA expression level was significantly upregulated after 48 h in both EDC‐treated fish groups compared with the control. Both groups also showed down‐regulation patterns after 48 h. The initial expression of PEPCK in both groups was down‐regulated after 24 h. In the bisphenol‐A treated group, the expression of FKBP52 showed substantially high up‐regulation from 6 to 48 h after exposure, and then strikingly reduced its expression level at 72 h. Our results provide insights into the EDCs metabolizing system of T. obscurus and offers baseline information for further research related to the possible use as a bio‐indicator of this commercially important aquaculture fish species.     

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.     

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.     

Protein‐sparing effects and LPL gene expressions of dietary lipids in the juvenile soft‐shelled turtle,Pelodiscussinensis

The aim of this study was to evaluate the effects of dietary lipids on protein‐sparing and lipoprotein lipase (LPL) mRNA expression in culture using 360 juvenile soft‐shelled turtles (Pelodiscussinensis) (initial weight 4.26 ± 0.14 g). The turtles were allotted to six diets with three duplicates for 60 days. A control diet with 46% protein and 55% fishmeal (CD) and five isonitrogenous diets with 41.3% protein and 45% fishmeal (F, S, L1, L2 and L3) were used, containing the following three lipid types: fish oil, soybean oil and mixed oils (soybean oil: fish oil = 1:1). The results showed that the survival rate was not affected by dietary lipids (P > 0.05). The highest weight gain and lowest feed coefficient ratio were seen in the L3 diets (P < 0.05). Turtles fed with L2 and L3 diets had lower superoxide dismutase activities, higher alanine aminotransferase activities and higher cholesterol concentrations than those exposed to other diets (P < 0.05). Hepatic LPL activity and LPL mRNA expression were higher in the L3 diets than in the other diets (P < 0.05). Overall, there were obvious protein‐sparing effects of dietary lipids and LPL mRNA expression was stimulated by high dietary lipids in soft‐shelled turtles in this study.     

Physiological,ionoregulatory, metabolic and immune responses of Persian sturgeon,Acipenser persicus (Borodin, 1897) to stress

The physiological ionoregulatory, metabolic and immune responses of Persian sturgeon, Acipenser persicus, to acute stress were investigated. Water levels were lowered to the fish dorsal scutes, and fish were blood‐sampled before stress (pre‐stress), and 0, 6, 24 and 72 h after stress. Results showed that serum cortisol rapidly increased after stress, returning to initial levels at 24 h. Serum glucose significantly increased at 6 h, declining to the pre‐stress levels at 72 h. Serum triglyceride and cholesterol showed significant decreases at 0 h, then increasing to higher than the initial levels at 72 h. Serum T₃ and T₄ significantly decreased at 0 h and recovered at 72 h. Serum chloride levels showed no significant changes while serum calcium showed a significant increase at 0 h and a further increase until 72 h poststress. Serum total protein and alternative complement activity showed a significant initial decrease and recovery at 24 h with further increase at 72 h. Serum lysozyme activity increased significantly at 24 and 72 h after stress. Serum total immunoglobulin significantly increased at 0 h and peaked at 24 h. This is the first work showing thyroid hormone and immunological responses in Persian sturgeon subjected to stress, and the results show that this species follows a classical hormonal and energetic stress response, although, in the low range among the sturgeons. Although transient and moderate, confinement stress can induce significant changes in the innate immune response.     

Expression and functional characterization of glutamine synthetase from giant freshwater prawn (Macrobrachium rosenbergii) under osmotic stress

The freshwater prawn, Macrobrachium rosenbergii naturally lives in the freshwater, though it migrates to the brackish water environment during spawning that claimed to be resistant on a broad range of saline fluxes. However, little is known about the osmoregulatory patterns and the effect of an enzyme glutamine synthetase (GS) in M. rosenbergii under stress. Here, we described the identification and functional characterization of GS from M. rosenbergii (Mr‐GS) at molecular and protein levels. The identified Mr‐GS was comprised of 361 amino acids that phylogenetically shared the highest identity with other crustaceans and predicted to contain Gln‐synt_C and Gln‐synt_N domains at the respective terminal regions. Tissue distribution analysis in M. rosenbergii revealed that the Mr‐GS was highly expressed in muscle, and commonly existed in other examined tissues in the following order gills > heart > stomach > brain > haemolymph. Whereas, the mRNA of Mr‐GS was significantly up‐regulated in the muscle and gill tissues following challenges with either hyper (0 → 13‰), or hypo (13 → 0‰) osmotic stress at 3, 6 and 12 hr. Furthermore, the level of Glutamine concentration was positively correlated with the GS mRNA and protein expression patterns in hyper‐osmotic stress, whereas in hypo‐osmotic stress a slight decrease in the gills and maintained a level in the muscle tissues at 3, 6 and 12 hr post‐treatments. Our findings suggest that Mr‐GS potentially exhibited the osmoregulation responses in the gill and muscle tissues of M. rosenbergii throughout the time of osmotic stress, which will benefit for future study on osmoregulation.     

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.     

Dietary microbial levan ameliorates stress and augments immunity in Cyprinus carpio fry (Linnaeus, 1758) exposed to sublethal toxicity of fipronil

A 45‐day feeding trial was conducted to study the stress ameliorating and immunomodulatory role of microbial levan in Cyprinus carpio fry exposed to sublethal dose (1/10th LC₅₀) of fipronil [(±)‐5‐amino‐1‐(2,6‐dichloro‐α,α,α‐trifluoro‐p‐tolyl)‐4‐trifluoromethylsulfinylpyrazole‐3‐carbonitrile]. Two hundred and twenty‐five fry were randomly distributed in five treatments in triplicates. Four purified diets were prepared with graded levels of microbial levan. Five different treatment groups were levan control L₀P₀ (basalfeed + 0% levan without exposure to pesticide); pesticide control L₀P₁ (basalfeed + 0% levan with exposure to pesticide); L_0.25P₁ (basalfeed + 0.25% levan with exposure to pesticide); L_0.50P₁ (basalfeed + 0.50% levan with exposure to pesticide) and L_0.75P₁ (basalfeed + 0.75% levan with exposure to pesticide). Lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and fructose‐1,6‐diphosphatase (FDPase) activites were significantly (P < 0.05) increased, whereas alkaline phosphatase (ALP), adenosine triphosphatase (ATPase) and acetyl choline esterase (AchE) activities were significantly (P < 0.05) reduced in higher levan‐fed groups. RBC, haemoglobin and WBC counts were significantly (P < 0.05) increased in the levan‐fed groups. Similar trends were also observed for the total serum protein, globulin, NBT and lysozyme activities. Blood glucose and serum cortisol exhibited a third order polynomial relationship with increasing level of dietary levan. Overall result showed stress ameliorating, immunostimulating and protective role of microbial levan against fipronil‐induced stress in C. carpio fry at 0.75% level of dietary levan supplementation.     

Comparison of L‐lysine·HCl and L‐lysine sulphate in the feed of Penaeus monodon and re‐evaluation of dietary lysine requirement for P. monodon

Two trials were conducted to compare L‐lysine HCl and L‐lysine sulphate regarding its availability to Penaeus monodon, and further evaluate the optimum dietary lysine requirement. In experiment 1, five experimental diets were formulated (D1, D2, D3, D4 and D5), a basal diet (D1), aimed at a low‐lysine concentration (2.22% dry matter), with lysine concentration of the other four diets increasing in two 0.25% L‐lysine intervals from either L‐lysine HCl (D2 and D3) or L‐lysine sulphate (D4 and D5). Each diet was fed at a restricted rate to three groups of 40 shrimp for 74 days. The highest values of growth performance (weight gain, WG; specific growth rate, SGR) and survival were observed with shrimp fed the L‐lysine HCl diet. Feed efficiency (FE) of shrimp fed D2 was significantly higher than that of shrimp fed D1 and D5 (P < 0.05), but without significant difference with shrimp fed D3 and D4 (P > 0.05). In experiment 2, six diets (d1, d2, d3, d4, d5 and d6) were formulated with six graded levels of lysine (2.21%, 2.41%, 2.59%, 2.87%, 3.11% and 3.29% of diet). Each diet was randomly assigned to triplicate groups of 40 shrimp for 74 days. WG, SGR and survival increased increasing levels of lysine up to 2.41% of diet and reached an apparent plateau. Broken‐line model analysis on WG and SGR indicated that the optimum dietary lysine level for optimal growth of shrimp was 2.37% of diet, corresponding to 5.78% of dietary protein. In conclusion, results of this trial suggest that L‐lysine HCl is superior to L‐lysine sulphate when fed to Penaeus monodon and optimal growth can be obtained at lysine levels corresponding to 2.37% of diet, or 5.78% of dietary protein in this specie.     

Molecular cloning and characterization of the cathepsin L gene in Pelodiscus sinensis and its expression in response to bacterial challenge

Cathepsin L is one of the most important lysosomal cysteine proteases for the initiation of protein degradation, and it is involved in the immune response in many vertebrates. However, its function in the innate immune system of turtles remains poorly understood. Here, we cloned the cathepsin L gene from the Chinese soft‐shelled turtle Pelodiscus sinensis. We then examined the mRNA expression in different tissues and at different time points after infection by Aeromonas hydrophila or Vibrio parahemolyticus. The full‐length cDNA sequence cloned from P. sinensis was 1,805 bp with a 1,071 bp open reading frame, which encodes a 40.39 kDa polypeptide of 356 amino acids. The deduced protein sequence contained an active triad of Cys, His and Asn, and conserved ERWNIN and GNFD motifs. Homology analysis showed that the deduced amino acid sequence shared 77%–96% identity with other known species. Sequence alignment, phylogenetic analysis and structural comparisons revealed that cathepsin L is a member of the cathepsin family. Real‐time PCR analyses showed that turtle cathepsin L mRNA is ubiquitously expressed in various tissues, and the expression level is higher in the liver than in other tissues. The expression level in the liver peaks 24 hr after A. hydrophila infection and at two times (12 and 72 hr) after V. parahemolyticus infection. These results suggest that the cathepsin L gene of P. sinensis is likely involved in the process of the antibacterial response to A. hydrophila and V. parahemolyticus. This study is of great importance for future work exploring the molecular mechanism of antibacterial immune responses in P. sinensis.     

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.