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An 8-week feeding trial was conducted to evaluate growth performance, whole-body composition, metabolism and digestion of Chinese soft-shelled turtle Pelodiscus sinensis fed with fish meal replacement by rice protein concentrate and squid paste supplementation diets. Three isonitrogenous and isoenergetic practical diets were formulated with the inclusion of 60% fishmeal (CT), 42% fish meal +18% rice protein concentrate (RP) and 42% fish meal +18% rice protein concentrate +1% squid paste (feeding attractant) (RPS), respectively. Microcapsule lysine was supplemented in RP and RPS diets to balance the amino acid profile. Turtles (initial weight 30.65 g) were fed thrice (6:00, 12:00 and 18:00) daily to apparent satiation. The results showed that growth performance in RP group showed no significant difference with that in CT group ( P > .05). In addition, daily squid paste supplementation significantly improved growth performance, nutrients retention and relative feed intake ( P P Pelodiscus sinensis with microcapsule lysine supplementation. Besides, diet with squid paste inclusion could improve feed intake and growth performance of Pelodiscus sinensis .

A 2 × 2 factorial design was conducted to elucidate the effect of dietary biotin on growth performance, body composition, tissue lipid content and lipid metabolism related genes expression of blunt snout bream, Megalobrama amblycephala fed with different dietary lipid levels. Juvenile fish (average initial weight: 36.0 ± 1.0 g) were fed with diets containing two lipid levels (5% and 11%) and two biotin levels (0.06 mg kg − 1 and 2.50 mg kg − 1 ) for 8 weeks, respectively. Specific growth rate (SGR), hepatosomatic index (HSI), mesenteric fat index (MFI), hepatopancreas lipid, total triacylglycerol (TG) and cholesterol (TC) contents, plasma TG and muscle TG contents were significantly ( P P P P P P P

The tuberous sclerosis complex 1 (TSC1) is a critical upstream regulator of autophagy. Nevertheless, the detailed molecular mechanism of TSC1 and its function in crustacean are still not well understood. In the present study, TSC1 was identified and characterized from Penaeus vannamei. Molecular feature analysis revealed that the open reading frame (ORF) of PvTSC1 was 3516 bp encoding a polypeptide of 1172 amino acids with a conserved N-terminal hamartin domain and two coiled-coil region domains in the C-terminus. The homologous alignment showed that the amino acid sequence of PvTSC1 shared 28.17% ~ 59.25% identity with that of TSC1 in other species. The mRNA transcripts of PvTSC1 were found in all detected tissues of shrimp, and PvTSC1 was mainly located in the cytoplasm of hemocytes. Antioxidant enzyme and PvTSC1 were significantly up-regulated in hemocytes, gill and hepatopancreas after high ammonia nitrogen stress. After PvTSC1 silencing, ROS accumulation increased and autophagy production decreased significantly. And the expression level of PvmTOR was increased significantly, on the contrary, autophagy-related genes (PvATG14 and PvBeclin-1) were decreased significantly. Finally, the mortality of shrimp increased significantly in the PvTSC1-silenced shrimp. These results indicated that ammonia nitrogen had a significant effect on oxidative stress injury of shrimp, and autophagy was a remarkable survival mechanism of shrimp with oxidative stress injury regulated by PvTSC1.

Males of the giant freshwater prawn (GFP), Macrobrachium rosenbergii, have been staged histologically into three morphotypes: small male (SM), orange claw (OC), and blue claw (BC). At present, the social population structure is the main limited factor of yield and profitability in commercial GFP production. The aim of this study is to investigate the relationship between the intestinal microbiota and the three male morphotypes of GFP. In this study, we quantified bacterial communities and abdominal muscle transcriptomes by parallel 16S rRNA gene amplicon sequencing and RNA-Seq in three male morphotypes of GFP. The abundance of bacteria varied significantly with three male morphotypes. Moreover, we identified 6062 differentially expressed genes and found a pattern of gene expression involved in developmental stages from the small male via the orange claw to the blue claw morphotype. Eleven signal transduction pathways classified into environmental information processing were related to the frequency of the different male morphotypes in the prawn population. Furthermore, the correlation analysis identified that p53, AMPK, MO25, and ATG1 gene expressions were positively correlated with the abundance of Candidatus Hepatoplasma and negatively correlated with the Lactococcus and Bacillus population in the giant freshwater prawn. We revealed an intimate linkage between the intestinal microbiome and morphotypic differentiation in the males of GFP. This study also provides a more effective method to manipulate the frequency of the different male morphotypes in the male prawn population.

Scientific evidence is increasing that intestinal microbial flora is closely related to organism health and immunity. The relationship between the shrimp midgut microbial community and blue body syndrome is, however, unclear. In this study, we collected midgut and seawater samples from ponds with healthy and diseased shrimp to understand variations in bacterial communities in the midguts of healthy or diseased shrimp from the same habitat. The expression level of penaeidin, lectin, crustins, and defensin in shrimp with blue body syndrome were decreased, compared to those of healthy shrimp, indicating that shrimp with blue body syndrome have a lower autoimmune. 16 s sequencing analysis found that total 37 phyla, 91 classes, 160 orders, 221 families and 373 genera was detected. Among them, Proteobacteria (55.7%), Cyanobacteria (30.5%), Bacteroides (5.4%), Actinomycetes (1.1%) and Trichomes (0.9%) are the core bacteria in the intestinal bacteria of Penaeus vannamei. The intestinal bacterial communities are closely associated with the severity of shrimp disease. This pattern was further evidenced by NMDS and LEfSe analyses that were significantly different in abundance between healthy and diseased shrimp. In addition, we found that within a given bacterial genus, the patterns of enrichment or decrease were inconsistent with the known probiotics functions of those bacteria. These results suggested that the composition disorders and immunity decrease of intestinal microbiota were closely associated with shrimp blue body syndrome. Moreover, the identified bacterial genus can be used as an indicator taxa to evaluate the occurrence probability of blue body syndrome in shrimp.

This study aimed to investigate the effects of dietary carbohydrate/lipid (CHO:L) ratios on immune responses, oxidative status, and liver histology of juvenile Pelteobagrus fulvidraco . Six isonitrogenous and isoenergetic diets were formulated, containing various CHO:L ratios from 1.11 to 9.50. Each diet was tested in four replicates for 8 weeks. The results indicated that the leucocyte count, plasma globulin content and lysozyme activity of fish fed the 5.58 CHO:L diet were significantly ( P P P P P

A full-length cDNA coding lipoprotein lipase (LPL) was cloned from the visceral adipose tissue of blunt snout bream Megalobrama amblycephala by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained covered 2372 bp with an open reading frame of 1524 bp encoding 507 amino acids, including a putative signal peptide of 22 amino acids long. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (63–97%) among most fish and higher vertebrates, retaining the consensus sequence, the polypeptide “lid”, the catalytic triad and eight cysteine residues at the N-terminal region. Then, a 10-week feeding trial was conducted to investigate the effects of dietary lipid levels (4%, 8% and 12%) on the activity and mRNA expression of LPL in M. amblycephala . LPL activities in the visceral adipose tissue and white skeletal muscle both increased significantly ( P P > 0.05) was also observed in that of the liver. LPL expressions in the visceral adipose tissue, liver and white skeletal muscles all increased significantly ( P liver > white skeletal muscle) was also observed. The results indicated that LPL gene of M. amblycephala showed a typical structure of the lipase family, and shared a high similarity with that of the other vertebrates. In addition, an up-regulation of both LPL expressions and activities was observed in various tissues by high dietary lipid levels.

High-fat diets may have favorable effects on growth partly based on protein-sparing, but high-fat diets often lead to excessive fat deposition in the liver of some fish. The goal of the study was therefore to investigate effects of fat-rich diet on mechanisms involved in fat deposition in the liver. Three hundred blunt snout bream (Megalobrama amblycephala) juveniles (initial weight 18.00 ± 0.05 g) were fed with two diets (5% or 15% fat) for 8 weeks. Histological and biochemical parameters were determined. The hepatic triacylglycerol (TG) secretion rate was measured by intraperitoneal injection with poloxamer 407 (a lipoprotein lipase inhibitor). Lipid transport was studied by determining components of lipoproteins. We also examined lipid uptake by assaying lipoprotein lipase (LPL) activities and mRNA expressions in tissues. Our results showed that ultrastructural lesions of organelles in hepatocytes occurred after intake of a high-fat diet. The hepatic TG secretion rate and plasma very low density lipoprotein (VLDL) amount of fish fed high-fat diet were significantly lower compared to those of fish fed control diet. Concomitantly, variations of components of lipoproteins were also apparent. In the liver, muscle and mesenteric fat tissue, the percentage of polyunsaturated fatty acid (PUFA) was significantly increased, while both saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) percentages decreased in high-fat group. Both activities and mRNA expressions of LPL were also increased in these tissues. On the whole, a low level of lipid transport out of the liver, alterations of lipid transport and elevated lipid uptake contributed to fatty liver of blunt snout bream fed high-fat diet.

A study was conducted to investigate the effects of dietary tribasic copper chloride on growth, copper status, antioxidant activities, immune responses and intestinal microflora in blunt snout bream, Megalobrama amblycephala. Eight practical diets containing different levels of copper (0, 3, 6, 9, 25, 50, 100 and 150 mg Cu kg− 1) from tribasic copper chloride were fed to blunt snout bream (31 g of initial average body weight) in quadruplicate groups in outdoor cages. Growth, copper status, antioxidant activities, immune responses and intestinal microflora were analyzed after a 56-day feeding period. Weight gain and specific growth rates in fish fed diets supplemented with 3–6 mg Cu kg− 1 were significantly higher than those in fish fed the control diet (which supplemented with 0 mg Cu kg− 1) (p

The experiment was conducted to compare the effects of dietary tribasic copper chloride (TBCC), copper amino acid complex (Cu–AA) and copper sulfate on performance, copper status and plasma antioxidant activities in crucian carp, Carassius auratus gibelio , fed a fish meal based diet. Two hundred and eighty crucian carps (18.10 ± 0.04 g average body weight) were randomly assigned to 10 treatments. Treatments consisted of 0, 3, 6 or 9 mg supplemental Cu/kg from TBCC, Cu–AA or copper sulfate. Growth performance, copper status and plasma antioxidant activities were analyzed after a 55-day feeding period. Fish fed diets supplemented with copper at levels of 3–6 mg/kg had obviously higher WG and SGR ( p 0.05) and relatively lower FCR ( p > 0.05) than fish fed the basal diet. Whole-body copper concentrations were significantly higher in fish fed diets supplemented with 3–9 mg Cu/kg (independent on copper sources) than fish fed the basal diet ( p 0.05). There was a trend that fish fed diets with Cu–AA had higher liver copper than those fed diets with equal levels of copper from copper sulfate or TBCC ( p > 0.05). Fish consuming diets supplemented with 6 or 9 mg Cu/kg from Cu–AA, or 9 mg Cu/kg from TBCC had significantly higher ceruloplasmin activities than those fed diets with 3 mg Cu/kg from either TBCC or copper sulfate ( p 0.05). GSH-Px activities were obviously higher in fish fed diets supplemented with 6 or 9 mg Cu/kg from copper sulfate than fish fed the basal diet ( p 0.05). Using linear regression and a slope ratio technique, relative bioavailability values (RBVs) of TBCC were 1.18, 1.31 and 1.73, based on plasma ceruloplasmin, plasma copper and liver copper, respectively, and RBV of Cu–AA were 1.04, 1.00 and 1.42, compared with copper sulfate (1.00). This study showed that TBCC is more bioavailable to crucian carp than copper sulfate and could be used as a new copper source.

In this study, the cell size, viability, respiratory burst (RB) and mitochondrial membrane potential (MMP) of lipopolysaccharide (LPS)-treated haemocytes fr om Litopenaeus vannamei were measured by flow cytometry, and phenoloxidase (PO) activity of the cell supernatants was also determined. Three concentrations of LPS (1, 3 and 5 µg ml − 1 ) were used to determine the effects of LPS on haemocytes. The results revealed that haemocytes treated by LPS in vitro showed conspicuous dose- and time-dependent decreases in cell size and viability. The addition of trypsin inhibitor (TI) which inhibits serine proteinases further delayed the cell size reduction and cell death caused by LPS, implying that serine proteinases were involved in the cell responses. PO activities of the supernatants from LPS-treated cells were significantly higher than that from the control. These results indicated that cell size reduction caused by LPS appeared to be related to degranulation of semigranular and granular cells to release components of proPO system. RB activities were induced by LPS, but not by PMA. Sustaining and tremendous increase of RB occurred during the course of experiment, suggesting that LPS is a more efficient activator to RB of L. vannamei haemocytes than PMA. Specially, MMP of the LPS-treated haemocytes rose at first and then dropped, and which was significantly lower than that of control after 120 min incubation with LPS. The initial increase in MMP may have a relation to energy supply for cellular processes, and the later decrease of MMP represent cell apoptosis. Decreases on cell viability and MMP may be related to the overfull product of ROS which oxidized mitochondrial membrane and cell constituents. Though LPS stimulated PO activity of L. vannamei , LPS at the concentrations used in the present study is cytotoxic. These facts suggest that LPS should be carefully used as immunomodulator for shrimp.

Ferritin is a storage protein that plays a key role in iron metabolism. In this study, the gene expression of ferritin in hepatopancreas, muscle and eye of white shrimp Litopenaeus vannamei were analysed by quantitative real-time PCR after exposure to pH stress. The results showed the expression level in the hepatopancreas and eye were about 5 times to those of beta-actin, whereas lower expression levels in muscle were about 1/2 to those of beta actin. The gene expressions of ferritin in hepatopancreas and muscle were up regulated at 1.5 h after exposure to pH stress, but down-regulated in eye slightly. The ferritin transcript in hepatopancreas of white shrimp L. vannamei at pH 9.3 and pH 5.6 treatments increased significantly in 1.5 h and 12 h post-challenge respectively, but both returned to the original values in 24h post-challenge. The present result showed that ferritin expression in tissue of L.vannamei was induced by acute pH stress and ferritin induction varied in response to duration of induction and acidic or alkaline pH treatment, and was more sensitive to high pH than low pH.

The copper plus zinc superoxide dismutase (Cu, Zn-SOD) was purified from haemolymph of the Oriental river prawn, Macrobrachium nipponense and partially characterized. Partial protein precipitation in crude extract was affected by using heat treatment and (NH 4 ) 2 SO 4 fractionated precipitation methods. Fractionation of superoxide dismutase was performed by DEAE-cellulose 32 ion-exchange chromatography and followed by CM-cellulose cation-exchange chromatography. The molecular weight of it was about 66.1 kDa, as judged by SDS polyacrylamide gel electrophoresis. The enzyme was sensitive to cyanide and H 2 O 2 , and contained 1.08 ± 0.14 atom of copper and 0.98 ± 0.11 zinc per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Cu, Zn superoxide dismutase. The purified enzyme had an absorption peak of 269 nm in ultraviolet region and the enzyme remained stable at 25–45 °C within 60 min. But it was rapidly inactivated at higher temperature (50 °C). The activity of purified shrimp Cu, Zn-SOD was remained stable over the range pH 5.8–8.3. Treated with 10 mM mercaptoethanol, the enzyme activity significantly increased. However, the enzyme activity was obviously inhibited by 10 mM CaCl 2 , ZnCl 2 , SDS, EDTA–Na 2 and 1 mM and 10 mM K 2 Cr 2 O 7 . The results showed that it might be a kind of EC-SOD. And it was the first report of some characterizations of this EC-SOD in M. nipponense .

This study examined the effects of dietary tocopheryl acetate (vitamin E) and salinity on muscle superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) activity and gill Na+/K+-ATPase activity in Litopenaeus vannamei. Three diets containing 0, 100 and 600 mg tocopheryl acetate kg-1 diet, respectively, were fed to shrimp (40-50 mg) for 35 days. Subsequently, shrimp were subjected to an acute salinity change (30[per thousand] to 5, 15, 30, and 50[per thousand]) for 24 h. Significantly (P 0.05). A similar trend was observed in Na+/K+-ATPase activity of shrimp fed the control diet. Na+/K+-ATPase activity of shrimp fed the vitamin E-supplemented diet showed no significant variation response to acute salinity changes. Supplementation of vitamin E in diet could enhance the resistance of shrimp to acute changes in salinity. However, the higher dose of vitamin E supplement was not significantly different from the moderate dose. The results demonstrated that vitamin E might have a potentially useful role as an effective antioxidant by regulating osmotic balance and resistance to salinity changes in shrimp.

The 24 h, 48 h and 96-h LC 50 of nitrite and the effects on reactive oxygen intermediates (ROIs) in haemocytes, superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GPX) activity, in muscle of Penaeus vannamei fed the basal and higher selenium diet exposed to ambient nitrite were investigated. The results showed that, in prawns that were fed higher selenium diet, there was a significant decrease in the 24 h, 48 h and 96-h LC 50 of nitrite, suggesting increase toxicity of nitrite to prawn when dietary higher selenium was added to feed. A significantly increased ROIs value was found in prawn fed the basal and higher selenium diet ( P P. vannamei , and that supplementation of diets with higher levels of selenium is harmful for prawn and increases toxicity of nitrite to prawn.

This study examined the effects of temperature on growth, adenosine phosphates, ATPase and cellular defense response of juvenile shrimps Macrobrachium nipponense. The results showed that specific growth rates in the temperature range 22–32 °C were significantly (P

The effects of supplemental vitamin C, in the form l -ascorbyl-2-polyphosphate (APP) in enriched live food dietary (Artemia) on reactive oxygen intermediates (ROIs) and free radical scavenging enzymes (such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione transferase) activities in muscle of Penaeus vannamei exposed to ambient ammonia-N were investigated. The results showed ROIs values of shrimps fed the starved and enriched Artemia increased with increased ammonia-N concentration. The ROIs value of shrimps fed the enriched Artemia exposed to increasing ammonia-N concentration were 37.4%, 26.4% and 31.1% lower (P

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme had been isolated, purified and partially characterized from muscle tissue of the shrimp Macrobrachium nipponense. The purification was achieved by heat treatment, ammonium sulfate fractionated precipitation and column chromatograph on DEAE-cellulose 32. Some physiological and biochemical characterization of it was tested. The molecular weight of it was about 21.7 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had an absorption peak of 278 nm in ultraviolet region, and the enzyme remained stable at 25-45 degreesC within 90 min. However, it was rapidly inactivated at higher temperature. Treatment of the enzyme with 1 mM ZnCl2, SDS and 1 mM or 10 mM mercaptoethanol showed some increasing activity. However, the enzyme activity was obviously inhibited by 10 mM CaCl2, CuSO4, ZnCl2 and 1 mM CaCl2 and 10 mM K2Cr2O7. SOD activity did not show significantly variation after incubated with 1 mM CaCl2, EDTA and 10 MM SDS. The enzyme was insensitive to cyanide and contained 1.03 +/- 0.14 atoms of manganese per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Mn superoxide dismutase. (C) 2004 Elsevier B.V. All rights reserved.

Growth, hemolymph osmolality, tissue water, ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) ratio, protein-bound amino acids and free amino acids (FAA) of Macrobrachium nipponense were investigated, after they were acclimated to 0‰, 7‰, 14‰ and 20‰ for 14 days. Growth was significantly (P

The 24-, 48- and 96-h LC50 of nitrite and the effects on reactive oxygen intermediates (ROIs) in haemocytes and muscle, superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GPX) activity, in muscle of Macrobrachium nipponense exposed to ambient nitrite were investigated. The 24-, 48- and 96-h LC50 values of nitrite at pH 8.0 were 46, 26 and 13.3 mg l−1. A significantly increased ROIs value was found in prawn (P

The 96-h LC50 of ammonia-N and the effects of dietary vitamin C on oxygen consumption, ammonia-N excretion and Na+/K+ ATPase activity of Macrobrachium nipponense exposed to ambient ammonia were investigated. The results showed that the 96-h LC50 of ammonia-N was 36.6 mg l−1 for the freshwater prawn, M. nipponense, at pH 8.0. When prawns were exposed to high ambient ammonia-N concentrations, the oxygen consumption rate increased and ammonia excretion decreased. Dietary vitamin C supplementation led to higher oxygen consumption and lower ammonia excretion. Na+/K+ ATPase activity increased with increased ambient ammonia-N exposure in the range of 0–18.3 mg l−1, and then was reduced at ambient ammonia-N 36.6 mg l−1. Na+/K+ ATPase activities of prawns fed a vitamin C-supplemented diet were significantly lower than those of prawns fed a diet which was not supplemented with vitamin C.

Interferon is an important cytokine in the host defense against infection by viral and microbial pathogens. We determined quantitatively the antibacterial activity of the virus-resistant shrimp (Marsupenaeus japonicus) recombinant protein IntIP against three Gram-negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio vulnificus, using a turbidmetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp protein IntIP against Vibrio sp is at least equal to the values against the Gram-positive Micrococus luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus. (C) 2008 Elsevier B.V. All rights reserved.