Effects of acute hyperglycaemia stress on indices of glucose metabolism and glucose transporter genes expression in cobia (Rachycentron canadum)

The objective of the present study is to preliminarily clarify the mechanism of carbohydrates metabolism in cobia (Rachycentron canadum) (85 ± 3 g) receiving injection of glucose solution. We examined plasma glucose (GLU), total protein (TP), triglyceride (TG), cholesterol (CHOL), insulin, liver glycogen and muscle glycogen, activities of hepatic hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), as well as relative expressions of glucose transporter 1 (GLUT1), GLUT2, GLUT3, GLUT4, GLUT5 and GLUT9 in hemocyte, liver and muscle of R. canadum when fish were injected with 200 μl of glucose solution (255 mg/ml) after 0, 1, 2, 4, 8, 12, 24 and 48 hr. Fish received injection of 0.68% saline served as control. Results indicated that the plasma GLU, TG and CHOL increased and reached peak at 1, 8 and 48 hr postinjection (hpi) respectively. The hepatic glycogen increased from 1 hpi, and reached peak at 8 hpi, plasma insulin increased at 1 hpi, and reached peak at 2 hpi, and activity of hepatic PK peaked at 8 hpi. Furthermore, the relative expressions of GLUT1, GLUT2, GLUT3, GLUT4 and GLUT5 in hemocytes reached peak at 1,4, 8, 4 and 8 hpi, respectively, relative expressions of GLUT2, GLUT3, GLUT5 and GLUT9 in liver reached peak at 24, 24, 12 and 24 hpi, respectively, and relative expressions of GLUT1 and GLUT3 in muscle were significantly higher at 2 and 2–4 hpi, respectively compared with those in controls. In conclusion, low ability of utilizing glucose in R. canadum may be attributed to insufficient insulin secretion, low activities of key glycolytic enzymes (HK, PFK and PK) regulated by glucose injection and slow increase of GLUTs.     

Changes in activities and mRNA expression of lipoprotein lipase and fatty acid synthetase in large yellow croaker,Larimichthys crocea (Richardson), during fasting

Over‐winter fasting and man‐made food deprivation to increase meat quality are common in the process of large yellow croaker (Larimichthys crocea) aquaculture. This study aimed to determine the changes in lipoprotein lipase (LPL) and fatty acid synthetase (FAS) activities and mRNA expressions, and the relationships between these changes and fat content in large yellow croaker liver and muscle tissues during fasting. A total of 2933 bp LPL cDNA, including an open reading frame of 1533 bp encoding 510 amino acids, and a 1233 bp fragment of FAS cDNA coding 411 amino acids were cloned. Expressions of both genes were ubiquitous. During a 35‐day fasting period, the hepatosomatic index and fat content in muscle and liver were significantly decreased (P < 0.05). mRNA levels of LPL increased significantly during the fasting period except the first 3 days, and FAS mRNA levels decreased significantly in both muscle and liver (P < 0.05) although there were some fluctuations. Muscle and liver LPL activities were significantly higher following fasting for 7 days, decreased to the initial value following fasting for 14 days, and elevating significantly afterwards (P < 0.05). FAS activities in muscle and liver maintained a significantly decreasing trend during the short‐term fasting (P < 0.05) and kept obviously rising thereafter (P < 0.05). Activities and mRNA levels of both LPL and FAS were not always consistent, which implied that both pre‐translational and post‐translational regulations existed during fasting. Our results suggest that the reasonable fasting time is 21 days before harvesting when fat content decreased significantly.     

Multilocus sequence analysis of Vibrionaceae isolated from farmed amberjack and the development of a multiplex PCR assay for the detection of pathogenic species

Since 2011, high mortality rates and symptoms consistent with vibriosis have been observed in farmed amberjack (Seriola dumerili) in Japan. To identify 41 strains isolated from diseased amberjack, a multilocus sequence analysis using nine concatenated genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) was conducted. Twenty‐seven strains were identified as Vibrio harveyi, suggesting an epidemic of V. harveyi infection in amberjack farms. Other strains were identified as Vibrio anguillarum, Vibrio owensii and Photobacterium damselae subsp. damselae. To develop an efficient diagnostic method for vibriosis in amberjack, a multiplex PCR system was developed to identify V. anguillarum, V. harveyi and P. damselae subsp. damselae. The method successfully discriminated between these three bacterial species, with amplification products of 350 bp for V. anguillarum, 545 bp for V. harveyi and 887 bp for P. damselae subsp. damselae and can be used for diagnosis in aquaculture farms.     

Effects of dietary tea polyphenols on growth,immunity and lipid metabolism of juvenile black carp Mylopharyngodon piceus

An 8‐week feeding experiment was aimed to investigate the effect of dietary tea polyphenols (TP) on growth, immunity and lipid metabolism in juvenile black carp Mylopharyngodon piceus (initial weight 5.90 ± 0.03 g). Tea polyphenols were added at different levels (0, 25, 50, 100 and 500 mg/kg; TP₀, TP₂₅, TP₅₀, TP₁₀₀ and TP₅₀₀). The results are as follows: the highest specific growth rate (SGR) and condition factor (CF) and activity of trypsin (TRS) of intestine in TP₅₀, but SGR and activities of lipase (LPS)and TRS of intestine and content of whole body crude protein in TP₅₀₀ were remarkably lower than TP₀. Compared with TP₀, content of serum superoxide dismutase (SOD) and glutamic oxalacetic transaminase (GOT) remarkably increased (p < .05), but contents of glutathione (GSH), glutathione peroxidase (GSH‐Pox), malondialdehyde (MDA), triglyceride (TG) and low‐density lipoprotein cholesterol (LDL‐C) were significantly decreased (p < .05), content of cortisol was remarkably lower in TP₅₀ and TP₁₀₀ (p < .05), expression of growth hormone (GH) and melanocortin 4 receptor (MC4R) in liver and GH in muscle were remarkably up‐regulated in TP₅₀, but expression of apolipoprotein A‐1 (ApoA1), GH and MC4R of intestine, ApoA1 of liver and MC4R of muscle in TP₅₀₀ were remarkably down‐regulated, contents of complement 3 (C3), complement 4 (C4), high‐density lipoprotein cholesterol (HDL‐C) and LDL‐C were significantly reduced in TP₅₀₀ (p < .05). In conclusion, TP could improve growth performance and oxidative capacity on juvenile black carp, and its optimal dosage was 50 mg/kg.     

Effects of dietary zinc level on performance,zinc status,tissue composition and enzyme activities of juvenile Siberian sturgeon,Acipenser baerii (Brandt 1869)

This study conducted to evaluate the effect of dietary zinc (Zn) levels on feed utilization, tissue Zn composition and serum enzyme activities of juvenile Siberian sturgeon, Acipenser baerii. Five isoenergetic and isonitrogenous semi‐purified diets were formulated with increasing Zn sulphate (ZnSO₄.5H₂O) level to provide the actual Zn values of 14.7 (control), 20.8, 27.3, 37.7 and 46.4 mg/kg diet. Each diet was assigned to three groups of 20 experimental Siberian sturgeons with uniform size (initial weight of 26.52 ± 0.94 g) for a period of 8‐week feeding trial. Results showed that growth performance and muscle protein content were significantly increased with increasing dietary Zn level up to 27.3 mg/kg (p < .05), beyond which they remained significantly unchanged (p > .05). Muscle lipid content significantly declined with increasing dietary Zn level. While muscle and serum Zn contents were not significantly changed among treatments (p > .05), liver Zn content tended to rise with increasing dietary Zn supplementation. Alkaline phosphatase, superoxide dismutase and glutathione peroxidase activities were also raised with increasing dietary Zn level. The adequate amount of dietary Zn requirements for the Siberian sturgeon was estimated to be 28.24 mg/kg based on the relative growth rate and 34.60 mg/kg based on the liver Zn content.     

Synergistic effect of sodium selenite and Pediococcus acidilactici on growth,intestinal bacterial counts,selenium bioavailability,hepatic enzymes and non‐specific immune response in rainbow trout (Oncorhynchus mykiss)

Lactic acid bacteria (LAB) have a crucial role in inorganic selenium metabolism as well as their known desirable effects on fish. In this study, the synergistic effects of dietary sodium selenite and Pediococcus acidilactici on growth performance, intestinal bacterial counts, selenium bioavailability, hepatic antioxidant enzyme thioredoxin reductase activity and hepatic glycolytic enzyme activity that is hexokinase, phosphofructokinase and pyruvate kinase, and non‐specific immune response such as serum lysozyme and complements C₃, C₄ and ACH₅₀ activity in rainbow trout (Oncorhynchus mykiss) were investigated. Thus, a 3 × 3 factorial design experiment was conducted with nine purified diets including three levels of sodium selenite (0, 1 and 2 mg/kg) and three levels of P. acidilactici (0, 7 and 9 log CFU/g). After 8 weeks of feeding, weight gain and specific growth rate were increased by increasing dietary sodium selenite and P. acidilactici levels compared to control (p < .05), whereas feed conversion ratio and condition factor was decreased by increasing dietary sodium selenite and P. acidilactici amounts in comparison with control (p < .05). Survival rate was not significantly affected among the experimental treatments (p > .05). Total cultivable bacterial populations after 4 and 8 weeks of the feeding trial were not significantly different among the dietary treatments, while LAB levels were higher in P. acidilactici‐fed groups than in control and selenium‐fed groups (p < .05). Selenocysteine, methylselenocysteine and selenomethionine levels in the intestine of rainbow trout were increased by increasing the sodium selenite and P. acidilactici levels (p < .05), and selenocysteine was found the most selenium species in the trout intestine. The quantity of total selenium in the whole body, intestine, blood, liver and muscle of rainbow trout were increased by increasing the amounts of sodium selenite and P. acidilactici compared to control (p < .05). Hepatic thioredoxin reductase and hexokinase activity were increased by increasing dietary selenium and P. acidilactici levels in comparison to control (p < .05), whereas phosphofructokinase and pyruvate kinase activity in the liver of rainbow trout were not significantly different between the dietary treatments. Serum lysozyme, complements C₃, C₄ and ACH₅₀ activity were enhanced by increasing dietary selenium and P. acidilactici levels compared to control (p < .05). The most synergistic effects of dietary supplements on growth and metabolism of rainbow trout were obtained at 2 mg/kg sodium selenite and 7 log CFU/g P. acidilactici. The findings revealed the synergistic effect of dietary selenium and P. acidilactici on growth and metabolism in rainbow trout (O. mykiss).     

Effects of dietary docosahexaenoic acid on growth performance,fatty acid profile and lipogenesis of blunt snout bream (Megalobrama amblycephala)

Six diets were designed to investigate the effects of dietary docosahexaenoic acid (22:6n‐3; DHA) levels (0.5, 1.3, 2.3, 4.2, 8.1 and 15.9 g/kg diets) on growth performance, fatty acid profile and expression of some lipogenesis‐related genes of blunt snout bream (Megalobrama amblycephala). Fish (average weight: 26.40 ± 0.11 g) were randomly fed one of six diets for 8 weeks. Results indicated that the final body weight (FBW) and specific growth rate (SGR) of fish fed 1.3 g/kg DHA were significantly higher than other groups except for the 2.3 g/kg DHA (p < .05). Compared with other groups, the number of lipid droplet clusters of the liver stained with oil red O in the 2.3 g/kg DHA group was the highest, which was consistent with the lipid contents of whole body and liver. The DHA proportion in liver and muscle significantly increased with the increasing dietary DHA levels (p < .05), which reflected fatty acid profiles of diets. The highest mRNA expressions of acetyl‐CoA carboxylase α (ACCα), fatty acid synthase (FAS) and sterol regulatory element‐binding protein‐1 (SREBP‐1) occurred in the 1.3 g/kg DHA group, followed by 2.3 g/kg DHA. In summary, the supplementation of 1.3–2.3 g/kg DHA could improve growth performance and lipogenesis, and the dietary DHA could improve DHA and PUFA proportion in liver and muscle.     

Bacillus subtilis CK3 used as an aquatic additive probiotics enhanced the immune response of crayfish Procambarus clarkii against newly identified Aeromonas veronii pathogen

To identify the aetiology of the disease outbreak in cultured Procambarus clarkii, a dominant bacteria strain from the diseased crayfish hepatopancreas was isolated. It was identified as Aeromonas veronii based on biochemical identification and 16S rDNA sequencing. The symptoms of artificially infected crayfish were similar to those of naturally infected P. clarkii. The A. veronii was sensitive to norfloxacin, ciprofloxacin, ceftazidime, cefuroxime and ceftriaxone antibiotics, while resistant to penicillin, ampicillin, oxacillin and piperacillin. Virulence genes such as the enterotoxin genes (act, alt and ast) and haemolytic toxin genes (hlyA and aerA) were detected. After A. veronii infection, the connections between some liver tubules disappeared, the vacuoles appeared in the brush border and the mitochondria were enlarged. The antagonistic action of previous identified B. subtilis CK3 against A. veronii was also detected. The supernatant of B. subtilis CK3 exhibits a significant bactericidal effect on A. veronii. After B. subtilis CK3 immersion, the antioxidant enzymes and immune-related enzyme activities in hepatopancreas were significantly higher than control. The accumulative mortality caused by infection of A. veronii can be significantly reduced by adding B. subtilis CK3 into the aquatic water. These results demonstrated that B. subtilis CK3 could act as a water additive to improve the immune response of P. clarkii against newly identified A. veronii. The present study will provide a new way for the prevention and control of crayfish bacterial diseases and also provide technical support for the healthy cultivation of P. clarkii.     

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.     

Effects of dietary Leucas aspera levels on growth performance,nutrient utilization,digestive enzymes and physio-metabolic and health status of bagrid catfish,Rita rita (Hamilton, 1822)

The study was to examine the effects of dietary L. aspera on growth performance, physio-metabolic response and health status of Rita rita fish reared in tanks for 60 days. Six experimental diets were formulated containing 0 g/kg, 3 g/kg, 6 g/kg, 9 g/kg and 12 g/kg L. aspera and designated as D0, D3, D6, D9 and D12 respectively. The inclusion of L. aspera at graded levels resulted in significantly varying effects on growth performance where the weight gain (%), specific growth rate (SGR) and feed conversion efficiency (FCE) were significantly higher (p<0.05) in D6 and D9 groups than others. The protease and amylase activities were elevated in L. aspera supplemented groups than the control group (p<0.05), whereas lactate dehydrogenase, malate dehydrogenase and serum transaminase enzyme activities were significantly reduced with the increasing level of L. aspera and comparatively higher activities of the respective enzymes were found in the control group. The activities of oxidative stress enzymes were significantly higher (p<0.05) in the D9 group and lower in control. The blood glucose level significantly decreased with the increasing level of L. aspera in the experimental diets. The L. aspera supplemented fish had significantly higher liver glycogen, serum protein, albumin and globulin than control group. Red blood cell count was significantly higher (p<0.05) in D6-D12, whereas white blood cell count and haemoglobin content were significantly higher (p<0.05) in D6 and D9 groups. Regression analysis showed that L. aspera at the level of 7.3–7.9 g/kg is optimum for better growth and feed efficiency.     

Growth performance,muscle biochemical constituents,amino acid and fatty acid compositions of the giant freshwater prawn,Macrobrachium rosenbergii,fed with herb‐incorporated diet

This study was conducted to evaluate the influence of herbs such as Ocimum sanctum, Phyllanthus amarus and Solanum trilobatum on the freshwater prawn, Macrobrachium rosenbergii, postlarvae (PL). These herbs were powdered and incorporated at 5% with the basal diet separately and in a combination. The M. rosenbergii was fed with these feeds for a period of 90 days. Results indicated that significant (P < 0.05) improvements were observed in the survival, nutritional indices (weight gain, specific growth rate and protein efficiency ratio), proximate muscle biochemical constituents (total protein, amino acid, carbohydrate and lipid), profiles of essential amino acids and fatty acids of prawns fed with herb‐incorporated feeds. Among these herbs supplemented, P. amarus produced significantly (P < 0.05) better performance followed by S. trilobatum and O. sanctum. However, prawns fed with these herbs combination showed an insignificant (P > 0.05) improvement in survival and growth performance when compared to individual supplementation. Hence, this study suggests that the herbs (O. sanctum, P. amarus and S. trilobatum) can be used as a supplementary feed for a sustainable development of freshwater prawn culture.     

Replacement of fish oil with a DHA‐rich Schizochytrium meal on growth performance,activities of digestive enzyme and fatty acid profile of Pacific white shrimp (Litopenaeus vannamei) larvae

This trial was conducted to evaluate the effects of replacing dietary fish oil with Schizochytrium meal for Pacific white shrimp (Litopenaeus vannamei) larvae (initial body weight 4.21 ± 0.10 mg). Six test microdiets were formulated using Schizochytrium meal to replace 0 g/kg, 250 g/kg, 500 g/kg, 750 g/kg, 1000 g/kg or 1500 g/kg fish oil DHA. No significant differences were observed in survival, growth, final body length and activities of digestive enzyme among shrimp fed different diets (p > .05). No significant differences were observed in C20:5n‐3 (EPA) in muscle samples (p > .05). C18:3n‐3 and C20:4n‐6 in muscle increased as Schizochytrium meal replacement level increased (p < .05). No significant differences were observed in C22:6n‐3 (DHA) and n‐3 fatty acids among shrimp fed diets that algae meal replaced 0 g/kg ‐ 1000 g/kg of fish oil. Shrimp fed diet R150 had higher DHA content than other groups and had higher n‐3 fatty acids than that of shrimp fed diets R50, R75 and R100 (p < .05). C18:2n‐6, PUFA and n‐6 fatty acids in muscle increased, while n‐3/n‐6 ratio decreased with increasing algae meal replacement level from 0 g/kg to 1000 g/kg (p < .05). In conclusion, Schizochytrium meal could replace 1500 g/kg fish oil DHA in the microdiets without negatively affecting shrimp larvae survival, growth and activities of digestive enzyme.     

Outbreaks of Streptococcosis associated with Streptococcus iniae in Siberian sturgeon (Acipenser baerii) in China

Streptococcus iniae has emerged as an important fish pathogen over the past few decades causing high losses in aquaculture farms all over the world. At least 27 species of fish have been documented to be infected by S. iniae, including cultured and wild populations. In August and October 2013, a serious infectious disease characterized by body ulcer, internal organs haemorrhages and nodules showing on epicardium occurred on the Acipenser baerii farms in Ya'an country, China. Histological examination revealed a multisystemic, necrotising inflammatory response that was particularly marked in liver, kidney, heart and brain. Mass mortality (>40%) was observed in infected fish and two Gram‐positive cocci (Ab130920 and Ab131025) were obtained from kidneys and livers of diseased fish. Experimental infections with these two isolates resulted in marked symptoms in the sturgeons similar to those observed in natural outbreaks, and the LD₅₀ values of the two isolates were 5.1 × 10⁵ and 6.4 × 10⁵ cfu per fish respectively. The two microorganisms were identified as S. iniae through physiological and biochemical tests, 16S rRNA and lctO gene sequence analysis. Both two isolates showed a similar antibiotic susceptibility, which were sensitive to ampicillin, amoxicillin, cefazolin, amikacin, deoxycycline, florfenicol, azithromycin, ciprofloxacin, vancomycin and resistant to streptomycin, gentamicin, kanamycin, norfloxacin and sinomin (SMZ/TMP). Multiplex PCR assay for virulence genes showed both isolates possessed six main virulence genes: simA, scpI, pdi, pgm, cpsD and sagA genes. These results indicated that S. iniae could act as a pathogen of farmed A. baerii. This is the first report of S. iniae infection associated with mass mortality in A. baerii.     

Effects of dietary Bacillus natto supplementation on growth performance and the growth‐related gene/microRNA expression in the skeletal muscle of grass carp (Ctenopharyngodon idella)

In this study, Bacillus natto NT was evaluated for use as a probiotic supplement in the feeds on the growth performance and the growth‐related genes' and microRNAs' (miRNAs) expression in the skeletal muscle of grass carp (Ctenopharyngodon idella). Grass carps (ave. wt. 43.96 ± 0.27 g) were fed diets supplemented with 1.87 × 10⁹ (Bn1), 3.73 × 10⁹ (Bn2), 5.60 × 10⁹ (Bn3), 7.47 × 10⁹ (Bn4) and 9.33 × 10⁹ (Bn5) B. natto NT cells per 100 g feed for 56 days. The control group (BnC) was not supplemented with the B. natto NT. The fish of Bn3, Bn4 and Bn5 groups displayed better growth performance and lower feed conversion ratio (FCR) than the other groups (P < 0.05). Compared with the fish of BnC group, miR‐1a, miR‐181a, miR‐23a and miR‐206 expressions increased (P < 0.05), and the myostatin and myocyte enhancer factor C (MEF2C) mRNAs were down‐regulated (P < 0.05) in the fish of Bn4 and Bn5 groups. The specific growth rate analysis and apparent expressional regulation of the growth‐related miRNAs and genes stimulated by B. natto suggest the potential application of B. natto in improving the growth performance on the grass carps.