Response of facilitative glucose transporter 1 to salinity stress and dietary carbohydrate nutrition in white shrimp Litopenaeus vannamei

Facilitative glucose transporter 1 (GLUT1) is a transporter protein for glucose transport via the plasma membrane of the cells to provide energy through carbohydrate metabolism. GLUT1 cDNA from Litopenaeus vannamei was obtained and analysed in this study. Full‐length GLUT1 cDNA is 2062 bp long and contained a 1506‐bp ORF encoding a 502 amino acid protein, a 270‐bp 5′UTR and a 284‐bp 3′UTR. When shrimp were under acute low salinity stress, the expression in hepatopancreas, muscle, gill and eyestalk was all up‐regulated at 12 h (P < 0.05) and 96 h (P < 0.05), while the expression in the four tissues was all down‐regulated at 6 h (P < 0.05) and 48 h (P < 0.05) . The expression in the muscle of shrimp at water salinity of 3 was lower than that at water salinity of 30 independent of dietary carbohydrate levels, while expression in hepatopancreas, gill and eyestalk was up‐regulated at 200 and 300 g kg^?1 carbohydrate levels. The expression in all tissues fed glucose was up‐regulated when compared to the expression in shrimp held at a water salinity of 30. This study suggests that GLUT1 is a conserved protein in L. vannamei, and changes in expression due to environmental salinity and dietary carbohydrate level and source.     

Characterization of a γ‐aminobutyrate type A receptor‐associated protein gene,which is involved in the response of Portunus trituberculatus to CO2‐induced ocean acidification

The γ‐aminobutyrate type A receptor‐associated protein (GABARAP) is a ubiquitin‐like modifier implicated in membrane trafficking and fusion events involving the γ‐aminobutyrate type A receptor, autophagy and apoptosis. In this study, the gene encoding GABARAP was cloned from swimming crab Portunus trituberculatus (PtGABARAP) based on the expression sequence tag (EST). The full‐length cDNA of 664 bp includes a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 223 bp with a poly(A) tail, and an open reading frame (ORF) of 354 bp encoding a polypeptide of 117 amino acids with a predicted molecular weight of 13.96 kDa. The deduced amino acid sequence shares high similarity (93%–100%) with GABARAPs from other species and includes a conserved Atg8 domain. In a phylogenetic analysis PtGABARAP clustered with GABARAPs from other species, and more widely with other GABARAP family proteins. The impact of elevated ocean acidification (OA) on P. trituberculatus behaviours was investigated, and real‐time RT‐PCR revealed that PtGABARAP expression was up‐regulated after OA exposure. Ocean acidification also caused crabs anxiety‐like behaviours, like the shoal average speed increase, preference for dark environment (scototaxis) and fast exploration. The results indicated that GABARAP might be involved in the interactions of GABA_A receptors and elevated‐CO₂ seawater.     

Characterization of β‐catenin 1 during the gonad development in the common carp (Cyprinus carpio)

β‐catenin gene is a pivotal gene for gonad development and maintenance of ovarian function in mammals. However, little is known about its expression and function in gonad development of fish. In this study, a complete cDNA (3342 bp) sequence of β‐catenin 1 was cloned from the common carp, Cyprinus carpio, by RACE PCR, which encodes a 780‐amino‐acid protein. Quantitative real‐time PCR demonstrated that β‐catenin 1 mRNA expressions were high in the testis and ovary tissue and the expression increased as the testes developed and the early stage ovaries developed. Western blot results revealed a single immunoreactive band with an estimated molecular weight of 90 kDa in testes. Immunohistochemistry analysis revealed that the β‐catenin 1 protein was concentrated mainly in the cytoplasm of early development stage of oocyte cells and in the cytomembrane of developing and mature sperm cells. 17β‐Ethinylestradiol injecting intraperitoneally into the fish decreased the relative β‐catenin 1 mRNA expression level except 1 μg/g 72 hr and 5 μg/g 48 hr of treatments in the ovary by real‐time PCR. These results suggest, for the first time, that β‐catenin 1 is an essential protein in gonad development and might be involved in ovarian early development of C. carpio.     

Effects of dietary L‐lysine supplementation on growth,body composition and muscle‐growth‐related gene expression with an estimation of lysine requirement of GIFT tilapia

An 8‐week feeding trial was conducted with extruded practical diets containing 320 g/kg of crude protein from plant‐based sources to determine the effects of L‐lysine on growth performances, muscle‐growth‐related gene (myoD, myogenine and myostatin) expression and haemato‐biochemical responses in juvenile genetically improved farmed tilapia (GIFT). Five isonitrogenous and isoenergetic diets were formulated to contain graded levels of lysine (14.3, 16, 17.5, 19 and 20.5 g/kg of diet). Each diet was randomly assigned to triplicate groups of 30 juvenile fish (5.2 ± 0.1 g), which were fed thrice a day (9:00, 13:00 and 17:00 hr). Maximum growth performances were observed in fish fed with lysine at 19 g/kg of diet. There was no significant (p > .05) effect on whole‐body composition and amino acids profile by dietary lysine supplementation. Significant (p < .05) changes were observed in relative expression of muscle‐growth‐related genes namely myoD and myogenine and in plasma metabolites by dietary lysine supplementation. In contrast, the relative expression of myostatin was not affected by dietary lysine supplementation. Broken‐line regression analysis and second‐order polynomial regression analysis of weight gain and N gain against dietary lysine levels showed that the dietary lysine requirement for juvenile GIFT tilapia was 18–18.3 g/kg of diet and 19.3–19.5 g/kg of diet, respectively.     

Glucose transporters in pearl gentian grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂): Molecular cloning,characterization, tissue distribution and their expressions in response to dietary carbohydrate level

The glucose transporters (GLUTs) play vital role in mediating the glucose uptake process, the movement of glucose across plasma membranes. In this study, three GLUTs, GLUT1, GLUT2 and GLUT4 were cloned and characterized form pearl gentian grouper, a hybrid grouper, and their expressions in response to dietary carbohydrate level (8.02%, 11.89% and 16.08%) were investigated after feeding. The full‐length cDNA of GLUT1, GLUT2 and GLUT4 were 2,104, 3,759 and 2,815 bp, respectively, encoding a putative protein of 491, 508 and 505 amino acids respectively. The results of sequence and phylogenetic analysis revealed that grouper GLUTs were highly conserved and clustered together with their corresponding teleost orthologues, rather than mammals. In addition, GLUT1 was ubiquitously expressed in all detected tissues with relative high expressions in heart and brain. GLUT2 is relatively abundant in some certain tissues that release glucose, such as liver and intestine, and GLUT4 was expressed primarily in muscle and eye. The elevated dietary carbohydrate level had no significant difference on the expression of GLUT1 in grouper liver. The expression of GLUT2 in grouper liver was significantly up‐regulated with the increasing dietary carbohydrate from 8.02% to 11.89%, and therefore down‐regulated significantly. Meanwhile, the expression of GLUT4 in grouper muscle increased significantly with increasing dietary carbohydrate. Results of this study indicate that the up‐regulation of GLUTs in fish contribute to maintain glucose equilibrium to some extent when fish were fed with high carbohydrate diets.     

Identification of SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene and their association with precocity of the Chinese mitten crab Eriocheir sinensis

Moult‐inhibiting hormone (MIH), an important regulator of steroidogenesis, inhibits the synthesis of ecdysteroid in Y‐organ (YO) and plays a significant role in the regulation of moulting and post‐embryonic development of crustacean. Because unsuccessful moulting have been widely observed in precocious crabs, we investigated whether genetic variants in the 5′‐flanking region and 3′‐untranslated region (3′‐UTR) of the MIH gene are associated with precocity of the Chinese mitten crab. Thirty individual DNA samples were sequenced to search for SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene. Five SNPs (g.196 T>A, g.230 C>T, g.305 T>C, g.323 C>A and g.372 C>T) in the 5′‐flanking region and 6 SNPs (g.2677 C>T, g.2759 T>A, g.2807 T>C, g.3042 A>G, g.3088 T>G and g.3295 T>G) in the 3′‐UTR of the MIH gene were selected for the individual genotyping in a two‐stage association study. We found that a SNP g.3088 T>G in the 3′‐UTR of MIH gene was consistently associated with precocity of the Chinese mitten crab in stage 1 and stage 2, with a per‐allele OR (Odds Ratio) of 1.469 (95% CI: 1.169–1.844) after two stages combined (P = 0.001). However, no significant associations were observed between the other 10 SNPs and precocity of the Chinese mitten crab. To our best knowledge, this is the first association study between various SNP genotypes and phenotype attributes in Chinese mitten crab. Our findings suggest that the SNP g.3088 T>G may be a candidate marker for effective marker‐assisted selection to decrease the precocity of the Chinese mitten crab in future studies.     

The optimum dietary isoleucine requirement of juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

A six‐week growth trial was conducted to evaluate the optimum dietary isoleucine requirement of juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂). Seven isoenergetic (3,400 kcal/kg of dry matter), isoproteic (496 g/kg of dry matter) and isolipidic (70 g/kg of dry matter) diets were formulated to contain graded Ile levels (7.3, 11.3, 15.7, 19.6, 23.5, 26.8 and 30.8 g/kg, dry‐matter basis). Each experimental diet was fed to triplicate groups of 12 hybrid grouper juveniles (average initial body weight: 6.00 ± 0.01 g/fish). Experimental fish were randomly distributed into 21 glass tanks (L 60 × W 45 × H 50 cm) connected to mechanical and biological water filters as a recycling system. Fish were fed twice daily (08:00 and 16:00) to apparent satiation. After the sampling of the growth trial, the remaining fish in each group were fed their corresponding diets for 2 d and then exposed to 4 mg Cu²⁺ · L^?1 water for 24 hr. Results showed that growth performance and feed utilization were significantly affected by different dietary Ile levels (p < .05). Weight gain percentage (WG%), protein productive value (PPV), protein efficiency ratio (PER) and feed efficiency (FE) were increased as dietary Ile level increased, reaching a peak value at 19.6 g/kg dietary Ile, and thereafter, these four parameters declined as dietary Ile level continued to increase. Daily feed intake (DFI) showed an opposite tendency of variations as FE. The quadratic regression analysis of WG%, PPV, PER and FE against dietary Ile levels indicated that the optimum dietary Ile requirement for hybrid grouper was estimated to be 19.8, 20.8, 19.4 and 19.1 g/kg dry matter, respectively. Among all experimental treatments, fish fed 19.6 g/kg dietary Ile had the highest expression of growth and protein synthesis‐related genes, including growth hormone (GH) in pituitary, insulin‐like growth factor 1 (IGF‐1), growth hormone receptor 1 (GHR1), target of rapamycin (TOR) and S6‐kinase 1 (S6K1) in liver. Gut micromorphology was significantly influenced by dietary Ile levels. After the exposure to 4 mg Cu²⁺ · L^?1 water for 24 hr, fish fed 19.6 g/kg dietary Ile had the highest survival and the best immunologic manifestation among all experimental treatments. Generally, the optimum dietary Ile requirement for maximum growth of hybrid grouper was estimated to be 19.8 g/kg dry matter, corresponding to 39.9 g/kg dietary protein.     

Effects of different dietary lipid sources on growth performance,body composition and lipid metabolism‐related enzymes and genes of juvenile golden pompano,Trachinotus ovatus

Three groups of juvenile golden pompano, Trachinotus ovatus (54.75 ± 0.25 g), were each fed one of three diets containing different lipid sources: fish oil (FO), soybean oil (SO) and lard oil (LO). Fish were reared in sea cages for 8 weeks, and the fish fed the FO diet had significantly higher specific growth rate (SGR) but lower condition factor (CF) than the other treatments. The fatty acid (FA) composition of whole‐body lipids was closely correlated with those in the diets. Although no differences can be found in hepatic fatty acid synthase (fasn) activity, the carnitine palmitoyl transferase 1 (cpt1) activity in fish fed the FO diet was significantly higher compared with other treatments. In addition, the relative gene expression of lipid metabolism‐related enzymes, such as cpt1, fas, apolipoprotein B100 (apoB100), delta‐6 fatty acyl desaturase (fadsd6) and fatty acid‐binding protein 1 (fabp1), was also influenced by the different dietary lipid sources. Serum triglyceride (TG) and glucose content in fish fed the LO and FO diets were significantly higher than those in the SO group. Accordingly, it can be concluded that FO could not be completely replaced by SO or LO in golden pompano diets. The lipid sources of a diet could impose significant influence on body condition factor and hepatic lipid metabolism of golden pompano.     

Influence of 5′‐inosine monophosphate and 5′‐guanosine monophosphate on growth,feed digestibility and activity of digestive enzymes in turbot Scophthalmus maximus

We evaluated the influence of different proportions of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on growth, feed digestibility and activity of digestive enzymes of turbot Scophthalmus maximus. Weight gain and daily feed intake were significantly higher in S. maximus fed with IMP or GMP, in comparison with fish fed with neither IMP nor GMP. The growth of 0.05% IMP + 0.05% GMP group was the best, and the intestinal digestive function was improved. The addition of IMP and GMP to fish diets significantly increased the apparent feed digestibility coefficient of dry matter and protein, as well as intestinal protease activity. The highest intestinal protease activity was observed in fish fed with 1 g/kg IMP. However, the lipase activity in hepatopancreas decreased significantly after addition of nucleic acid. According to our results, the optimal level of dietary IMP is 1 g/kg, which is in line with most of the growth performance and feed digestibility.     

Effect of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on the growth,immunity and muscle composition of turbot,Scophthalmus maximus (Linnaeus, 1758)

We evaluated the effect of different concentrations of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on the growth, immunity and muscle composition of turbot Scophthalmus maximus. Eight diets (containing no IMP or GMP, or 0.5 g/kg IMP, 1.0 g/kg IMP, 2.0 g/kg IMP, 0.5 g/kg GMP, 1.0 g/kg GMP, 2.0 g/kg GMP, or 0.5 g/kg IMP plus 0.5 g/kg GMP) were prepared. A total of 360 fish (average body weight of 105 g) were randomly selected and placed in groups into 24 plastic aquaria (8 treatments × 3 replicates × 15 individuals per plastic aquaria). The tanks were maintained at the temperature of 15 ± 2°C. The experimental diets were fed for 60 days. The specific growth rate (SGR) was significantly higher in S. maximus fed with IMP or GMP compared with fish fed neither IMP nor GMP. The highest SGR was observed in fish fed with 1.0 g/kg IMP. Supplementation with these dietary nucleotides had a positive, but not significant effect on the activity of superoxide dismutase, alkaline phosphatase and acid phosphatase. There was a significant difference in the moisture and crude lipid content of muscle from S. maximus fed the different diets compared with control fish. The highest moisture content was 83.44 for a diet of 0.5 g/kg IMP plus 0.5 g/kg GMP, which was also significantly higher when compared to fish fed alternative diets. The crude lipid content of S. maximus fed diets containing either IMP or GMP was significantly higher than those fed diets without IMP or GMP. Thus, according to these results, the optimal level of dietary IMP is 1.0 g/kg, which correlates with the largest increase in growth performance of S. maximus.     

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.     

A novel 79‐bp insertion/deletion polymorphism in 3′‐flanking region of IGF‐I gene is associated with growth‐related traits in common carp (Cyprinus carpio L.)

Insulin‐like growth factor‐I (IGF‐I) participates in the modulation of the cellular development, differentiation and proliferation in various vertebrate tissues. Therefore, variations in IGF‐I gene are good candidates as growth regulators. In this study, a novel 79‐bp insertion/deletion (I/D) polymorphism was identified in 3′‐flanking region of IGF‐I gene by polymerase chain reaction (PCR) and sequencing approaches in common carp Cyprinus carpio. To gain insight into the association of the 79‐bp I/D polymorphism with growth‐related traits, a total of 747 individuals from Heilongjiang carp Cyprinus cario haematopterus (YL) (n = 263), selective line of German mirror carp Cyprinus carpio L. mirror (JL) (n = 229), and cold‐resistance line of Hebao red carp Cyprinus carpio var. wuyuanensis (HL) (n = 255) were genotyped. The DD genotype of the polymorphism was significantly associated with higher body length (135), body weight (325, 385, and 445 days), net weight (445 days) and lower overwintering body loss rate in JL. In addition, in pooled population (YL+JL+HL), fish with DD genotype also showed significantly higher body weight (445 days) and net weight (445 days) than those with II genotype. Taken together, these results suggest that the novel polymorphism in IGF‐I gene could be considered as a potential genetic marker for selection of growth performance in common carp.