Silencing the fatty acid elongase gene elovl6 induces reprogramming of nutrient metabolism in male Oreochromis niloticus.

Elongation of very long-chain fatty acids protein 6 (ELOVL6) plays a pivotal role in the synthesis of endogenous fatty acids, influencing energy balance and metabolic diseases. The primary objective of this study was to discover the molecular attributes and regulatory roles of ELOVL6 in male Nile tilapia, Oreochromis niloticus. The full-length cDNA of elovl6 was cloned from male Nile tilapia, and was determined to be 2255-bp long, including a 5'-untranslated region of 193 bp, a 3'-untranslated region of 1252 bp, and an open reading frame of 810 bp encoding 269 amino acids. The putative protein had typical features of ELOVL proteins. The transcript levels of elovl6 differed among various tissues and among fish fed with different dietary lipid sources. Knockdown of elovl6 in Nile tilapia using antisense RNA technology resulted in significant alterations in hepatic morphology, long-chain fatty acid synthesis, and fatty acid oxidation, and led to increased fat deposition in the liver and disrupted glucose/lipid metabolism. A comparative transcriptomic analysis (elovl6 knockdown vs. the negative control) identified 5877 differentially expressed genes with significant involvement in key signaling pathways including the peroxisome proliferator-activated receptor signaling pathway, fatty acid degradation, glycolysis/gluconeogenesis, and the insulin signaling pathway, all of which are crucial for lipid and glucose metabolism. qRT-PCR analyses verified the transcript levels of 13 differentially expressed genes within these pathways. Our findings indicate that elovl6 knockdown in male tilapia impedes oleic acid synthesis, culminating in aberrant nutrient metabolism.

Integrated transcriptome and miRNA sequencing analyses reveal that hypoxia stress induces immune and metabolic disorders in gill of genetically improved farmed tilapia (GIFT, Oreochromis niloticus).

The survival and growth of fish are significantly impacted by a hypoxic environment (low dissolved oxygen). In this study, we compared tissue structure, physiological changes, and mRNA/miRNA transcriptome, in gills of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) between the hypoxic group (DO: 0.55 mg/L, HG) and the control group (DO: 5 mg/L, CG). The results showed that the gill filaments in the hypoxic group showed curling, engorgement, and apoptotic cells increased, and that exposure for 96 h resulted in a reduction in the antioxidant capacity. We constructed and sequenced miRNA and mRNA libraries from gill tissues of GIFT at 96 h of hypoxia stress. Between the HG and CG, a total of 14 differentially expressed (DE) miRNAs and 1557 DE genes were obtained. GO and KEGG enrichment showed that DE genes were mainly enriched in immune and metabolic pathways such as natural killer cell mediated cytotoxicity, steroid biosynthesis, primary immunodeficiency, and synthesis and degradation of ketone bodies. Based on the results of mRNA sequencing and screening for miRNA-mRNA pairs, we selected and verified six DE miRNAs and their probable target genes. The sequencing results were consistent with the qRT-PCR validation results. The result showed that under hypoxia stress, the innate immune response was up-regulated, and the adaptive immune response was down-regulated in the gill of GIFT. The synthesis of cholesterol in gill cells is reduced, which is conducive to the absorption of solvent oxygen. These findings offer fresh information about the processes of fish adaptation to hypoxic stress.

Effects of acute heat stress on haemato-biochemical parameters, oxidative resistance ability, and immune responses of hybrid yellow catfish (pelteobagrus fulvidraco × P. vachelli) juveniles.

This study investigated the effect of heat stress on the physiological parameters, oxidation resistance ability and immune responses in juvenile hybrid yellow catfish. Heat stress group exposed to 35 °C and control to 28 °C. Blood and liver were sampled at different hours' post-exposure. Results showed that red blood cell (RBC), white blood cell (WBC) counts, Hemoglobin (HGB) levels and hematocrit (HCT) values increased significantly (P < 0.05) post-exposure to heat stress. This indicates the increase of cell metabolism. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities, total cholesterol (TC), total protein (TP), triglyceride (TG) and glucose increased significantly (P < 0.05) indicating the need to cope with stress and cell damage. Liver TC, TG, COR hormone, C3 complement increased significantly from 24 to 96 h. Heat stress mostly affects the hepatic antioxidant and immune resistance functions, resulting in increments of cortisol levels, lysozyme, superoxide dismutase (SOD), and catalase (CAT) enzyme activities. The increase of Malondialdehyde (MDA), alkaline phosphatase (AKP) indicate stimulation of the immune responses to protect the liver cells from damage. The decrease in Liver TP indicated liver impairment. Decrease in Glycogen content from 6 to 96 h indicated mobilization of more metabolites to cope with increased energy demand. Interestingly, results showed that heat stress trigged costly responses in the experimental fish like accelerated metabolism and deplete energy reserves, which could indirectly affect ability of fish to set up efficient long term defense responses against stress. These results provide insight into prevention and management of stress in juvenile hybrid yellow catfish.

[Effects of Biochar and Straw Returning on Soil Fungal Community Structure Diversity in Cotton Field with Long-term Brackish Water Irrigation].

Brackish water irrigation increases soil salinity and changes the soil environment, which affects the structure and diversity of soil fungi. In this study, the effects of biochar and straw (3.7 t·hm-2 and 6 t·hm-2, respectively) on soil physical and chemical properties and fungal community structure diversity were investigated on the basis of long-term brackish water irrigation. The results showed that compared to the absence of biochar and straw application (control), biochar application significantly increased pH and the contents of total carbon, available potassium, and available phosphorus in soil but significantly decreased the soil conductivity by 20.71%. Straw treatment significantly increased the content of available potassium and phosphorus but significantly decreased the soil bulk density and conductivity by 4.17% and 64.50%, respectively. The biochar and straw treatment showed an increasing trend in the Chao1 index and ACE index of the fungal community but a decreasing trend in the Shannon index and Simpson index. The dominant fungal phyla in the soil were Ascomycota, Mortierellomycota, Basidiomycota, Chytridiomycota, and Glomeromycota. The dominant fungal genera were Chaetomium, Gibberella, Fusarium, Idriella, and Mortierella. Biochar and straw were applied to increase the relative abundance of Ascomycota, Mortierellomycota, Basidiomycota, Glomeromycota, and Chaetomium. However, the relative abundance of Chytridomycota, Gibberella, and Idriella decreased. LEfSe analysis showed that biochar application and straw returning decreased the number of potential biomarkers in fungal communities. RDA results showed that soil fungal community structure was significantly correlated with EC1:5 and TN. Brackish irrigation had adverse effects on soil, in which EC1:5and TN were the main factors driving the change in soil fungal community structure. The soil fungal community adapted to a salt-stress environment through the improvement of soil by biochar and straw.

Knock-down of amh transcription by antisense RNA reduces FSH and increases follicular atresia in female Oreochromis niloticus.

Anti-Müllerian hormone (Amh) plays an important role in regulating gonad development in teleosts. However, little is known about the effects of Amh on follicle development. In this study, we transfected the vector containing antisense RNA fragments of the amh gene to produce Nile tilapia, Oreochromis niloticus, with knocked-down Amh function in vivo. The results confirmed that the antisense RNA effectively inhibited amh transcription and Amh protein expression in female tilapia ovarian tissue. At 180 days of age, compared with control fish, female tilapia with knocked-down Amh function showed significantly increased growth and significantly decreased ovary weight and gonadosomatic index (P < 0.05). Female fish in the control group had ruddy-colored external genitalia, eggs extruded from the abdomen when gently squeezed, and most oocytes were developmental stage V. In contrast, the external genitalia of female fish with knocked-down Amh function did not have the ruddy color, no eggs extruded from the abdomen when squeezed, most oocytes were at developmental stages II and III, and considerable follicular atresia was apparent. At 180 days of age, the transcript levels of amhrII, cyp19a1a, foxl2 and sox9b in ovarian tissue, and the titers of luteinizing hormone, follicle stimulating hormone, and estradiol in the serum, were significantly lower in fish with knocked-down Amh function than in control fish (P < 0.05). We concluded that decreased serum hormone levels and an abnormal AMH signal delayed development and caused follicular degeneration in Nile tilapia with knocked-down Amh function. These findings show that antisense RNA is a feasible approach for gene silencing in fish, and represents an accurate and effective strategy to study gene function.

Role of Astaxanthin as a Stimulator of Ovarian Development in Nile Tilapia (Oreochromis niloticus) and Its Potential Regulatory Mechanism: Ameliorating Oxidative Stress and Apoptosis.

A 60-day feeding experiment was performed to evaluate the effect of dietary astaxanthin on gonad development, the antioxidant system, and its inherent mechanism in female Nile tilapia (Oreochromis niloticus). Fish were fed with diets containing astaxanthin at five levels [0 mg/kg (control), 50 mg/kg, 100 mg/kg, 150 mg/kg, and 200 mg/kg]. At the end of experiment, the group fed with 150 mg/kg astaxanthin showed significantly increased specific growth rate, feed utilization, viscerosomatic index, and hepatosomatic index compared with the control group (P < 0.05). Gonad development was stimulated in the groups fed with 100 mg/kg and 150 mg/kg astaxanthin, and their gonadosomatic index and egg diameter were significantly higher than those of the control group and the group fed with 200 mg/kg astaxanthin. The ovaries of females in the groups fed with 100 mg/kg and 150 mg/kg astaxanthin were fully developed, the eggs were gray-yellow and uniform in size, and a large number of oocytes developed to stages IV and V. The serum levels of 17 ß-estradiol, follicle-stimulating hormone, and luteinizing hormone were significantly higher in the groups fed with 100 mg/kg and 150 mg/kg astaxanthin than in the group fed with 200 mg/kg astaxanthin. Compared with the control and the other groups, the group fed with 150 mg/kg astaxanthin showed significantly higher transcript levels of genes encoding hormone receptors and higher catalase activity in ovarian tissues, lower malondialdehyde content, decreased apoptosis (reduced granulosa cell apoptosis and lower transcript levels of bax and caspase-3), and reduced follicular atresia. Gene ontology analyses revealed that cell division and the cell cycle were enriched with differentially expressed genes in the group fed with 150 mg/kg astaxanthin, compared with the control group. We concluded that dietary astaxanthin at a concentration of 150 mg/kg activates follicle development by inhibiting expression of mapk1 (involved in MAPK signaling) and increasing the expression genes involved in oocyte meiosis (chp2, ppp3ca, map2k1, and smc1a1) and progesterone-mediated oocyte maturation (igf1, plk1, and cdk1). In conclusion, female Nile tilapia fed with 150 mg/kg astaxanthin showed increased growth, reduced oxidative stress in ovarian tissue, lower levels of cell apoptosis, and improved oocyte development.

Transcriptional inhibition of steroidogenic factor 1 in vivo in Oreochromis niloticus increased weight and suppressed gonad development.

Steroidogenic factor 1 (sf1) (officially designated as nuclear receptor subfamily 5 group A member 1 [NR5A1]) is an important regulator of gonad development. Previous studies on sf1 in fish have been limited to cloning and in vitro expression experiments. In this study, we used antisense RNA to down-regulate sf1 transcription and sf1 protein expression. Down-regulation of sf1 resulted in an increase in body weight and inhibition of gonadal development in both males and females with the consequent lower gonadosomatic index compared to fish in the control group. Hematoxylin-eosin staining of the gonads of fish with down-regulated sf1 revealed fewer seminiferous tubules and sperm in the testis of males. In addition, the oocytes were mainly stage II and many of them were atretic follicle. We conducted comparative transcriptome and proteome analyses between the sf1-down-regulated group and the control group. These analyses revealed multiple gene-protein pairs and pathways involved in regulating the observed changes, including 44 and 74 differently expressed genes and proteins in males and females, respectively. The results indicated that dysfunctional retinal metabolism and fatty acid metabolism could be causes of the observed weight gain and gonad abnormalities in sf1-down-regulated fish. These findings demonstrate the feasibility of using antisense RNA for gene editing in fish. This methodology allows the study gene function in species less amenable to gene editing as for example aquaculture species with long life cycles.

Transcriptome profiling reveals differential expression of immune-related genes in gills of hybrid yellow catfish (Tachysurus fulvidraco ♀ × Pseudobagrus vachellii ♂) under hypoxic stress: Potential NLR-mediated immune response.

Fish gills are the primary organ that respond to sudden changes in the dissolved oxygen (DO) level in the aquatic environment. Hypoxic stress impairs the normal function of gill tissues. However, little is known about the mechanisms of the response of yellow catfish gills to hypoxic stress. In this study, we compared transcriptomic and physiological changes in gill tissues of hybrid yellow catfish (Tachysurus fulvidraco ♀ × Pseudobagrus vachellii ♂) between a hypoxia-treated group (DO: 1.5 mg/L) and a control group (DO: 6.5 mg/L). In fish in the hypoxia-treated group, gill filaments underwent adaptive changes, and the number of vacuoles in gill tissues increased. Exposure to hypoxic conditions for 96 h resulted in increased anaerobic metabolism and decreased antioxidant and immune capacity in gill tissues. Transcriptome analyses revealed 1556 differentially expressed genes, including 316 up-regulated and 1240 down-regulated genes, between fish in the hypoxia-treated and control groups. Functional analyses indicated that the main pathway enriched with differentially expressed genes was immune response, followed by energy metabolism and signal transduction. Under hypoxic stress, the transcript levels of genes involved in the NOD-like receptor signaling pathway initially increased rapidly but then decreased over time, suggesting that the NOD-like receptor-mediated immune response plays an essential role in hypoxia tolerance and resistance in hybrid yellow catfish. Our results provide novel insights into which immune-related genes and pathways are activated under hypoxic stress, and reveal details of early adaptation of the immune response and defense mechanisms under hypoxic stress.

Optimal combination of temperature and photoperiod for sex steroid hormone secretion and egg development of Oreochromis niloticus as determined by response surface methodology.

For successful reproduction of farmed fish, it is important to understand the relationship between gonadal development and environmental factors such as temperature and photoperiod. In this study, we determined the effects of temperature (T) and photoperiod (Pp) on serum estradiol-17ß (E2) and progesterone (P) contents, gonadosomatic index (GSI), and oocyte development in female tilapia. We used a central composite experimental design and response surface methodology. The experimental ranges were 18-36 °C for T and 0-24 h for Pp. The results show that the quadratic effects of T and Pp were highly significant for serum E2 and P contents, GSI, and the ratio of stage III to stage II oocytes (P < 0.01), and that the linear effects of T and Pp were also significant for these indicators (P < 0.05). The T × Pp interaction significantly affected serum E2 content (P < 0.05). Serum E2 and P content, GSI, and the ratio of stage III to stage II oocytes increased and then decreased with increasing T or Pp. The best combination of T and Pp for egg development was 28.6 °C/14.29 h. We observed the part of ovarian tissue containing stage V oocytes that are about to be discharged. Shortening the photoperiod or lowering the water temperature delayed the development of ovarian tissue so that most oocytes remained at stage II, and there were many atretic follicles. There were significant positive correlations between female GSI and serum E2, P, and the ratio of stage III to stage II oocytes. The results of this study provide a reference for the regulation of temperature and photoperiod to control broodstock gonadal maturation and hormone-induced broodstock spawning.

Multi-omics analysis reveals the glycolipid metabolism response mechanism in the liver of genetically improved farmed Tilapia (GIFT, Oreochromis niloticus) under hypoxia stress.

BACKGROUND: Dissolved oxygen (DO) in the water is a vital abiotic factor in aquatic animal farming. A hypoxic environment affects the growth, metabolism, and immune system of fish. Glycolipid metabolism is a vital energy pathway under acute hypoxic stress, and it plays a significant role in the adaptation of fish to stressful environments. In this study, we used multi-omics integrative analyses to explore the mechanisms of hypoxia adaptation in Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus). RESULTS: The 96 h median lethal hypoxia (96 h-LH50) for GIFT was determined by linear interpolation. We established control (DO: 5.00 mg/L) groups (CG) and hypoxic stress (96 h-LH50: 0.55 mg/L) groups (HG) and extracted liver tissues for high-throughput transcriptome and metabolome sequencing. A total of 581 differentially expressed (DE) genes and 93 DE metabolites were detected between the CG and the HG. Combined analyses of the transcriptome and metabolome revealed that glycolysis/gluconeogenesis and the insulin signaling pathway were down-regulated, the pentose phosphate pathway was activated, and the biosynthesis of unsaturated fatty acids and fatty acid metabolism were up-regulated in GIFT under hypoxia stress. CONCLUSIONS: The results show that lipid metabolism became the primary pathway in GIFT under acute hypoxia stress. Our findings reveal the changes in metabolites and gene expression that occur under hypoxia stress, and shed light on the regulatory pathways that function under such conditions. Ultimately, this information will be useful to devise strategies to decrease the damage caused by hypoxia stress in farmed fish.

Growth performance, physiological parameters, and transcript levels of lipid metabolism-related genes in hybrid yellow catfish (Tachysurus fulvidraco ♀ × Pseudobagrus vachellii ♂) fed diets containing Siberian ginseng.

In high-density aquaculture, fish health can suffer because of excessive feeding, which causes fatty liver disease. Siberian ginseng (Acanthopanax senticosus) has been used as a feed additive to promote animal growth, immunity, and lipid metabolism. In this study, we explored the effects of A. senticosus on the physiology of hybrid yellow catfish (Tachysurus fulvidraco ♀ × Pseudobagrus vachellii ♂). A control group and five groups fed diets containing A. senticosus (0.5, 1, 2, 4, and 8 g A. senticosus/kg feed) were established and maintained for 8 weeks. Dietary supplementation with A. senticosus at 4 g/kg promoted growth of the hybrid yellow catfish. Serum total cholesterol (TC) and triacylglycerol (TG) levels at 2 g/kg A. senticosus (TC: 1.31 mmol/L; TG: 1.08 mmol/L) were significantly lower than in the control group (TC: 1.51 mmol/L; TG: 1.41 mmol/L), and 4 g/kg A. senticosus (17.20 µmol/g tissue) reduced the liver TG level compared with the control group (21.36 µmol/g tissue) (P <0.05). Comparative transcriptomic analysis of liver tissue between the control group and the group showing optimum growth (4 g/kg A. senticosus) revealed 820 differentially expressed genes and 44 significantly enriched pathways, especially lipid metabolism pathways such as unsaturated fatty acid and fatty acid metabolism. The transcript levels of five lipid metabolism-related genes were determined by quantitative real-time PCR. The results showed that 2-4 g/kg A. senticosus supplementation reduced the FADS2, ELOVL2, CYP24a, and PLPP3 transcript levels and 4 g/kg A. senticosus increased the DIO2 transcript level (P <0.05), leading to altered synthesis of TG and thyroxine and reduced fat deposition in the liver. Our results show that dietary A. senticosus affects the regulation of fat metabolism and promotes the growth of hybrid yellow catfish. A. senticosus is a healthy feed additive, and the appropriate dietary supplementation rate is 2-4 g/kg.

Hypoxia-induced miR-92a regulates p53 signaling pathway and apoptosis by targeting calcium-sensing receptor in genetically improved farmed tilapia (Oreochromis niloticus).

miR-92a miRNAs are immune molecules that regulate apoptosis (programmed cell death) during the immune response. Apoptosis helps to maintain the dynamic balance in tissues of fish under hypoxia stress. The aim of this study was to explore the role and potential mechanisms of miR-92a in the liver of tilapia under hypoxia stress. We first confirmed that CaSR (encoding a calcium-sensing receptor) is a target gene of miR-92a in genetically improved farmed tilapia (GIFT) using luciferase reporter gene assays. In GIFT under hypoxia stress, miR-92a was up-regulated and CaSR was down-regulated in a time-dependent manner. Knocked-down CaSR expression led to inhibited expression of p53, TP53INP1, and caspase-3/8, reduced the proportion of apoptotic hepatocytes, and decreased the activity of calcium ions induced by hypoxia in hepatocytes. GIFT injected in the tail vein with an miR-92a agomir showed up-regulation of miR-92a and down-regulation of CaSR, p53, TP53INP1, and caspase-3/8 genes in the liver, resulting in lower serum aspartate aminotransferase and alanine aminotransferase activities under hypoxia stress. These findings suggest that stimulation of miR-92a interferes with hypoxia-induced apoptosis in hepatocytes of GIFT by targeting CaSR, thereby alleviating liver damage. These results provide new insights into the adaptation mechanisms of GIFT to hypoxia stress.

Cloning of the gene encoding acyl-CoA thioesterase 11 and its functional characterization in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) under heat stress.

Members of the ACOT (acyl-CoA thioesterase) family hydrolyze fatty acyl-CoA to form free fatty acids (FFAs) and coenzyme A (CoA). These enzymes play important roles in fatty acid metabolism. Here, we report the cloning and functional analysis of acot11ß in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂). The open reading frame of acot11ß was found to be 594 bp in length, encoding 198 amino acids. We determined the transcript levels of acot11ß in ten tissues of hybrid yellow catfish by qRT-PCR and found that it was highly expressed in the liver, so we chose the liver for further analysis. We determined the transcript levels of acot11ß in hybrid yellow catfish under heat stress conditions, and analyzed the changes in serum biochemical parameters, liver biochemical parameters, and transcript levels of lipid metabolism-related genes. Healthy yellow catfish were subjected to heat stress at 35 °C for 96 h, and the experimental results were compared with those from fish in a control group (28 °C). The levels of glucose (GLU), total cholesterol (TC), and triglyceride (TG) in serum were significantly increased in the heat-stressed group compared with the control group (P < 0.05). Acute heat stress led to decreased liver glycogen contents, but significantly increased TC and TG contents in the liver (P < 0.05). The transcript levels of acot11ß, acc, and fas were significantly reduced, while that of pparα was significantly increased in hybrid yellow catfish exposed to heat stress (P < 0.05). Our results indicate that acot11ß plays an important role in regulating lipid metabolism in hybrid yellow catfish, and this metabolic process is greatly affected by temperature. These results may be useful for developing effective strategies to prevent or reduce metabolic disorders of yellow catfish caused by high temperature.

Selenium-Cultured Potamogeton maackianus in the Diet Can Alleviate Oxidative Stress and Immune Suppression in Chinese Mitten Crab (Eriocheir sinensis) Under Copper Exposure.

Selenium (Se) is an essential trace element for aquatic animals. The aquatic plant Potamogeton maackianus is an important natural food of Chinese mitten crab (Eriocheir sinensis). The aim of this study was to determine whether the antioxidant and immune responses of Chinese mitten crab are affected by including Se-cultured P. maackianus in the diet. Three groups of P. maackianus were cultured at levels of 0.02 mg/kg Se, 8.83 mg/kg Se, and 16.92 mg/kg Se, and the plants in these groups were used in experimental diets fed to crabs (dietary Se content of 0.05, 0.43, and 0.82 mg/kg, respectively). Compared with crabs in the 0.05 mg/kg group, those in the 0.82 mg/kg group showed significantly increased specific growth rate, protease and lipase activities, triglyceride and cholesterol contents, and Se content in the hepatopancreas and muscle (P < 0.05); increased activities of glutathione peroxidase, glutathione reductase, and catalase in the antioxidant system; increased transcript levels of MT (encoding metallothionein); and decreased malondialdehyde content (P < 0.05). At the end of the rearing experiment, the crabs in the different groups were exposed to copper (Cu2+) stress for 96 h. All the juvenile crabs in the 0.43 and 0.82 mg/kg groups survived 96 h of Cu2+ stress. Crabs in the 0.82 mg/kg group showed enhanced antioxidant responses under Cu2+ stress, increased transcript levels of MT and LYZ, and increased resistance. Therefore, supplementation of the diet of Chinese mitten crab with increased levels of Se-cultured P. maackianus can reduce oxidative stress under Cu2+ exposure, activate the immune response, and benefit growth.

miR-34a Regulates the Activity of HIF-1a and P53 Signaling Pathways by Promoting GLUT1 in Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) Under Hypoxia Stress.

In fish under hypoxia stress, homeostasis can become imbalanced, leading to tissue and organ damage and decreased survival. Therefore, it is useful to explore the molecular and physiological regulation mechanisms that function in fish under hypoxia stress. The microRNA miR-34a is involved in fat and glycogen metabolism, and in apoptosis. In this study, we first verified that GLUT1, the gene encoding glucose transporter 1, is a potential target gene of miR-34a in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) by dual luciferase reporter assays. Then, we clarified the regulatory relationship between miR-34a and GLUT1 by qRT-PCR analyses. We analyzed the regulatory effects of knockdown or promotion of GLUT1 expression in vitro and in vivo in GIFT under hypoxia stress. The results confirm that GLUT1 is a target gene of miR-34a in GIFT. Down-regulation of miR-34a significantly promoted GLUT1 expression. Knockdown of GLUT1 reduced the glycogen content in GIFT liver cells, inhibited HIF-1a gene expression, up-regulated the expression of genes involved in P53 signaling pathways (P53 and CASPASE-3 genes), and accelerated hepatocyte apoptosis under hypoxia stress. Compared with the control group, the group injected in the tail vein with miR-34a antagomir showed up-regulated expression of GLUT1 in the liver, increased liver glycogen content at 96 h of hypoxia stress, down-regulated expression of P53 and CASPASE-3, and decreased serum aspartate aminotransferase and alanine aminotransferase enzyme activities. Our results provide information about the molecular regulation mechanism of miRNAs and their target genes in fish during the response to hypoxia stress.

Physiological and gut microbiome changes associated with low dietary protein level in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) determined by 16S rRNA sequence analysis.

The aim of this study was to determine the effects of different dietary protein levels on the growth, physiological parameters, and gut microbiome of genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Two pellet feed diets with low (25%, LPD) and normal (35%, NPD) protein levels were fed to GIFT in aquaria at 28°C for 8 weeks. The LPD reduced trypsin activity and inhibited the growth of GIFT. The serum alanine amino transferase and aspartate transaminase activities, hepatic malondialdehyde content, and superoxide dismutase, glutathione peroxidase, and catalase activities were significantly higher in LPD GIFT than in NPD GIFT (p < .05). The LPD led to decreased lysozyme activity and increased levels of C3 (p < .05). A 16S rRNA gene profiling analysis showed that the LPD significantly affected the gut microbial composition. Compared with the NPD, the LPD significantly decreased intestinal microbial diversity (p < .05). The macronutrient distribution affected the taxonomic profile of gut bacteria, mainly the phyla Bacteroidetes, Proteobacteria, and Firmicutes. The LPD favored growth of the genus Bacteroides. The NPD appeared to increase the abundance of the genera Lawsonia, Romboutsia, and Sphingomonas. Our results showed that, compared with NPD GIFT, the LPD GIFT had weakened nonspecific immune function, altered microbial community structure, and decreased gut microbial diversity.

Effects of dietary supplementation with apple peel powder on the growth, blood and liver parameters, and transcriptome of genetically improved farmed tilapia (GIFT, Oreochromis niloticus).

High-density aquaculture and nutritional imbalances may promote fatty liver in genetically improved farmed tilapia (GIFT, Oreochromis niloticus), thus reducing the gains achieved by breeding. In this study, apple peel powder (APP) was used as a feed additive for GIFT. A control group (fed on a diet without APP) and five groups fed on diets supplemented with APP (at 0.05%, 0.1%, 0.2%, 0.4%, or 0.8% of the diet, by weight) were established to investigate the effects of APP on GIFT growth performance and physiological parameters, and on gene expression as determined by transcriptomic analysis. Dietary supplementation with APP at 0.2% promoted GIFT growth, reduced total cholesterol and triacylglycerol levels in the serum and liver, and decreased alanine aminotransferase and aspartate aminotransferase activities in the serum. Gene expression profiles in the liver were compared among the control, 0.2% APP, and 0.8% APP groups, and differentially expressed genes among these groups were identified. Annotation analyses using tools at the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases showed that the differentially expressed genes were mainly involved in the regulation of immunity and fat metabolism. The results showed that excessive supplementation with APP in the diet significantly inhibited the expression of insulin-like growth factor 2 and liver-type fatty acid-binding protein, and stimulated the expression of fatty acid desaturase 2, heat shock protein 90 beta family member 1, and nuclear factor kappa B. This resulted in disordered lipid metabolism and increased pro-inflammatory reactions, which in turn caused liver damage. Therefore, APP has good potential as an environmentally friendly feed additive for GIFT at levels of 0.1%-0.2% in the diet, but excessive amounts can have adverse effects.

Synergistic effect of water temperature and dissolved oxygen concentration on rates of fertilization, hatching and deformity of hybrid yellow catfish (Tachysurus fulvidraco♀×Pseudobagrus vachellii♂).

In the process of selecting and developing freshwater aquaculture species, yellow catfish (Tachysurus fulvidraco) have received widespread attention from Chinese farmers, fishery scientists and technologists. Achieving full artificial breeding of yellow catfish would help improve the quantity and quality of fingerlings supplied for large-scale production of this species. Temperature (T) and dissolved oxygen (DO) are the most important abiotic factors affecting the breeding efficiency of aquatic organisms. In this study, the synergistic effects of T and DO on fertilization rate (FR, %), hatching rate (HR, %) and deformity rate (DR, %) of hybrid yellow catfish (T. fulvidraco♀ × Pseudobagrus vachellii♂) were studied by central composite design (CCD) and response surface methodology. A quadratic regression model for the effects of T and DO on FR, HR and DR was established, and the combination of T and DO was optimized. The first and second order effects of T and DO on FR and HR were significant under the conditions of this experiment (P < 0.05). The first and second order effects of T on DR were significant (P < 0.05) but there was no significant effect of DO on DR (P > 0.05). T and DO had significant interaction effects on FR (P < 0.05). High T and high DO environments reduced FR and HR of yellow catfish eggs and increased DR of the newly hatched larvae. The optimal combination of T and DO was 26.0 °C and 8.3 mgL-1, respectively. Maximum FR and HR coincided with minimal DR whose predicted values were 87.2%, 89.1% and 2.7%, respectively, with reliability of 0.979. Maintaining T and DO in the best combination will help to improve breeding efficiency and ensure production of the highest quantity and quality of fingerlings.

Dietary vitamin E deficiency inhibits fat metabolism, antioxidant capacity, and immune regulation of inflammatory response in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) fingerlings following Streptococcus iniae infection.

Vitamin E plays an important role in maintaining normal metabolism and physiological functions in animals. The health of fish fingerlings directly affects the rate of disease incidence in adult fish, and healthy fingerlings ultimately result in better breeding outcomes for cultured fish. To date, no previous studies have focused on the effects vitamin E deficiency on tilapia at the fingerling stage. In this study, we investigated the effects of dietary vitamin E on the growth, fat metabolism, antioxidant capacity, and inflammatory response of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) fingerlings. Vitamin E at different concentrations (0, 20, 40, 80, 160, and 320 mg/kg) was added to the diet and GIFT were fed for 55 days. Then, the GIFT were intraperitoneally injected with Streptococcus iniae and tested for infection. Vitamin E deficiency decreased growth and increased the food conversion ratio of GIFT fingerlings. Vitamin E deficiency also reduced the white blood cell count, increased hematocrit and hemoglobin contents in the blood, increased serum aspartate aminotransferase and alanine aminotransferase activities, and increased liver stress (P < 0.05). Vitamin E deficiency inhibited fat metabolism, down-regulated the expression of genes encoding lipoprotein lipase and heart-type and liver-type fatty acid-binding proteins, and increased serum total protein and fat deposition. Vitamin E deficiency significantly decreased superoxide dismutase, glutathione peroxidase, and catalase activities, increased malondialdehyde content, and caused oxidative damage. Vitamin E deficiency also up-regulated the expression of genes encoding interleukin 1ß and tumor necrosis factor α in the head kidney, and stimulated a pro-inflammatory response. Overall, vitamin E deficiency inhibited growth, impaired fat metabolism, and disrupted the inflammatory response of GIFT fingerlings, whereas vitamin E supplementation in the diet reversed these negative effects. The diets with high concentrations of vitamin E (160-320 mg/kg) led to vitamin E accumulation in the fish tissues and rapid activation of the inflammatory response and antioxidant capacity in GIFT fingerlings exposed to S. iniae.

Changes in Physiological Parameters, Lipid Metabolism, and Expression of MicroRNAs in Genetically Improved Farmed Tilapia (Oreochromis niloticus) With Fatty Liver Induced by a High-Fat Diet.

Tilapia is susceptible to hepatic steatosis when grown in intensive farming systems. The aim of this study was to explore the mechanism of fatty liver induced by a high-fat diet (HFD) in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Juvenile GIFT were fed with HFD or a normal-fat diet (NFD) for 60 days. Substantial fat deposition in the liver of HFD-fed GIFT on days 20, 40, and 60 was observed using hematoxylin - eosin staining and oil red O staining. The increased fat deposition was consistent with increased triglyceride (TG) and total cholesterol (TC) levels in the liver of HFD-fed GIFT. There were significant differences (P < 0.05) in serum biochemical indexes (TG, TC, low density lipoprotein-cholesterol, and insulin contents, and alanine aminotransferase activity) between GIFT fed a HFD and GIFT fed a NFD on days 20, 40, and 60. Furthermore, 60 days of a HFD significantly changed (P < 0.05) the hepatic fatty acid composition, and led to increased polyunsaturated fatty acid levels and decreased saturated fatty acid and monounsaturated fatty acid levels. Hepatic antioxidant enzyme activities increased by day 20 and then declined, which led to an increase in malondialdehyde contents in the liver of HFD-fed GIFT. Molecular analyses revealed that the microRNAs miR-122, miR-29a, and miR-145-5p were upregulated, whereas miR-34a was downregulated in HFD-fed GIFT. SCD, ELOVL6, and SRD5A2 encode three important enzymes in lipid metabolism, and were identified as potential targets of miRNAs. The transcript levels of hepatic SCD and ELOVL6 were decreased and that of hepatic SRD5A2 was increased in GIFT fed a HFD. Overall, the results of this study revealed a potential link between miRNAs and fatty liver induced by HFD, and suggest that a HFD could lead to excess fat deposition in the GIFT liver, which may disrupt hepatic lipid metabolism and reduce the antioxidant defense capacity.