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.

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.

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.

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.

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.

The effects of temperature and dissolved oxygen on the growth, survival and oxidative capacity of newly hatched hybrid yellow catfish larvae (Tachysurus fulvidraco♀ × Pseudobagrus vachellii♂).

Demand for yellow catfish fry, an economically important farmed fish in China, has increased dramatically. Newly hatched larvae are highly sensitive to changes in environmental conditions, with water temperature (T) and dissolved oxygen (DO) being two important factors that affect their early development. We investigate optimal T (between 19.0 and 33.0 °C) and DO (between 2.0 and 12.0 mg L-1) concentrations on growth and antioxidant enzyme activity of newly hatched hybrid yellow catfish larvae (Tachysurus fulvidraco × Pseudobagrus vachellii) using a central composite design. We use a response surface method to optimize the response variables for survival (S) and growth, and the reduction of oxidative stress, over a 50-day experimental duration. T has a significant effect on specific growth rate (SGR), hepatic malondialdehyde (MDA) content, and superoxide dismutase (SOD) and catalase (CAT) activities (P < 0.05). DO concentration has a significant effect on SGR, S, hepatic MDA content, and SOD and CAT activities (P < 0.05). T and DO also have significant second order effects on SGR, S, SOD, and CAT activities (P < 0.05). Increased DO at low T stimulates SOD and CAT activities and alleviates oxidative damage. Adjusted R2 values for SGR, S, CAT, SOD, and MDA models are 0.734, 0.937, 0.916, 0.894 and 0.826, respectively. A combination of 26.8 °C and 7.3 mg L-1 represents optimal rearing conditions, in that larval growth and antioxidant ability is improved. Results show that T and DO during larviculture of yellow catfish have important implications for aquaculture.

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.

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.

High Fat Diet-Induced miR-122 Regulates Lipid Metabolism and Fat Deposition in Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) Liver.

The liver is an important organ for the regulation of lipid metabolism. In genetically improved farmed tilapia (GIFT, Oreochromis niloticus), fat deposition in the liver occurs when they are fed high-lipid diets over a long term. This can affect their growth, meat quality, and disease resistance. MicroRNAs (miRNAs) are known to be crucial regulatory factors involved in lipid metabolism; however, the mechanism by which they regulate lipid deposition in GIFT remains unclear. Comparative miRNA expression profiling between GIFT fed a normal diet and those fed a high-lipid diet showed that miR-122 is closely related to lipid deposition. Using miR-122 as a candidate, we searched for a binding site for miR-122 in the 3'-untranslated region (UTR) of the stearoyl-CoA desaturase gene (SCD) using bioinformatics tools, and then confirmed its functionality using the luciferase reporter gene system. Then, the regulatory relationship between this miRNA and its target gene SCD was analyzed using real-time polymerase chain reaction (qRT-PCR) and western blotting analyses. Last, we investigated the effect of the loss of miR-122 expression on lipid metabolism in GIFT. The results showed that a sequence in the 3'-UTR region of SCD of GIFT was complementary to the miR-122 seed region, and there was a negative relationship between the expression of miRNA and SCD expression. Inhibition of miR-122 up-regulated SCD, increased the expression of fat synthesis-related genes, increased hepatic triglyceride and cholesterol contents, and promoted weight gain in fish. Our results showed that miR-122 targets SCD to mediate hepatic fat metabolism. These results provide new insights for the prevention and treatment of fatty liver disease in GIFT.

miR-205-5p negatively regulates hepatic acetyl-CoA carboxylase ß mRNA in lipid metabolism of Oreochromis niloticus.

MicroRNAs (miRNAs) are non-coding RNAs that function as post-transcriptional gene regulators and that play vital roles controlling lipid metabolism. miR-205 is an important miRNA related to adipogenesis and lipid metabolism. However, little is known about the potential role of miR-205-5p in genetically improved farmed tilapia (GIFT, Oreochromis niloticus). In this study, we used miRanda software to search for potential miR-205-5p target genes and found a lipid-metabolism-related gene called acetyl-CoA carboxylase ß (ACACß). Quantitative real-time polymerase chain reaction data indicated that there may be a negative regulation relationship between miR-205-5p and ACACß gene expression under HFD rearing. Using luciferase reporter assays, we verified the binding site of miR-205-5p in the 3'-untranslated region of the ACACß mRNA. Furthermore, an in vivo functional analysis of miR-205-5p was performed by injecting GIFT juveniles with a miR-205-5p antagomir. Reduced levels of miR-205-5p in GIFT liver increased ACACß mRNA expression 12 h post-injection. miR-205-5p suppression also increased fatty acid synthase and peroxisome proliferator-activated receptor-α mRNA levels 48 h and 120 h post-injection, respectively. Taken together, our results indicate that miR-205-5p negative regulates hepatic ACACß mRNA expression, and may serve as an important regulator in controlling hepatic lipid metabolism in GIFT.

Responses of blood biochemistry, fatty acid composition and expression of microRNAs to heat stress in genetically improved farmed tilapia (Oreochromis niloticus).

We investigated the effects of heat stress on genetically improved farmed tilapia, focusing on metabolic and immune responses. Differences in blood parameters, serum biochemistry, muscle fatty acid composition, and microRNA (miRNA) expression were analyzed in fish under heat stress. Fish were exposed to heat stress at 35 °C and sampled at 0, 6, 12, 24, and 48 h after exposure and compared with a control group maintained at 28 °C. The results showed that red and white blood cell counts, hemoglobin levels, and hematocrit values tended to increase (P < 0.05) and reached their maximum levels after 24 h, then declined. Acute heat stress enhanced serum glucose, total protein, and total cholesterol levels, and muscle fatty acid components were also altered. Serum alanine aminotransferase (ALT) activity was significantly increased after heat stress for 6 and 12 h. Polyunsaturated fatty acids levels were increased after heat stress for 12 and 24 h, whereas levels of monounsaturated fatty acids decreased in response to heat stress. Expression of hepatic miR-1 and miR-122 was significantly upregulated, and expression of miR-10c was significantly increased (P < 0.05) only after heat stress for 48 h. Acute heat stress altered metabolism closely related to the immune system and the liver of tilapia. These findings contribute to a theoretical framework for tilapia breeding at high temperatures.

miR-1338-5p Modulates Growth Hormone Secretion and Glucose Utilization by Regulating ghitm in Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus).

MicroRNAs (miRNAs) are endogenous, non-coding small RNA molecules about 22 nt in length, which could regulate the expressions of target genes and participate in growth and development of organisms. Genetically improved farmed tilapia (GIFT, Oreochromis niloticus) is an important economic freshwater species in China and the growth performance is one of the main breeding indicators. Growth hormone inducible transmembrane protein (ghitm) plays an important role in growth and development of both mammals and invertebrates; however, little studies have been reported on fish. Our previous experiments indicated that miR-1338-5p expression may be negatively correlated with ghitm expression. In this study, we firstly used qRT-PCR and northern blot to verify the expression of miR-1338-5p and ghitm, and determined the binding site of miR-1338-5p in the ghitm 3'-untranslated region (UTR) by luciferase reporter assay. Secondly, juveniles GIFT injected with miR-1338-5p antagomir were used to analyze the regulatory function of the miR-1338-5p-ghitm pair in vivo. The results showed that the ghitm 3'-UTR was complementary to the 5' 2-8-nt site of miR-1338-5p. Inhibition of miR-1338-5p promoted ghitm expression in the pituitary and liver of GIFT. ghitm could interfere in the growth hormone (Gh)-growth hormone receptor (Ghr)-insulin-like growth factor (Igf) signaling pathway by competing with the ghr1 for combination with Gh, and then reduce the growth of GIFT. Moreover, the reduction of Gh in serum may regulate insulin secretion and result in the increasing sugar and fat storage in serum and liver. Our results suggest that miR-1338-5p participates in the growth and development of GIFT through the regulation of ghitm, which provides theoretical support for the study of the fish growth mechanism.

Identification and characterization of lipid metabolism-related microRNAs in the liver of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) by deep sequencing.

MicroRNAs (miRNAs) play vital roles in modulating diverse metabolic processes in the liver, including lipid metabolism. Genetically improved farmed tilapia (GIFT, Oreochromis niloticus), an important aquaculture species in China, is susceptible to hepatic steatosis when reared in intensive culture systems. To investigate the miRNAs involved in GIFT lipid metabolism, two hepatic small RNA libraries from high-fat diet-fed and normal-fat diet-fed GIFT were constructed and sequenced using high-throughput sequencing technology. A total of 204 known and 56 novel miRNAs were identified by aligning the sequencing data with known Danio rerio miRNAs listed in miRBase 21.0. Six known miRNAs (miR-30a-5p, miR-34a, miR-145-5p, miR-29a, miR-205-5p, and miR-23a-3p) that were differentially expressed between the high-fat diet and normal-fat diet groups were validated by quantitative real-time PCR. Bioinformatics tools were used to predict the potential target genes of these differentially expressed miRNAs, and Gene Ontology enrichment analysis indicated that these miRNAs may play important roles in diet-induced hepatic steatosis in GIFT. Our results provide a foundation for further studies of the role of miRNAs in tilapia lipid homeostasis regulation, and may help to identify novel targets for therapeutic interventions to reduce the occurrence of fatty liver disease in farmed tilapia.