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.

Integrative analysis of miRNA-mRNA expression in the brain during high temperature-induced masculinization of female Nile tilapia (Oreochromis niloticus).

Temperature is one of the most important non-genetic sex differentiation factors for fish. The technique of high temperature-induced sex reversal is commonly used in Nile tilapia (Oreochromis niloticus) culture, although the molecular regulatory mechanisms involved in this process remain unclear. The brain is an essential organ for the regulation of neural signals involved in germ cell differentiation and gonad development. To investigate the regulatory roles of miRNAs-mRNAs in the conversion of female to male Nile tilapia gender under high-temperature stress, we compared RNA-Seq data from brain tissues between a control group (28 °C) and a high temperature-treated group (36 °C). The result showed that a total of 123,432,984 miRNA valid reads, 288,202,524 mRNA clean reads, 1128 miRNAs, and 32,918 mRNAs were obtained. Among them, there were 222 significant differentially expressed miRNAs (DE miRNAs) and 810 differentially expressed mRNAs (DE mRNAs) between the two groups. Eight DE miRNAs and eight DE mRNAs were randomly selected, and their expression patterns were validated by qRT-PCR. The miRNA-mRNA co-expression network demonstrated that 40 DE miRNAs targeted 136 protein-coding genes. Functional enrichment analysis demonstrated that these genes were involved in several gonadal differentiation pathways, including the oocyte meiosis signaling pathway, progesterone-mediated oocyte maturation signaling pathway, cell cycle signaling pathway and GnRH signaling pathway. Then, an interaction network was constructed for 8 miRNAs (mir-137-5p, let-7d, mir-1388-5p, mir-124-4-5p, mir-1306, mir-99, mir-130b and mir-21) and 10 mRNAs (smc1al, itpr2, mapk1, ints8, cpeb1b, bub1, fbxo5, mmp14b, cdk1 and hrasb) involved in the oocyte meiosis signaling pathway. These findings provide novel information about the mechanisms underlying miRNA-mediated sex reversal in female Nile tilapia.

Ultrastructural, Antioxidant, and Metabolic Responses of Male Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) to Acute Hypoxia Stress.

Tilapia tolerate hypoxia; thus, they are an excellent model for the study of hypoxic adaptation. In this study, we determined the effect of acute hypoxia stress on the antioxidant capacity, metabolism, and gill/liver ultrastructure of male genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Fish were kept under control (dissolved oxygen (DO): 6.5 mg/L) or hypoxic (DO: 1.0 mg/L) conditions for 72 h. After 2 h of hypoxia stress, antioxidant enzyme activities in the heart and gills decreased, while the malondialdehyde (MDA) content increased. In contrast, in the liver, antioxidant enzyme activities increased, and the MDA content decreased. From 4 to 24 h of hypoxia stress, the antioxidant enzyme activity increased in the heart but not in the liver and gills. Cytochrome oxidase activity was increased in the heart after 4 to 8 h of hypoxia stress, while that in the gills decreased during the later stages of hypoxia stress. Hypoxia stress resulted in increased Na+-K+-ATP activity in the heart, as well as hepatic vacuolization and gill lamella elongation. Under hypoxic conditions, male GIFT exhibit dynamic and complementary regulation of antioxidant systems and metabolism in the liver, gills, and heart, with coordinated responses to mitigate hypoxia-induced damage.

Single nucleotide polymorphism SNP19140160 A > C is a potential breeding locus for fast-growth largemouth bass (Micropterus salmoides).

BACKGROUND: Largemouth bass (Micropterus salmoides) has significant economic value as a high-yielding fish species in China's freshwater aquaculture industry. Determining the major genes related to growth traits and identifying molecular markers associated with these traits serve as the foundation for breeding strategies involving gene pyramiding. In this study, we screened restriction-site associated DNA sequencing (RAD-seq) data to identify single nucleotide polymorphism (SNP) loci potentially associated with extreme growth differences between fast-growth and slow-growth groups in the F1 generation of a largemouth bass population. RESULTS: We subsequently identified associations between these loci and specific candidate genes related to four key growth traits (body weight, body length, body height, and body thickness) based on SNP genotyping. In total, 4,196,486 high-quality SNPs were distributed across 23 chromosomes. Using a population-specific genotype frequency threshold of 0.7, we identified 30 potential SNPs associated with growth traits. Among the 30 SNPs, SNP19140160, SNP9639603, SNP9639605, and SNP23355498 showed significant associations; three of them (SNP9639603, SNP9639605, and SNP23355498) were significantly associated with one trait, body length, in the F1 generation, and one (SNP19140160) was significantly linked with four traits (body weight, height, length, and thickness) in the F1 generation. The markers SNP19140160 and SNP23355498 were located near two growth candidate genes, fam174b and ppip5k1b, respectively, and these candidate genes were closely linked with growth, development, and feeding. The average body weight of the group with four dominant genotypes at these SNP loci in the F1 generation population (703.86 g) was 19.63% higher than that of the group without dominant genotypes at these loci (588.36 g). CONCLUSIONS: Thus, these four markers could be used to construct a population with dominant genotypes at loci related to fast growth. These findings demonstrate how markers can be used to identify genes related to fast growth, and will be useful for molecular marker-assisted selection in the breeding of high-quality largemouth bass.

Transcriptional Knock-down of mstn Encoding Myostatin Improves Muscle Quality of Nile Tilapia (Oreochromis niloticus).

Myostatin (encoded by mstn) negatively regulates skeletal muscle mass and affects lipid metabolism. To explore the regulatory effects of mstn on muscle development and lipid metabolism in Nile tilapia (Oreochromis niloticus), we used antisense RNA to transcriptionally knock-down mstn. At 180 days, the body weight and body length were significantly higher in the mstn-knock-down group than in the control group (p < 0.05). Additionally, fish with mstn-knock-down exhibited myofiber hyperplasia but not hypertrophy. Oil red O staining revealed a remarkable increase in the area of lipid droplets in muscle in the mstn-knockdown group (p < 0.05). Nutrient composition analyses of muscle tissue showed that the crude fat content was significantly increased in the mstn-knock-down group (p < 0.05). The contents of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids were all significantly increased in the mstn-knock-down group (p < 0.05). Comparative transcriptome analyses revealed 2420 significant differentially expressed genes between the mstn-knock-down group and the control group. KEGG analysis indicates that disruptions to fatty acid degradation, glycerolipid metabolism, and the PPAR signaling pathway affect muscle development and lipid metabolism in mstn-knock-down Nile tilapia: acaa2, eci1, and lepr were remarkably up-regulated, and acadvl, lpl, foxo3, myod1, myog, and myf5 were significantly down-regulated (p < 0.05). These results show that knock-down of mstn results in abnormal lipid metabolism, acceleration of skeletal muscle development, and increased adipogenesis and weight gain in Nile tilapia.

Vitamin E Ameliorates Impaired Ovarian Development, Oxidative Stress, and Disrupted Lipid Metabolism in Oreochromis niloticus Fed with a Diet Containing Olive Oil Instead of Fish Oil.

Aquaculture feed containing olive oil (OO) instead of fish oil (FO) can cause oxidative stress and impair gonad development in fish. We determined the effect of dietary OO-induced oxidative stress on ovarian development, and explored whether vitamin E (VE) could mitigate negative effects. Female Nile tilapia (Oreochromis niloticus) were fed for 10 weeks with four diets: 5% OO + 70 mg/kg VE, 5% OO + 200 mg/kg VE, 5% FO + 70 mg/kg VE, or 5% FO + 200 mg/kg VE. Dietary OO reduced the specific growth rate and gonadosomatic index, inhibited superoxide dismutase and catalase, delayed ovarian development, decreased serum sex hormone levels, and reduced ovarian triglyceride and n-3 highly unsaturated fatty acid contents. The transcript levels of genes encoding sex hormone receptors (erα, fshr, lhr) and components of the lipid metabolism pathway (pparα, pparγ, hsl, accα, elovl6), the nrf2 signaling pathway (nrf2, keap1), and the nf-κb signaling pathway (nf-κb, tnfα, infγ, il1ß) differed between the 70VE/OO and 70VE/FO groups. Supplementation with 200 mg/kg VE mitigated the adverse effects of OO by improving antioxidant capacity and alleviating inflammation and abnormal lipid metabolism. This may be because VE is an antioxidant and it can regulate the nrf2-nf-κb signaling pathway.

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.

Transcriptional Inhibition of AGPAT2 Induces Abnormal Lipid Metabolism and Oxidative Stress in the Liver of Nile Tilapia Oreochromis niloticus.

The enzyme 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) is an intermediate enzyme in triglyceride synthesis. The aim was to study the regulatory mechanism of AGPAT2 on Nile tilapia, Oreochromis niloticus. In this study, antisense RNA technology was used to knock-down AGPAT2 in Nile tilapia. Compared with the control groups (transfected with ultrapure water or the blank expression vector), the AGPAT2 knock-down group showed a significantly higher weight gain rate, special growth rate, visceral somatic index, and hepatopancreas somatic index; and significantly increased the total cholesterol, triglycerides, glucose, low-density lipoprotein cholesterol, and insulin levels in serum. In addition, the contents of total cholesterol and triglycerides and the abundance of superoxide dismutase, catalase, and glutathione peroxidase in the liver significantly increased, while the malondialdehyde content significantly decreased. The liver cells became severely vacuolated and accumulated lipids in the AGPAT2 knock-down group. Comparative transcriptome analyses (AGPAT2 knock-down vs. control group) revealed 1789 differentially expressed genes (DEGs), including 472 upregulated genes and 1313 downregulated genes in the AGPAT2 knock-down group. Functional analysis showed that the main pathway of differentially expressed genes enrichment was lipid metabolism and oxidative stress, such as steroid biosynthesis, unsaturated fatty acid biosynthesis, the PPAR signaling pathway, and the P53 pathway. We used qRT-PCR to verify the mRNA expression changes of 13 downstream differential genes in related signaling pathways. These findings demonstrate that knock-down of AGPAT2 in tilapia leads to abnormal lipid metabolism and oxidative stress.

Transport Stress Induces Oxidative Stress and Immune Response in Juvenile Largemouth Bass (Micropterus salmoides): Analysis of Oxidative and Immunological Parameters and the Gut Microbiome.

Transport is essential in cross-regional culturing of juvenile fish. Largemouth bass (Micropterus salmoides) often exhibit decreased vitality and are susceptible to disease after transportation. To study the effects of transport stress on juvenile largemouth bass, juveniles (average length: 8.42 ± 0.44 cm, average weight 10.26 ± 0.32 g) were subjected to a 12 h simulated transport, then subsequently, allowed to recover for 5 d. Liver and intestinal tissues were collected at 0, 6 and 12 h after transport stress and after 5 d of recovery. Oxidative and immunological parameters and the gut microbiome were analyzed. Hepatocytic vacuolization and shortened intestinal villi in the bass indicated liver and intestinal damage due to transport stress. Superoxide dismutase, lysozyme and complement C3 activities were significantly increased during transport stress (p < 0.05), indicating that transport stress resulted in oxidative stress and altered innate immune responses in the bass. With the transport stress, the malondialdehyde content first increased, then significantly decreased (p < 0.05) and showed an increasing trend in the recovery group. 16S rDNA analysis revealed that transport stress strongly affected the gut microbial compositions, mainly among Proteobacteria, Firmicutes, Cyanobacteria and Spirochaetes. The Proteobacteria abundance increased significantly after transport. The Kyoto Encyclopedia of Genes and Genomes functional analysis revealed that most gut microbes played roles in membrane transport, cell replication and repair. Correlation analyses demonstrated that the dominant genera varied significantly and participated in the measured physiological parameter changes. With 5 days of recovery after 12 h of transport stress, the physiological parameters and gut microbiome differed significantly between the experimental and control groups. These results provide a reference and basis for studying transport-stress-induced oxidative and immune mechanisms in juvenile largemouth bass to help optimize juvenile largemouth bass transportation.

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.

Integrated Transcriptome and 16S rDNA Analyses Reveal That Transport Stress Induces Oxidative Stress and Immune and Metabolic Disorders in the Intestine of Hybrid Yellow Catfish (Tachysurus fulvidraco♀ × Pseudobagrus vachellii♂).

Live fish are often transported in aquaculture. To explore the effects of transport stress, hybrid yellow catfish (Tachysurus fulvidraco♀ × Pseudobagrus vachellii♂) were subjected to simulated transport treatments (0-16 h) with 96 h of recovery after the 16-h transport treatment, and intestinal biochemical parameters, the transcriptome, and gut microbiota were analyzed. Transportation affected the number of mucus cells and led to oxidative stress in the intestine, which activated immune responses. Changes in lipid metabolism reflected metabolic adaptation to oxidative stress. Toll-like receptor signaling, peroxisome proliferator-activated receptor signaling, and steroid biosynthesis pathways were involved in the transport stress response. Gene expression analyses indicated that transport-induced local immune damage was reversible, whereas disordered metabolism recovered more slowly. A 16S rDNA analysis revealed that transport stress decreased the alpha diversity of the gut microbiota and disrupted its homeostasis. The dominant phyla (Fusobacteria, Bacteroidetes) and genera (Cetobacterium, Barnesiellaceae) were involved in the antioxidant, immune, and metabolic responses of the host to transportation stress. Correlation analyses suggested that gut microbes participate in the transport stress response and the host-microbiota interaction may trigger multiple events in antioxidant, immune, and metabolic pathways. Our results will be useful for optimizing transport processes.

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.

Transport stress induces innate immunity responses through TLR and NLR signaling pathways and increases mucus cell number in gills of hybrid yellow catfish (Tachysurus fulvidraco ♀ × Pseudobagrus vachellii ♂).

Transport stress poses a threat to most teleost fish in production, causing mass losses to the aquaculture industry. Fish gills are a mucosa-associated lymphoid tissue in direct contact with water, and they represent an ideal tissue type to study mechanisms of transport stress. In this study, hybrid yellow catfish (Tachysurus fulvidraco ♀ × Pseudobagrus vachellii ♂) were exposed to simulated transport stress for 16 h and then allowed to recover for 96 h. Gill tissues and blood samples were collected at 0 h, 2 h, 4 h, 8 h, and 16 h of transport stress and after 96 h of recovery, as well as from fish in a control group at the same sampling times. The activities of alkaline phosphatase, acid phosphatase, and superoxide dismutase and the total antioxidant capacity first increased and then decreased during the 16 h transport treatment. Exposure to 16 h of transport stress resulted in decreased serum triglyceride and total cholesterol contents, increased serum glucose content, increased activities of alanine aminotransferase and aspartate transaminase, and more mucus cells, compared with the control group. Transcriptome analysis revealed differential expression of 1525 genes (803 down-regulated and 722 up-regulated) between the control and 16 h transportation groups. Functional analyses revealed that the differentially expressed genes were enriched in immune response, signal transduction, and energy metabolism pathways. We found that tlr5, tnfɑ, hsp90ɑ, il-1ß, map2k4, il12ba were clearly up-regulated and arrdc2, syngr1a were clearly down-regulated following 8 h and/or 16 h simulated transport after qRT-PCR validation. These findings suggested that Toll- and NOD-like receptor signaling pathways potentially mediate transport stress. Transport stress altered innate immunity responses and energy use in the gill tissues of hybrid yellow catfish. After 96 h of recovery, only alanine aminotransferase and alkaline phosphatase activities and the number of mucus cells had returned to control levels. We speculate that for juvenile yellow catfish to recover to a normal state, a recovery period of more than 96 h is required after 16 h of transportation. These results provide new perspectives on the immune response of yellow catfish under transport stress and theoretical support for future optimization of their transportation.

Comparative Ionomics and Metabolic Responses and Adaptive Strategies of Cotton to Salt and Alkali Stress.

Soil salinization and alkalization severely inhibit agriculture. However, the response mechanisms of cotton to salt stress or alkali stress are unclear. Ionomics and metabolomics were used to investigate salt and alkali stresses in cotton roots and leaves. Compared with the control, salt-treated and alkali-treated cotton plants showed 51.8 and 53.0% decreases in biomass, respectively. Under salt stress, the concentration of N decreased in roots but increased in leaves, and the concentrations of P and K increased in roots but decreased in leaves. Salt stress inhibited Ca, B, N, and Fe uptake and Mg, K, P, S, and Cu transport, but promoted Mo, Mn, Zn, Mg, K, P, S, and Cu uptake and Mo, Mn, Zn, B, N, and Fe transport. Under alkali stress, the concentrations of N and P in roots and leaves decreased, while the concentrations of K in roots and leaves increased. Alkali stress inhibited P, Ca, S, N, Fe, and Zn uptake and N, P, Mg and B transport, but promoted K, Mn, Cu, Mo, Mg, and B uptake and K, Mn, Cu, Mo, Fe, and Zn transport. Under salt stress in the leaves, 93 metabolites increased, mainly organic acids, amino acids, and sugars, increased in abundance, while 6 decreased. In the roots, 72 metabolites increased, mainly amino acids, organic acids, and sugars, while 18 decreased. Under alkali stress, in the leaves, 96 metabolites increased, including organic acids, amino acids, and sugars, 83 metabolites decreased, including organic acids, amino acids, and sugars; In the roots, 108 metabolites increased, including organic acids, amino acids, and sugars. 83 metabolites decreased, including organic acids and amino acids. Under salt stress, cotton adapts to osmotic stress through the accumulation of organic acids, amino acids and sugars, while under alkali stress, osmoregulation was achieved via inorganic ion accumulation. Under salt stress, significant metabolic pathways in the leaves and roots were associated with amino acid and organic acid metabolism, sugar metabolism was mainly used as a source of energy, while under alkali stress, the pathways in the leaves were related to amino acid and linoleic acid metabolism, ß-Oxidation, TCA cycle, and glycolysis were enhanced to provide the energy needed for life activities. Enhancing organic acid accumulation and metabolism in the roots is the key response mechanism of cotton to alkalinity.

TGF-ß1/SMADs signaling involved in alleviating inflammation induced by nanoparticulate titanium dioxide in BV2 cells.

There are increasing safety concerns accompanying the widespread use of nanoparticulate titanium dioxide (nano-TiO2). It has been demonstrated that nano-TiO2 can cross the blood-brain barrier and enter the brain, causing damage to the nervous system, consisting mainly of neuroinflammation and neuronal apoptosis. Several studies have linked the TGF-ß1/SMADs signaling to the development of inflammatory response in various organs. However, no studies have connected the induction of microglial inflammation by nano-TiO2 to this signaling. Therefore, this study aimed to investigate the role of TGF-ß1/SMADs signaling in microglia inflammatory response induced by nano-TiO2. The results showed that nano-TiO2 increased the secretions of pro-inflammatory cytokines (IL-1α, IL-6, and TNF-α) and decreased the expressions of TGF-ß1 and SMAD1/2/3 proteins in BV2 cells. When TGF-ß1/SMADs signaling was inhibited, the inflammatory effect induced by nano-TiO2 increased, suggesting a suppressive effect of this signaling on the inflammation. In addition, exogenous TGF-ß1 upregulated the expressions of TGF-ß1 and SMADs1/2/3 proteins as well as decreased the secretions of pro-inflammatory cytokines (IL-1α, IL-6, and TNF-α) compared to BV2 cells treated with only nano-TiO2. Our results suggest that nano-TiO2 may inhibit the TGF-ß1/SMADs signaling by suppressing the intracellular secretion of active TGF-ß1, leading to microglial activation and the induction or exacerbation of inflammatory responses.

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 and 16S rRNA Analyses Reveal That Hypoxic Stress Affects the Antioxidant Capacity of Largemouth Bass (Micropterus salmoides), Resulting in Intestinal Tissue Damage and Structural Changes in Microflora.

Dissolved oxygen (DO) is a key factor affecting the health of aquatic organisms in an intensive aquaculture environment. In this study, largemouth bass (Micropterus salmoides) were subjected to acute hypoxic stress for 96 h (DO: 1.00 mg/L) followed by recovery under sufficient DO conditions (DO: 7.50 mg/L) for 96 h. Serum biochemical indices, intestinal histomorphology, the transcriptome, and intestinal microbiota were compared between hypoxia-treated fish and those in a control group. The results showed that hypoxia caused oxidative stress, exfoliation of the intestinal villus epithelium and villus rupture, and increased cell apoptosis. Transcriptome analyses revealed that antioxidant-, inflammation-, and apoptosis-related pathways were activated, and that the MAPK signaling pathway played an important role under hypoxic stress. In addition, 16S rRNA sequencing analyses revealed that hypoxic stress significantly decreased bacterial richness and identified the dominant phyla (Proteobacteria, Firmicutes) and genera (Mycoplasma, unclassified Enterobacterales, Cetobacterium) involved in the intestinal inflammatory response of largemouth bass. Pearson's correlation analyses showed that differentially expressed genes in the MAPK signaling pathway were significantly correlated with some microflora. The results of this study will help to develop strategies to reduce damage caused by hypoxic stress in aquacultured fish.