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.

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.

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.

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.

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.

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.

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.

α2A -AR antagonism by BRL-44408 maleate attenuates acute lung injury in rats with downregulation of ERK1/2, p38MAPK, and p65 pathway.

Acute respiratory distress syndrome (ARDS), characterized by acute hypoxic respiratory dysfunction or failure, is a manifestation of multiple organ failure in the lung, and the most common risk factor is sepsis. We previously showed that blocking α2 -adrenoceptor (α2 -AR) could attenuate lung injury induced by endotoxin in rats. α2A -adrenoceptor (α2A -AR), a subtype of α2 -AR plays a key role in inflammatory diseases, but the mechanism remains unknown. Here, we explored the effect of BRL-44408 maleate (BRL), a specific α2A -AR antagonist, on cecal ligation puncture (CLP)-induced ARDS in rats and the underlying mechanism. Preadministration of BRL-44408 maleate significantly alleviated CLP-induced histological injury, macrophage infiltration, inflammatory response, and wet/dry ratio in lung tissue. However, there was no statistical difference in survival rate between the CLP and CLP+BRL groups. Extracellular regulated protein kinase (ERK1/2), p38MAPK, and p65 were activated in the CLP group, and BRL-44408 maleate inhibited the activation of these signal molecules, c-Jun N-terminal kinase (JNK) and protein kinase A (PKA) showed no changes in activation between these two groups. BRL-44408 maleate decreased lipopolysaccharide (LPS)-induced expression of cytokines in NR8383 rat alveolar macrophages and reduced phosphorylation of ERK1/2, p38MAPK, and p65. JNK and PKA were not influenced by LPS. Together, these findings suggest that antagonism of α2A -AR improves CLP-induced acute lung injury and involves the downregulation of ERK1/2, p38MAPK, and p65 pathway independent of the activation of JNK and PKA.

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.

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.

The prognostic role of tumour-infiltrating lymphocytes in oral squamous cell carcinoma: A meta-analysis.

It has been suggested that tumour-infiltrating lymphocytes (TILs) are associated with the progression of oral squamous cell carcinoma (OSCC). However, the prognostic value of TILs is inconclusive due to the heterogeneity of immune cells within the tumour microenvironment. In this meta-analysis, we aimed to assess the prognostic value of TILs in OSCC. The PubMed, Cochrane, Embase, Scopus and Web of Science databases were searched up to April 20, 2019, and 33 studies were ultimately included in this meta-analysis. Our pooled meta-analysis showed that high infiltration of CD8+ TILs, CD45RO+ TILs and CD57+ TILs favoured better overall survival (OS). However, high infiltration of CD68+ macrophages and CD163+ macrophages was associated with poor prognosis in OSCC. These findings suggest that CD8+ TILs, CD45RO+ TILs, CD57+ TILs, CD68+ macrophages and CD163+ macrophages might serve as novel prognostic factors and therapeutic targets in OSCC.

CCD and RSM optimization approach for antioxidative activity and immune regulation in head kidney of yellow catfish (Pelteobagrus fulvidraco) based on different lipid levels and temperatures.

Yellow catfish (Pelteobagrus fulvidraco) is an important economic cultured fish in China. Here we report antioxidative activity and immune regulation in head kidney using a central composite design based on water temperature (20-34 °C) and dietary lipid (2-17%). Response values were optimized using response surface methodology to maximize the immune response and relieve oxidative stress. The experiment was conducted under laboratory conditions and lasted for seven weeks. The results showed that the linear effects of lipid level on superoxide dismutase (SOD, and lysozyme (LYZ) activity, and malondialdehyde (MDA) content in head kidney, respiratory burst activity (RBA) of head kidney macrophages, and cumulative mortality of fish infected by Streptococcus iniae (S. iniae) were significant (P < 0.05). Similarly, the linear effects of water temperature on SOD activity, MDA content, and cumulative mortality were significant (P < 0.05). In addition, the quadratic effects of water temperature and lipid level on all experimental response values were significant (P < 0.05), and no interactive effect was found between water temperature and lipid level (P > 0.05). High water temperature and high lipid diet significantly reduced the antioxidative activity and immune response in head kidney, and increased MDA content, which caused increased mortality of the S. iniae-infected fish. The adjusted R2 values for SOD activity, MDA content, LYZ activity, RBA, phagocytic activity, and cumulative mortality regression models were 0.76, 0.85, 0.87, 0.79, 0.64, and 0.87, respectively. The optimal combination of water temperature and lipid level was 26.9 °C and 7.7%, at which good antioxidative activity and immune regulation were achieved, with reliability of 0.878. This combination was close to the optimal combination of water temperature and lipid level for growth performance (27.5 °C and 9.2%) reported previously. Thus, the optimal combination may not only promote growth, but also enhance antioxidant and immune levels.

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.

Metabolic-Activity-Based Assessment of Antimicrobial Effects by D2O-Labeled Single-Cell Raman Microspectroscopy.

To combat the spread of antibiotic resistance, methods that quantitatively assess the metabolism-inhibiting effects of drugs in a rapid and culture-independent manner are urgently needed. Here using four oral bacteria as models, we show that heavy water (D2O)-based single-cell Raman microspectroscopy (D2O-Raman) can probe bacterial response to different drugs using the Raman shift at the C-D (carbon-deuterium vibration) band in 2040 to 2300 cm-1 as a universal biomarker for metabolic activity at single-bacterial-cell resolution. The "minimum inhibitory concentration based on metabolic activity" (MIC-MA), defined as the minimal dose under which the median ΔC-D-ratio at 8 h of drug exposure is ≤0 and the standard deviation (SD) of the ΔC-D ratio among individual cells is ≤0.005, was proposed to evaluate the metabolism-inhibiting efficacy of drugs. In addition, heterogeneity index of MIC-MA (MIC-MA-HI), defined as SD of C-D ratio among individual cells, quantitatively assesses the among-cell heterogeneity of metabolic activity after drug regimens. When exposed to 1× MIC of sodium fluoride (NaF), 1× MIC of chlorhexidine (CHX), or 60× MIC of ampicillin, the cariogenic oral pathogen Streptococcus mutans UA159 ceased propagation yet remained metabolically highly active. This underscores the advantage of MIC-MA over the growth-based MIC in being able to detect the "nongrowing but metabolically active" (NGMA) cells that underlie many latent or recurring infections. Moreover, antibiotic susceptible and resistant S. mutans strains can be readily discriminated at as early as 0.5 h. Thus, D2O-Raman can serve as a universal method for rapid and quantitative assessment of antimicrobial effects based on general metabolic activity at single-cell resolution.

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.

Inhibition of miR-92d-3p enhances inflammation responses in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) with Streptococcus iniae infection by modulating complement C3.

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate target gene expression by binding to the 3'-untranslated regions (3'-UTRs) of their target mRNAs. The miR-92 family is an important miRNA family, which was discovered to be related to regulation of tumor proliferation, apoptosis, invasion, and metastasis. Inhibition of miR-92d-3p was found previously in head kidney of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) exposed to Streptococcus iniae infection. In this study, we found that miR-92d-3p regulated complement C3 mRNA levels by binding to its 3'-UTR by 3'-UTR luciferase reporter assay, and reduced miR-92d-3p expression resulted in increased C3 mRNA levels. We detected a negative relationship between the expression levels of miR-92d-3p and C3 in GIFT injected with miRNA antagomir. We performed in vivo functional analysis by miR-92d-3p silencing. Inhibition of miR-92d-3p levels in GIFT head kidney caused a significant increase in C3 expression, which consequently increased the white blood cell counts and interleukin-1ß, tumor necrosis factor-α, and interferon-γ mRNA levels, all of which may help to activate the inflammatory response in GIFT post-infection with S. iniae. Our findings indicate that miR-92d-3p regulated C3 levels by binding with the C3 mRNA 3'-UTR, and this interaction affected S. iniae infection induction and the immune response in GIFT. We concluded that miR-92d-3p plays an important role in modulating the inflammatory response in GIFT head kidney. Our findings may contribute to understanding the mechanisms of miRNA-mediated gene regulation in tilapia in response to S. iniae infection.

Influences of dietary lipid and temperature on growth, fat deposition and lipoprotein lipase expression in darkbarbel catfish (Pelteobagrus vachellii).

Darkbarbel catfish (Pelteobagrus vachellii) is an important freshwater fish in China. Water temperature greatly influences the absorption and utilization of dietary lipid by fish. Response values (including growth, hepatic fat deposition, and gene expression) for darkbarbel catfish mediated by two factors (water temperature 20-34°C; dietary lipid level 2-17%) were the focus of this study. The relationship between the two factors and the response values was evaluated by the response surface method using the central composite design. The experiment was conducted under laboratory conditions and lasted for seven weeks. A total of 975 experimental fish (average weight 11.75 ± 0.17g) were selected and placed in 39 plastic tanks. The results showed that the linear effects of lipid level on feed conversion rate (FCR), hepatopancreas somatic index (HSI), hepatic triglycerides (TG), cholesterol (TC), and lipoprotein lipase (LPL) gene expression were significant (P < 0.05). The linear effects of water temperature on specific growth rate (SGR), HSI, TC level, and LPL mRNA expression were significant (P < 0.05). The quadratic effects of water temperature and lipid level on SGR and FCR were significant (P < 0.05). Low water temperature and low lipid diets significantly inhibited growth, increased HSI, and reduced hepatic TG and TC levels, and LPL mRNA expression. The adjusted R2 values for the SGR, FCR, HSI, TC, TG, and LPL mRNA regression models were 0.77, 0.85, 0.62, 0.73, 0.85, and 0.91, respectively. The optimal combination of water temperature and dietary lipid level was 27.5°C and 9.2%, at which the greatest growth and FCR were 2.13%.d-1 and 1.31 respectively, with desirability of 0.904. These results indicated that water temperature may mediate the requirement and utilization of dietary lipid, and intervene in hepatic fat deposition. The results of this study can be used to help optimize the culture conditions of darkbarbel catfish.

Reverse and Multiple Stable Isotope Probing to Study Bacterial Metabolism and Interactions at the Single Cell Level.

The interactions between microorganisms driven by substrate metabolism and energy flow are important to shape diversity, abundance, and structure of a microbial community. Single cell technologies are useful tools for dissecting the functions of individual members and their interactions in microbial communities. Here, we developed a novel Raman stable isotope probing (Raman-SIP), which uses Raman microspectroscopy coupled with reverse and D2O colabeling to study metabolic interactions in a two-species community consisting of Acinetobacter baylyi ADP1 and Escherichia coli DH5α-GFP. This Raman-SIP approach is able to detect carbon assimilation and general metabolic activity simultaneously. Taking advantage of Raman shift of single cell Raman spectra (SCRS) mediated by incorporation of stable-isotopic substrates, Raman-SIP with reverse labeling has been applied to detect initially 13C-labeled bands of ADP1 SCRS reverting back to 12C positions in the presence of 12C citrate. Raman-SIP with D2O labeling has been employed to probe metabolic activity of single cells without the need of cell replication. Our results show that E. coli alone in minimal medium with citrate as the sole carbon source had no metabolic activity, but became metabolically active in the presence of ADP1. Mass spectrometry-based metabolite footprint analysis suggests that putrescine and phenylalanine excreted by ADP1 cells may support the metabolic activity of E. coli. This study demonstrates that Raman-SIP with reverse labeling would be a useful tool to probe metabolism of any carbon substrate, overcoming limitations when stable isotopic substrates are not readily available. It is also found that Raman-SIP with D2O labeling is a sensitive and reliable approach to distinguish metabolically active cells but not quiescent cells. This novel approach extends the application of Raman-SIP and demonstrates its potential application as a valuable strategic approach for probing cellular metabolism, metabolic activity, and interactions in microbial communities at the single cell level.