Metabolomic profile of muscles from tilapia cultured in recirculating aquaculture systems and traditional aquaculture in ponds and protein stability during freeze-thaw cycles.

Muscle protein stability during freeze-thaw (F-T) cycles was investigated with tilapia cultured in recirculating aquaculture systems (RAS) and traditional aquaculture in ponds (TAP). This study found that fatty acids (eg., palmitic acid) were enriched in TAP, while antioxidants (eg., glutathione) were enriched in RAS. Generally, proteins in the RAS group exhibited greater stability against denaturation during the F-T cycle, suggested by a less decrease in haem protein content (77% in RAS and 86% in TAP) and a less increase in surface hydrophobicity of sarcoplasmic protein (63% in RAS and 101% in TAP). There was no significant difference in oxidative stability of myofibrillar protein between the two groups. This study provides a theoretical guide for the quality control of tilapia cultured in RAS during frozen storage.

Combined effects of copper and cadmium exposure on ovarian function and structure in Nile Tilapia (Oreochromis niloticus).

With the rapid development of industrialization and urbanization, the issue of copper (Cu) and cadmium (Cd) pollution in aquatic ecosystems has become increasingly severe, posing threats to the ovarian tissue and reproductive capacity of aquatic organisms. However, the combined effects of Cu and Cd on the ovarian development of fish and other aquatic species remain unclear. In this study, female Nile tilapia (Oreochromis niloticus) were individually or co-exposed to Cu and/or Cd in water. Ovarian and serum samples were collected at 15, 30, 60, 90, and 120 days, and the bioaccumulation, ovarian development, and hormone secretion were analyzed. Results showed that both single and combined exposure significantly reduced the gonadosomatic index and serum hormone levels, upregulated estrogen receptor (er) and progesterone receptor (pr) gene transcription levels, and markedly affected ovarian metabolite levels. Combined exposure led to more adverse effects than single exposure. The data demonstrate that the Cu and Cd exposure can impair ovarian function and structure, with more pronounced adverse effects under Cu and Cd co-exposure. The Cu and Cd affect the metabolic pathways of nucleotides and amino acids, leading to ovarian damage. This study highlights the importance of considering combined toxicant exposure in aquatic toxicology research and provides insights into the potential mechanisms underlying heavy metal-induced reproductive toxicity in fish.

Transcriptome analysis of the Nile tilapia (Oreochromis niloticus) reveals altered expression of immune genes by cadmium.

Nile tilapia (Oreochromis niloticus) is a worldwide farmed fish and has been widely used for the study on comparative immunology in teleosts. It is well known that cadmium (Cd) can cause a variety of adverse effects in fish. However, data on the effects of Cd in fish liver and the defensive mechanisms of these effects using transcriptome approach are relatively scarce to date. In this study, by using an RNA sequencing approach, the gene expression profiling was performed in livers of tilapia exposed to 0 (control), 50, 100, and 200 µg/L of Cd for 2 months. The results showed that exposure to 50 µg/L Cd altered the expressions of 911 genes, while exposure to 100 and 200 µg/L Cd resulted in 4318 and 3737 differentially expressed genes compared to the control. Weighted correlation network analysis (WGCNA) and gene ontology (GO) analysis identified a 14-gene network linked to the immune system development. Further, in a fuzzy analysis, the GO term immune system development was enriched in cluster 3, and gene expression decreased with increasing Cd levels in a concentration-dependent manner. The qPCR and RNA-seq results identified 4 genes, i.e., dnmt3bb.1, sf3b1, SMARCAL1, and zap70, as convenient potential biological indicators for detecting waterborne Cd. The present results help systematically understand the effects of Cd on the hepatic transcriptome in tilapia.

Nonadditive and allele-specific expression of ghrelin in hybrid tilapia.

Background: Interspecies hybridization is an important breeding method to generate fishes with heterosis in aquaculture. Using this method, hybrid Nile tilapia (Oreochromis niloticus, ♀) × blue tilapia (Oreochromis aureus, ♂) has been produced and widely farmed due to its growth and appetite superiorities. However, the genetic mechanism of these advanced traits is still not well understood. Ghrelin is a crucial gene that regulates growth and appetite in fishes. In the present study, we focused on the expression characteristics and its regulation of ghrelin in the hybrid. Results: The tissue distribution analysis showed that ghrelin was predominantly expressed in the stomach in the hybrid. Ghrelin was more highly expressed in the stomach in the hybrid and Nile tilapia, compared to blue tilapia, showing a nonadditive pattern. Two single-nucleotide polymorphism (SNP) sites were identified including T/C and C/G from the second exon in the ghrelin gene from Nile tilapia and blue tilapia. By pyrosequencing based on the SNP sites, the allele-specific expression (ASE) of ghrelin in the hybrid was assayed. The result indicated that ghrelin in the hybrid showed higher maternal allelic transcript ratios. Fasting significantly increased ghrelin overall expression at 4, 8, 12, 24, and 48 h. In addition, higher maternal allelic transcript ratios were not changed in the fasting hybrids at 48 h. The cis and trans effects were determined by evaluating the overall expression and ASE values in the hybrid. The expression of ghrelin was mediated by compensating cis and trans effects in hybrid. Conclusion: In summary, the present lines of evidence showed the nonadditive expression of ghrelin in the hybrid tilapia and its regulation by subgenomes, offering new insight into gene expression characteristics in hybrids.

Essential oil from Artemisia argyi alleviated liver disease in zebrafish (Danio rerio) via the gut-liver axis.

The popularity of intensive fish farming has led to the emergence of fish diseases characterized by hepatobiliary syndrome. Artemisia argyi (A. argyi) essential oils have anti-inflammatory and anti-oxidant effects. However, their alleviating effects and mechanism on liver disease in fish are still unclear. Thus, adult zebrafish were used to construct an animal model to observe histopathological damages, determine biochemical parameters and expression of inflammatory cytokines and mRNAs in the PPAR-γ/NF-κB pathway, and conduct 16 S sequencing of intestinal microbiota. The results found that after treatment with A. argyi essential oil, the histopathological damage caused by ethanol was relieved; the CAT, SOD, and GSH levels were remarkably elevated, while the MDA level was obviously lowered (P < 0.05); the expression levels of IL-10 and IFN-γ mRNAs were enhanced, but the levels of IL-1ß, IL-6, PPAR-γ, NF-κB, and TNF-α mRNAs were reduced (P < 0.05) relative to the EtOH group. A. argyi essential oil remarkably attenuated the damage to intestinal tissue structure, and elevated the levels of Muc2, ZO-1, Claudin-1, and Occludin mRNA (P < 0.05). Sequencing of the gut flora showed that A. argyi essential oil significantly altered the composition of gut microbes compared with the EtOH group. In addition, KEGG and COG analyses also showed significant (P < 0.05) changes in acetate cycling metabolism in the EtOH group, catechol 2, 3-dioxygenase and nitroreductase were significantly increased (P < 0.001), and lipid metabolism and terpenoid synthesis were significantly elevated (P < 0.001) in A. argyi essential oil group. The results indicate that A. argyi essential oil could effectively relieve ethanol-caused histopathological damage of livers by modulating the composition of gut microbiota, thus inhibiting the level of IL-1ß and mRNAs in the PPAR-γ/NF-κB pathway, increasing the IL-10 level, reducing the oxidative stress. This may offer a rationale for further research on the rationality of A. argyi as a substitute for feed antibiotics in aquaculture.

The Requirement and Protective Effects of Dietary Protein against Chronic Ammonia Exposure in Juvenile Genetically Improved Farmed Tilapia (Oreochromis niloticus).

Ammonia is a key risk factor in intensive aquaculture systems. This experiment is aimed at investigating the influence of dietary protein levels on genetically improved farmed tilapia (GIFT, Oreochromis niloticus) under chronic ammonia stress. GIFT juveniles of 4.00 ± 0.55 g were exposed to high ammonia level at 0.88 mg/L and fed with six diets comprising graded protein levels at 22.64%, 27.26%, 31.04%, 35.63%, 38.47%, and 42.66% for 8 weeks. The fish in negative control was fed the diet with 31.04% protein in normal water (0.02 mg ammonia/L water). Our results showed that high ammonia exposure (0.88 mg/L) caused significant decrease in fish growth performance, hematological parameters, liver antioxidant enzymes (catalase and glutathione peroxidase), and gill Na+- and K+-dependent adenosine triphosphatase (Na+/K+-ATP) activity. When fish were under high ammonia exposure, the weight gain rate, special growth rate, feed efficiency, and survival rate elevated significantly with dietary protein supplementation increase to 35.63%, whereas protein efficiency ratio, hepatosomatic index, and viscerosomatic index showed a decreased tendency. Dietary protein administration significantly enhanced crude protein but reduced crude lipid contents in the whole fish. Fish fed diets with 35.63%-42.66% protein had higher red blood cell counts and hematocrit percentage than fish fed 22.64% protein diet. The values of serum biochemical indices (lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase), hepatic antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), and gill Na+/K+-ATP activity were all elevated with the increment of dietary protein. Moreover, histological analysis indicated that dietary protein administration could prevent the ammonia-induced damages in fish gill, kidney, and liver tissues. Based on weight gain rate as a response criterion, the optimal dietary protein requirement for GIFT juveniles under chronic ammonia stress was 37.9%.

Effects of four hormones on the mitigation of ovarian damage in tilapia (Oreochromis niloticus) after copper and cadmium exposure.

Female tilapia of the Genetic Improvement of Farmed Tilapia (GIFT) strain were selected as an animal model to study the effects of four hormonal drugs in mitigating ovarian damage following exposure to copper and cadmium. After combined exposure to copper and cadmium in aqueous phase for 30 d, tilapia were randomly injected with oestradiol (E2), human chorionic gonadotropin (HCG), luteinizing hormone releasing hormone (LHRH), or coumestrol and raised in clear water for 7 d Ovarian samples were collected after combined exposure to heavy metals for 30 d and after recovery for 7 d Gonadosomatic index (GSI), copper and cadmium levels in the ovary, reproductive hormone levels in serum, and mRNA expression of key reproductive regulatory factors were determined. After 30 d of exposure to the combined copper and cadmium in aqueous phase, the Cd2+ content in tilapia ovarian tissue increased by 1,242.46% (p < 0.05), whereas the Cu2+ content, body weight, and GSI decreased by 68.48%, 34.46%, and 60.00% (p < 0.05), respectively. Additionally, E2 hormone levels in tilapia serum decreased by 17.55% (p < 0.05). After drug injection and recovery for 7 d, compared to the negative control group, the HCG group exhibited an increase of 39.57% (p < 0.05) in serum vitellogenin levels. Increases of 49.31%, 42.39%, and 45.91% (p < 0.05) in serum E2 levels were observed, and mRNA expression of 3ß-HSD increased by 100.64%, 113.16%, and 81.53% (p < 0.05) in the HCG, LHRH, and E2 groups, respectively. The mRNA expression of CYP11A1 in tilapia ovaries increased by 282.26% and 255.08% (p < 0.05) and mRNA expression of 17ß-HSD increased by 109.35% and 111.63% in the HCG and LHRH groups, respectively (p < 0.05). All four hormonal drugs, particularly HCG and LHRH, promoted the restoration of tilapia ovarian function to varying degrees after injury induced by combined exposure to copper and cadmium. This study presents the first hormonal treatment protocol for the mitigation of ovarian damage in fish exposed to combined aqueous phases of copper and cadmium as a strategy to prevent and treat fish ovarian damage induced by heavy metals.

Alleviating effects of essential oil from Artemisia vulgaris on enteritis in zebrafish via modulating oxidative stress and inflammatory response.

Artemisia vulgaris (A. vulgaris) is a traditional Chinese medicine widely distributed in China and contains many bioactive compounds with pharmacological effects. However, the anti-inflammatory effects and mechanism of essential oil from A. vulgaris on enteritis in fish are still unclear. In this study, in order to elucidate the underlying mechanism of essential oil from A. vulgaris on zebrafish enteritis, zebrafish were used for establishing animal models to observe the histopathological changes of intestines, determine the activities of immune-related enzymes and oxidative stress indicators, and the mRNA expression of genes in MyD88/TRAF6/NF-KB signaling pathways. The results showed that different doses of A. vulgaris essential oil could effectively alleviate zebrafish enteritis in a dose- and time-dependent manner by improving the intestinal histopathological damage, decreasing the intestinal oxidative stress, repairing the intestinal immune ability, changing the expression levels of IL-1ß, IL-10 and genes in MyD88/TRAF6/NF-κB pathway. In addition, co-treatment with oxazolone and MyD88 inhibitor could alleviate the morphological damage, the induction of oxidative stress, and the levels of immune-related enzymes and the mRNA expression of genes in MyD88/TRAF6/NF-κB signaling pathway. Moreover, essential oil from A. vulgaris had more significantly therapeutic effects on enteritis of male zebrafish than that of female zebrafish. This result will clarify the therapeutic effect and anti-inflammatory mechanism of essential oil from A. vulgaris on zebrafish enteritis, and provide a theoretical basis for further research on the rationality of A. vulgaris to replace feed antibiotics.

Self-recovery study of fluoride-induced ferroptosis in the liver of zebrafish (Danio rerio).

Ferroptosis plays a key role in fluorosis in aquatic organisms, but whether it is involved in fluoride-induced liver damage remains unclear. Previous studies have indicated that fluoride toxicity has the reversible tendency, but the mechanism of self-recovery after fluorosis in aquatic animals has not been elucidated. In this study, adult zebrafish and embryos were exposed to 0, 20, 40, 80 mg/L of fluoride for 30, 60 and 90 d and 3, 4 and 5 d post-fertilization (dpf), respectively. After 90 d, adult zebrafish were transferred to clean water for self-recovery of 30 d. The results showed that fluoride induced the prominent histopathologial changes in liver of adults, and the developmental delay and dark liver area in larvae. Fluoride significantly increased the iron overload, while decreased the expression levels of transferrin (tf), transferrin receptor (tfr), ferroportin (fpn), membrane iron transporter (fpn), and ferritin heavy chain (fth) in adults and larvae. Fluoride also induced the oxidative stress in adults and larvae by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), while decreasing the glutathione (GSH) content and the levels of glutathione peroxidase 4 (gpx4) and solute carrier family 7 member 11 (slc7a11). Self-recovery relieved fluoride-induced ferroptosis by reducing the histopathological damage and oxidative stress, reversing the expression levels of fth and slc7a11, Fe2+ metabolism and GSH synthesis. Lipid peroxidation and Fe2+ metabolism may be the key factor in alleviating effects of self-recovery on fluoride toxicity. Moreover, males are more sensitive than females. Our results provide a theoretical basis for studying the alleviating effects of self-recovery on fluoride toxicity and the underlying mechanism of its protective effect.

Interactive effects of fluoride and seleno-l-methionine at environmental related concentrations on zebrafish (Danio rerio) liver via the gut-liver axis.

Fluoride (F) is a ubiquitous aquatic environmental pollutant and co-exists with other pollutants to form combined pollution. Selenium (Se) is beneficial at low levels yet toxic at high levels and can interact with some metals. However, the interactive effects of F and Se on the liver in fish remains enigmatic. In this study, zebrafish (Danio rerio) were exposed to F (80 mg/L) and dietary seleno-l-methionine (Se-Met, 0.25, 0.5 and 1.0 µg/g dry weight) alone or in combination for 90 d. The results indicated that co-treatment to F and Se-Met attenuated the histopathological damage, oxidative stress, and inflammatory in the liver, compared with the F treatment alone. Meanwhile, dietary Se-Met treatment improved F-induced intestinal barrier dysfunction, increased the transcripts of tight junction proteins (ZO-1, Claudin-1 and Occludin), and restored the homeostasis of intestinal microbiota. Moreover, dietary Se-Met ameliorated F-induced intestinal and liver inflammation by inhibiting lipopolysaccharide (LPS) levels and transcripts of TLR4 and p65 in the intestine and liver. This study manifested that Se-Met alleviates F-induced liver and intestinal injury when both co-occur at specific concentrations, and that the gut-liver axis pathway may serve as a mechanistic base for these alleviative effects.

Comprehensive Characterization of Circular RNAs in Ovary and Testis From Nile Tilapia.

Circular RNA (circRNA) is an endogenous biomolecule in eukaryotes. It has tissue- and cell-specific expression patterns and can act as a microRNA sponge or competitive endogenous RNA. Although circRNA has been found in several species in recent years, the expression profiles in fish gonad are still not fully understood. We detected the expression of circRNA in the ovary, testis, and sex-changed gonad of tilapia by high-throughput deep sequencing, and circRNA-specific computing tools. A total of 20,607 circRNAs were obtained, of which 141 were differentially expressed in the testis and ovary. Among these circRNAs, 135 circRNAs were upregulated and 6 circRNAs were downregulated in female fish. In addition, GO annotation and KEGG pathway analysis of the host genes of circRNAs indicated that these host genes were mainly involved in adherens junction, androgen production, and reproductive development, such as ZP3, PLC, delta 4a, ARHGEF10, and HSD17b3. It is worth noting that we found that circRNAs in tilapia gonads have abundant miRNA-binding sites. Among them, 935 circRNAs have a regulatory effect on miR-212, 856 circRNAs have a regulatory effect on miR-200b-3p, and 529 circRNAs have a regulatory effect on miR-200b-5p. Thus, our findings provide a new evidence for circRNA-miRNA networks in the gonads in tilapia.

Transcriptomics integrated with metabolomics reveals the effect of Lycium barbarum polysaccharide on apoptosis in Nile tilapia (Oreochromis niloticus).

Lycium barbarum polysaccharide (LBP) is one of the main active ingredients in the fruit of L. barbarum L. It has been used as herbal medicine for thousands of years in China. In this study, Nile tilapia (Oreochromis niloticus) was taken as the research object. After feeding tilapia with 5 different doses of LBP (0 mg/kg, 500 mg/kg, 1000 mg/kg, 1500 mg/kg, 2000 mg/kg) for 55 d, it was found that LBP could promote the growth of tilapia, and this effect was the strongest at Group 1500 mg/kg. Apoptosis analysis in the liver and spleen showed that dietary supplementation with 1000 mg/kg LBP had the best protective effect on the spleen and liver in tilapia. Combined transcriptomics and metabolomics of the spleen in tilapia at Group 0 mg/kg and 1000 mg/kg showed that the differentially expressed genes (DEGs) such as NT5C2L1, pmm1, FasL and the differentially metabolites such as xanthine, dGMP, guanine and glutamate were mainly concentrated in signaling pathways such as Purine metabolism and FoxO signaling pathway. In conclusion, LBP regulates the metabolic waste levels of tilapia mainly through Purine metabolism and the FoxO signaling pathway, thereby inhibiting cell apoptosis, improving the utilization of nutrients, and promoting the growth of tilapia. This study not only provides a theoretical basis for the application of LBP in aquatic animals but also provides useful information for the healthy development of the aquaculture.

Cyclophosphamide affects eye development and locomotion in zebrafish (Danio rerio).

Cyclophosphamide (CP) is a broad-spectrum anticancer drug and has been frequently detected in aquatic environments due to its incomplete removal by wastewater treatment facilities and slow degradation in waters. Its toxicity in fish remains largely unknown. In this study, zebrafish eggs <4 h post fertilization (hpf) were exposed to CP at the concentrations from 0.5 to 50.0 µg/L until 168 hpf, and its toxicity was evaluated by biochemical, transcriptomic, and behavioral approaches. The results showed that malformation and mortality rates increased with CP concentrations. The 7-day malformation EC50 and mortality (LC30) by CP were calculated to be 86.8 µg/L and 7.5 mg/L, respectively. Inhibited startle response (light to dark) (a minimal of 19%) and reduced swimming velocity (a minimal of 30%) were observed in the CP-exposed larvae. The thicknesses of retinal ganglion layer, inner plexiform layer, and inner nuclear layer in the retina were increased after exposure to CP. Meanwhile, exposure to CP increased karyorrhexis and karyolysis in the liver tissue. Transcriptomic analysis identified 607 differentially expressed genes (DEGs) (159 up-regulated and 448 down-regulated). A significant reduction in the transcripts of sgk1 (the FoxO pathway), jun (the MAPK pathway), and diabloa (apoptosis pathway) were observed in the CP-treated larvae. This study has demonstrated that low concentrations of CP cause malformation, reduced swimming capacity, histopathological alterations in the retina and liver tissues, and interference on transcriptional expressions of key genes associated with different pathways. The ecological risk of CP and other anticancer drugs to aquatic organisms merits future investigation.

Low expression of miR-19a-5p is associated with high mRNA expression of diacylglycerol O-acyltransferase 2 (DGAT2) in hybrid tilapia.

DGAT2 (acyl CoA:diacylglycerol acyltransferase 2) is a key and rate-limiting enzyme that catalyzes the final step of triglyceride (TG) synthesis. In this study, hybrid tilapia were generated from Nile tilapia (♀) and blue tilapia (♂) crossing. The TG content levels in the liver of these tilapia were measured. The results showed that the TG content was higher in the hybrid tilapia. In addition, protein and mRNA expression levels in the tilapia livers were determined. Higher hepatic mRNA and protein expression of DGAT2 in the hybrid fish was found. A luciferase reporter assay with HEK293T cells revealed that miRNA-19a-5p targeted the 3'UTR of DGAT2, suggesting a direct regulatory mechanism. Using qRT-PCR, we found that DGAT2 mRNA levels had a negative correlation with miRNA-19a-5p expression in Nile tilapia and hybrid. Taken together, these findings provide evidence that miRNA-19a-5p is involved in TG synthesis in the regulation of lipid metabolism in tilapia.

Fluoride exposure changed the expression of microRNAs in gills of male zebrafish (Danio rerio).

Fluoride has been found to cause detrimental effects on fish gills. Despite essential roles in various metabolism activities, whether and how miRNAs participate in the course of toxicity caused by fluoride in gills is still unclear. In this study, male zebrafish were exposed to 0, 20, 40 mg/L fluoride for 60 days to study the underlying osmotic regulatory mechanism by determining the influences of fluoride on the miRNAs and regulated genes in gills. mRNAs were isolated from the gills and the expression profiles were analyzed by using Illumina Hiseq 2500 platforms. Expressions of 7 differentially miRNAs and some related-genes in gills were validated by qRT-PCR. The results showed that miRNAs expressions were notably altered by fluoride. A total of 584 and 327 miRNAs were remarkably changed after 20 and 40 mg/L fluoride exposure, of which 322 were increased and 262 were decreased in 20 mg/L fluoride group, whereas 219 were elevated and 108 were reduced in 40 mg/L fluoride group. The differentially expressive miRNAs confirmed by qRT-PCR were consistent with micro-assay data. Cluster of Orthologous Groups of proteins (COG) function classification showed that the target genes of differentially expressive miRNAs are mainly related to signal transduction mechanisms, replication, transcription, inorganic ion transport and metabolism, repair and recombination, and energy formation and transformation. In addition, fluoride disturbed the expressions of target genes involved in the osmoregulation of the gill in the fluoride-exposed zebrafish, such as the increased expressions of OSTF1 and the decreased expressions of Na+-K+-ATPase, CFTR, and AQP-3, which provides a possibility that miRNAs regulation induced by fluoride has an effects on osmotic regulation, providing new hints to the osmotic regulatory mechanism of the toxicity caused by fluoride in zebrafish, and distinguishes new biomarkers of miRNAs for fluoride toxicity.

Sesamin attenuates histological alterations, oxidative stress and expressions of immune-related genes in liver of zebrafish (Danio rerio) exposed to fluoride.

Sesamin is the main lignan in sesame and is reported to have many benefits and medicinal properties. However, its protective effects against fluoride-induced damage in the liver of zebrafish have not been elucidated. Our previous studies found that fluoride exposure caused damage to the liver of zebrafish. In the study, the effects of sesamin on oxidative stress and immune damage in liver of zebrafish exposed to fluoride were measured. The results indicated that fluoride exposure damaged the microstructures of liver, increased significantly the oxidative stress, decreased remarkably the activities of ACP, AKP, and LZM, and affected obviously the expressions of immune-related genes. Treatment with sesamin remarkably attenuated fluoride-induced liver damage in a dose-dependent manner, indicated by the histopathological observation. Furthermore, sesamin treatment also significantly inhibited the production of ROS and oxidative stress, such as the decrease of lipid peroxidation level and the increase of CAT and SOD activities in liver. Sesamin treatment reversed the activities of immune-related enzymes and the expressions of immune-related genes in liver exposed to fluoride. These findings suggested that sesamin could protect the liver from fluoride-induced immune damage by oxidative stress downstream-mediated changes in reversing the activities of immune-related enzymes and the expressions of immune-related genes. Taken together, sesamin plays an important role in maintaining hepatic health and preventing liver from toxic damage caused by fluoride.

The effects of norethindrone on the ontogeny of gene expression along the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes in zebrafish (Danio rerio).

Little is known about the molecular effects of progestins on the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes in fish prior to sexual differentiation. In this study, the effects of norethindrone (NET) on the ontogeny of HPG- and HPA-related genes in zebrafish embryo/early larvae prior to sexual differentiation were evaluated. Embryo/larvae were exposed to different concentrations (5, 50, 500 ng/L) of NET for 6 days. The levels of the transcripts of the genes closely related to the HPG and HPA axes were determined daily during 3 stages (embryo, embryo/larvae transition, and early larvae). The results showed that most genes were up-regulated and the ontogeny of genes in the HPA axis was earlier than that of HPG axis, especially for the upstream genes of both the HPG (gnrh2, gnrh3, fshb, lhb) and the HPA (crh, pomc, star) axes. In contrast, the transcriptional expressions of genes of the cortisol/stress pathway (cyp11b, mr) were inhibited and those of the progesterone pathway were not affected. More importantly, NET exposure induced the expressions of the genes (esr1, vtg1, hsd17b3, hsd11b2, ar) that are closely related to the steroid hormone pathways in the embryos/larvae stages, implying a precocious effects of NET in zebrafish. This study demonstrates that NET alters the expression of HPA- and HPG-axes related genes in zebrafish at early stages, pointing to the need for the same type of analysis during the zebrafish gonadal differentiation window.

Genome-wide analysis revealed the virulence attenuation mechanism of the fish-derived oral attenuated Streptococcus iniae vaccine strain YM011.

Streptococcus iniae has become one the most serious aquatic pathogens causing invasive diseases in farmed marine and freshwater fish worldwide, and orally attenuated vaccine is still the best option in protecting these invasive diseases. In this study, the safety, stability, immunogenicity of the S. iniae attenuated strain YM011 were evaluated, and comprehensively analyzed its virulence weakening mechanism at whole genome level. The results shown that attenuated S. iniae strain YM011 completely lost its pathogenicity to tilapia and had good immunogenicity with relative percent survival being 93.25% at 15 days and 90.31% at 30 days via IP injection, respectively, and 76.81% at 15 days and 56.69% at 30 days via oral gavage, respectively. Back-passage safety assay indicated that YM011 did not cause diseases or death in tilapia after 100 generations of serial passaging. Comparative genome-wide sequencing shown that YM011 had a 0.4 M large inversion fragment compared with its parental strain virulent strain GX005, which encoded 372 genes including drug resistance genes pbp2A and tet, as well as known virulence factors including hemolysin transport system gene, recA, and mutator family transposase. The attenuated S. iniae strain YM011 is an ideal attenuated oral vaccine candidate with good immunogenicity, safety and stability. Abnormal expression of important drug resistance genes as well as known virulence factors due to inversion of a 0.4 M large fragment is the leading mechanism underlying its attenuated virulence.

Genome-Wide DNA Methylation and RNA Analysis Reveal Potential Mechanism of Resistance to Streptococcus agalactiae in GIFT Strain of Nile Tilapia (Oreochromis niloticus ).

Streptococcus agalactiae is an important pathogenic bacterium causing great economic loss in Nile tilapia (Oreochromis niloticus) culture. Resistant and susceptible groups sharing the same genome showed significantly different resistance to S. agalactiae in the genetically improved farmed tilapia strain of Nile tilapia. The resistance mechanism is unclear. We determined genome-wide DNA methylation profiles in spleen of resistant and susceptible O. niloticus at 5 h postinfection with S. agalactiae using whole-genome bisulfite sequencing. The methylation status was higher in the spleen samples from resistant fish than in the susceptible group. A total of 10,177 differentially methylated regions were identified in the two groups, including 3725 differentially methylated genes (DMGs) (3129 hyper-DMGs and 596 hypo-DMGs). The RNA sequencing showed 2374 differentially expressed genes (DEGs), including 1483 upregulated and 891 downregulated. Integrated analysis showed 337 overlapping DEGs and DMGs and 82 overlapping DEGs and differentially methylated region promoters. By integrating promoter DNA methylation with gene expression, we revealed four immune-related genes (Arnt2, Nhr38, Pcdh10, and Ccdc158) as key factors in epigenetic mechanisms contributing to pathogen resistance. Our study provided systematic methylome maps to explore the epigenetic mechanism and reveal the methylation loci of pathogen resistance and identified methylation-regulated genes that are potentially involved in defense against pathogens.

Effects of dietary Cu and Zn on the accumulation, oxidative stress and the expressions of immune-related genes in the livers of Nile tilapia (Oreochromis niloticus).

Excess Cu and Zn can cause many adverse effects in fish. However, few studies have addressed the effects of dietary Cu and Zn on antioxidant physiology and immunity and the underlying mechanisms in fishes. In this study, accumulation of Cu and Zn, effects on the antioxidant enzymes and the transcriptional expressions of immune-related genes were examined in the Oreochromis niloticus fed the Cu and/or Zn enriched duckweed. The results showed that the liver and intestine had the highest accumulation of Cu2+ and Zn2+ while the muscle had the lowest accumulation of these two metals. The activities of SOD, CAT, GPx and the contents of GSH, GSSG in the liver of all treatment groups were significantly decreased compared to the control group. MDA content was significantly elevated in all treatment groups after feeding for 21 days, implying lipid peroxidation in the liver. In the Cu + Zn group, the activities of SOD, GPx and the GSSG content in the liver were significantly decreased. Compared with the Zn group, the LZM activity in the Cu + Zn group was reversed after feeding for 42 days (P < 0.05). The transcriptional expressions of immune-related genes (TNF-α, INF-γ and IL-1ß) in Cu, Zn, Cu + Zn groups were significantly inhibited compared with the control group after treatment for 21 days. Compared with the Cu + Zn group, the level of INF-γ transcripts was significantly reduced in the Cu and Zn group, while the TNF-α expression was elevated after treatment for 42 days. Cu and Zn had synergistic effects on the antioxidant system. Cu has greater effects than Zn on the immunity of O. niloticus. This study demonstrates that dietary Cu and Zn may pose a potential threat to the tilapia populations.