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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-1β, 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.

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.

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 (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.

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.

Streptococcus agalactiae is a common pathogen in aquatic animals, especially tilapia, that hinders aquaculture development and leads to serious economic losses. Previously, a S. agalactiae strain named HN016 was identified from infected tilapia, and the attenuated strain YM001 was subsequently obtained by continuous passaging in Tryptic Soy Broth (TSB) medium. YM001 has been demonstrated as a safe vaccine for S. agalactiae infection in tilapia. To understand the molecular bases of the virulence of these two strains, we performed proteomic and transcriptomic analysis to reveal the protein and gene expression changes in the liver and intestine during the infection process. HN016 significantly decreased the contents of white blood cells (WBCs), neutrophils (NEUs), red blood cells (RBCs) and hematocrit (HCT) and increased the levels of total protein (TP), albumin (ALB) and globulin (GLO), while no such significant differences were observed when comparing the control with YM001. During the infection process, pathogenic peptidoglycan hydrolase, CSPA and membrane proteins were significantly differentially expressed between YM001 and HN016. Furthermore, both proteome and transcriptome data showed that the complement and coagulation cascades pathway and the antigen processing and presentation pathway were stimulated in the liver and intestine, respectively, by YM001 infection compared to HN016 infection. The interaction network analysis of key virulence genes from pathogens suggested that CSPA, as a key node, affects the expression of DOLPP1, MIPEP, PA2G4, OCIAD1, G3BP1 and CLIC5 with a positive correlation. The present evidence suggests that during the infection process, CSPA was the key genes contributing to low virulence in YM001.

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

Ghrelin is a peptide hormone secreted by gastrointestinal tract which regulates multiple physiological processes such as appetite, metabolism, growth and gonad development in fish. In the present study, the effects of ghrelin on hybrid tilapia infected with Aeromonas hydrophila are elucidated. Juvenile hybrid tilapia fish (20.0 ± 5.0 g) were intraperitoneally injected with 0, 0.1, 1.0, or 10.0 ng/g ghrelin/body weight synthetic ghrelin alone or in combination with A. hydrophila (0.5 × 106 CFU). At 10 days post treatment, the survival rate in the group that received 1.0 ng/g ghrelin/body weight ghrelin in combination with A. hydrophila was higher (66.66%) than that of the Ah group (13.33%) that received A. hydrophila alone. In tilapia that received ghrelin injections, reactive oxygen species (ROS) levels tended to increase at 5 h, while injection of 10.0 ng/g ghrelin/body weight ghrelin resulted in a significant decrease in ROS levels at 10 h. No changes in serum immune or antioxidant-related indicators were observed in fish injected with A. hydrophila compared to controls. However, ghrelin injection decreased Albumin (ALB), glutathione peroxidase (GSH-Px), lysozyme (LZM) and superoxide dismutase (SOD). Histological analysis showed that ghrelin injection alleviated the pathological changes in liver and spleen caused by A. hydrophila infection. Overall, the expression of HSP70, IL-1β, and TGF-β in the liver tended to upregulate compared to the control. In the kidney, HSP70, IL-1β and TGF-β levels were increased, and TNF-α expression levels were decreased compared to the control. The HSP70 level in the spleen was decreased, and IL-1β, TGF-β, and TNF-α were expressed at significantly higher levels in the spleen in the tilapia that received ghrelin injections. Taken together, our results indicate that injection with 1.0 ng/g ghrelin/body weight ghrelin may effectively protect juvenile hybrid tilapia against A. hydrophila infection by improving hematological indicators, maintaining normal histology and regulating cytokine gene expression.

In this study, the effect of Lycium barbarum polysaccharides (LBP) on immunological parameters, apoptosis, and growth performance of Nile tilapia (Oreochromis niloticus) was investigated. Dietary supplementation with LBP significantly increased complement 3 (C3) activity and promoted interleukin IL-1β gene expression in spleen tissue, significantly reduced apoptosis in spleen tissue, increased the specific growth rate (SGR), relative length gain (LG), and relative weight gain (WG) of Nile tilapia. However, dietary supplementation with LBP did not have a significant effect on serum alkaline phosphatase (AKP), malondialdehyde (MDA), and superoxide dismutase (SOD), blood constituents, apoptosis, or gene expression of IL-1β in liver tissue. Overall, the results showed that dietary supplementation with LBP increased the nonspecific immunity of Nile tilapia and reduced the apoptosis rate to promote growth and development. Thus, LBP has potential for use as a new immunostimulant in aquaculture.

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, Fe

In mammals, Galectin-3 has been revealed to be widely expressed in immune cells and played important role in immune reactions. However, Galectin-3 is frequently less reported in teleost. In the present study, a molecular characterization and expression analysis of galectin-3 were conducted in GIFT strain Nile tilapia. The full-length cDNA is 1034 bp with 690 bp of protein coding sequences. The result of qRT-PCR showed that the mRNA of galectin-3 was widely expressed in various tissues (heart, liver, spleen, gill, kidney, brain, intestine, skin, muscle, and ovary), and the higher expression was observed in immune-related tissues (liver and spleen). The time-course expression analysis revealed that galectin-3 was significantly up-regulated in intestine (5 h, 50 h, and 7 d), liver (5 h, 50 h, and 7 d), spleen (5 and 50 h), head-kidney (5 and 50 h), gill (5 h and 7 d) after Streptococcus agalactiae challenge, and significantly up-regulated in intestine (18, 24, 36, 72, and 96 h), liver (6, 18, 24, 96 h, and 6 d), spleen (18, 24, 36, 72, and 96 h), head-kidney (6, 12, 18, 24, 36, 72, and 96 h), and gill (12, 18, 24, and 36 h) after Aeromonas hydrophila challenge. Taken together, these data suggest that galectin-3 plays a role in immune responses in Nile tilapia after bacterial challenge.

Parachromis managuensis is a native cichlid fish from Central America that has been recently introduced to Southern China. The present study aimed to identify the adaptive evolution of P. managuensis using transcriptome data and provided genetic information regarding this novel fish in China. We obtained the transcriptome sequences from a mixed cDNA library of P. managuensis. A total of 19,419,739 raw reads were obtained from the cDNA library. The de novo assembly generated a total of 102,977 unigenes. The genomic data of Atlantic cod (Gadus morhua) and Nile tilapia (Oreochromis niloticus) were used to identify orthologs by phylogenetic analysis. Based on the phylogeny, we detected 105 positively selected genes in P. managuensis from 6197 orthologous unigenes. The GO annotation revealed that the T-box protein 20 (TBX20) in the category ‘Immune system process’ was positively selected and four positively selected genes were found in the ‘Immune system’ pathway by KEGG analysis. Among them, the genes encoding for phosphoinositide 3-kinase adapter protein 1, ras-related protein Rap-1b, complement factor I and probable ATP-dependent RNA helicase DHX58 were associated with immune adaptation. These findings can provide additional information regarding the natural selection of P. managuensis and can be used as a representative example for future comparative transcriptome and immunological studies.

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.

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.

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.

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 eggs4 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 EC

Selenite(IV) and selenate(VI) are the major forms of Se in aquatic ecosystem. In this study, Pseudorasbora parva were exposed to 10, 200 and 1000 μg L−1 selenite and selenate for 28 days. Selenium accumulation, antioxidant enzyme levels, glutathione concentrations, lipid peroxidation and histology were evaluated in livers following exposure. Our results showed that Se(IV) and Se(VI) caused different accumulation patterns in the liver, with a more rapid accumulation of Se with Se(IV) treatment. Both Se species increased hepatic lipid peroxidation after 14 and 28 d (~ 30%). Among the antioxidants examined, the activity of SOD (except day 28) and the cellular levels of GSH were induced by 72–137% at lower concentrations, while the activity of GST was at least 24% lower than that of the control at 200 and 1000 μg L−1 for both Se species at all sampling points. Both forms of Se reduced the hepatosomatic index at 1000 μg L−1 after 28 d. In addition, marked histopathological alterations (10–31%) were observed in the liver of P. parva after exposure to both Se species, with higher frequency in the Se(IV) exposed fish. Liver local necrosis was observed only in the liver of fish exposed to 1000 μg L−1 of Se(IV) (~ 20%). Our results suggest that the ecological impacts of dissolved Se in this freshwater species may also contribute to overall toxicity.

In recent years, many new chemicals have been synthesized from biomass with an aim for sustainable development by replacing the existing toxic chemicals with those having similar properties and applications. However, the effects of these new chemicals on aquatic organisms remain relatively unknown. In this study, the effects of bisphenol A (BPA) and lignin-derived bisphenol (LD-BP, a BPA analogue) on Daphnia magna were evaluated. The animals were exposed to BPA, LD-BP, and their binary mixture at concentrations (2-2000 μg L-1) for 21 days. The expression of various biochemical markers and the effects on growth, molting, and reproduction parameters were examined. The results showed that the weight of daphnids significantly increased after exposure to BPA, LD-BP, and the binary mixture relative to that of the control animals. The activity of superoxide dismutase was significantly inhibited by LD-BP and the binary mixture. At the highest exposure concentration of the binary mixture, the activities of acetylcholinesterase and α-glucosidase, fecundity, and the number of neonates per brood were significantly altered. Our results showed that the effects of BPA and LD-BP on D. magna were generally comparable, except for the effect on the weight at their environmentally relevant concentrations (e.g.

Scenic cycleway leads a physically active way of recreation, introduces a low carbon tourist economy to remote and less developed areas. Its construction and designation are increasingly been considered as valid policies for public health and sustainable development. Aiming at encouraging more and better usage of scenic cycleway, this paper adopts affordance theory to explore the complex interaction between cyclists and cycleway environments. Based on previous theoretical work on environmental affordances, we have developed a three-dimensional analytical framework to examine the supply and demand, patterns, and factors of the actualisation of affordances, using a cycleway in the outskirts of Kunming, China as a case study. Results show that 1) scenic cycleway usage includes recreational, incidental, consumptive and sportive categories, consists of 18 types of affordances, and their supply-demand relationships indicate the general usage condition of the cycleway; 2) environment’s function meanings and physical attributes both affect the actualisation of affordances: attractions, amenities, natural areas, and locations with cultural significance invite cyclists to perceive and utilise affordances, whilst everyday environments discourage the actualisation of affordances; 3) Roadway, roadside and climate are the main factors of affordance actualisation, hereinto, natural landscape, traffic operation, and climatic factors are the most significant features that influence recreational cycling. The above findings present a pathway for the evidence-based planning and design of scenic cycleways, so as to consolidate the health and sustainable benefits from recreational cycling.

Innate immune system is the primary defense mechanism against pathogen infection in teleost, which are living in pathogen-rich aquatic environment. It has been long hypothesized that the disease resistance in teleost are strongly correlated to the activities of innate immune genes. Tilapia is an important economical fish around the world, especially in China, where the production accounts for nearly half of the global production. Recently, S. agalactiae has become one of the most serious bacterial diseases in southern China, resulted in high cumulative mortality and economic loss to tilapia industry. Therefore, we sought here to characterize the expression profiles of tilapia against S. agalactiae infection at whole transcriptome level by RNA-seq technology. A total of 2822 genes were revealed significantly expressed in tilapia spleen with a general trend of induction. Notably, most of the genes were rapidly the most induced at the early timepoint. The significantly changed genes highlighted the function of pathogen attachment and recognition, antioxidant/apoptosis, cytoskeletal rearrangement, and immune activation. Collectively, the induced expression patterns suggested the strong ability of tilapia to rapidly recognize the invasive bacteria, and activation of downstream immune signaling pathways to clear the bacteria and prevent the tissue damage and bacteria triggered cell apoptosis. Our results heighted important roles of novel candidate genes which were often missed in previous tilapia studies. Further studies are needed to characterize the molecular relationships between key immune genes and disease resistance, and to identify the candidate genes for molecular-assistant selection of disease-resistant broodstock and evaluation of disease prevention and treatment measures.

Selenium is an essential element but toxic at high levels in animals. The effects of Se on growth performance and the immune system in Nile tilapia remain inconclusive. In this study, Nile tilapia Oreochromis niloticus was fed on selenium yeast (Se(Y))- and selenite (Se(IV))-enriched feed at 0, 3, 6, and 12 μg/g (dry wt) for 45 and 90 d. The growth, bioaccumulation, biochemical markers related to antioxidant, immunological, nervous and digestive systems were evaluated in various fish tissues (liver, intestine, kidney, muscle, brain, spleen, gills). The results showed that the accumulation of Se(Y) was 1.3–2 folds of Se(IV) in most tissues. The growth of tilapia was enhanced by both Se(Y) and Se(IV) at 3 μg/g after 90 d, with Se(Y) better than Se(IV) in tilapia feed. After 45 d, the levels of lipid peroxidation, the activity of the antioxidant enzymes, and the transcriptional levels of the immune related genes (IL-1β, IFN-γ and TNF-α) and stress proteins (HSP70 and MT) were enhanced in all treatments, except that of MT in the 12 μg/g Se(Y) group. In addition, both Se species inhibited the activity of acetylcholinesterase (AChE) in the brain and one digestive enzyme α-glucosidase (α-Glu) in the intestine at 12 μg/g. However, after 90 d, the effects on most biochemical markers were less pronounced, implying a possible acclimation after prolonged duration. The results demonstrate Se is beneficial to O. niloticus at low levels and toxic at elevated levels. The immunostimulation by Se might be greatly weakened after long term feeding Se-enriched feed. This study helps to better understand the effects of Se on the antioxidant and immune systems and to establish the optimal Se levels in the feed and duration for O. niloticus.

Estradiol (E2) is a sex steroid hormone that modulates multiple physiological processes in teleosts. The aim of this study was to explore the role of E2 in the hepatic lipid metabolism of hybrid tilapia. The hybrid tilapias were injected with different concentrations of E2 (0 mg/kg, 10 mg/kg, 25 mg/kg and 50 mg/kg) and ICI 182,780 (ICI) (35 mg/kg) (an E2 receptor antagonist). Subsequently, the liver lipid depositions were analyzed by tissue sections with oil red O staining. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and nonesterified fatty acids (NEFAs) were assayed from the fish in different groups. Genes related to very low-density lipoprotein (VLDL) assembly, lipoprotein lipase and lipoprotein receptors, fatty acid uptake and triacylglycerol metabolism were determined by quantitative RT-PCR. The results showed that 50 mg/kg E2 injections enlarged the lipid droplets significantly. Simultaneously, the E2 injections tended to upregulate TC, TG, LDL, and HDL in the serum. The 50 mg/kg E2 group showed a significantly higher expression of the VLDL assembly genes but depressed levels of LDLR and LRP1. In addition, FABP3, FABP11a and DGAT2 were significantly elevated, while CD36 and ACO1 decreased in the 50 mg/kg E2 injection. The ICI injection inhibited the expression of MTP, LPL, LRP1, CD36, FABP11a, ACO1 and FAS in tilapia livers. These results demonstrated that by stimulating the expression of genes associated with the VLDL assembly, inhibiting lipoprotein lipase and lipoprotein receptor-related genes and promoting the rate-limiting enzyme in the synthesis of the TG, E2 induced deposition of lipids in the livers of hybrid tilapia. Overall, the results suggest a role for E2 in fish lipid metabolisms that provide new clues to illustrate the sex steroid function in energy metabolism in livers.

Selenite(IV) and selenate(VI) are the major species of Se in the seleniferous aquatic ecosystem. The redistribution of Se in the water/sediment microcosm by bioturbation remains largely unknown. In this study, the redistribution of Se in the water/sediment microcosm by the benthic oligochaete Limnodrilus hoffmeisteri was assessed. The worms were exposed to 2–40 μg/g dry weight of Se(IV) or Se(VI) in the sediment (diet) for 2 months. The changes in the Se levels in different compartments of the microcosm (sediment, overlying water, and worms) were quantified after 2 weeks and 2 months. The subcellular distribution of Se in the worms were also evaluated. Finally, the volatilization of Se from the two Se sources was estimated. The results showed that Se concentration in the overlying water and Se bioaccumulation in the worms were increased with Se levels in the sediments. Approximately 1.6–9.8% of Se was volatilized in the absence of the worms and was intensified in the presence of the worms (2.1–25.7%). The subcellular distribution witnessed high levels of Se in the cell debris (>60%). Se(IV) and Se(VI) differ in their bioaccumulation, redistribution and the effects on the growth of the worms. Our results suggest that the bioturbation by benthos play an essential role in the redistribution of Se in the water/sediment microcosm.

Excessive perfluorooctane sulfonate (PFOS) in natural water ecosystem has the potential to detrimentally affect immune system, but little is known of such effects or underlying mechanisms in fish. In the present study, we evaluated the effects of PFOS on growth performance, organizational microstructure, activities of immune-related enzymes and expressions of immune-related genes in male zebrafish (Danio rerio) exposed to different concentrations of 0, 0.02, 0.04 and 0.08 mg/L of PFOS for 7, 14, and 21 days or cotreatment with PFOS and PDTC to investigate the effects of PFOS on immune system and the potential toxic mechanisms caused by PFOS. The results indicated that PFOS accumulated in livers after exposure, and remarkably elevations were found in three exposure groups compared with the control group at three stages. The growth of the adult zebrafish in the experiments was significantly inhibited, the microstructures of liver were serious damaged. The ROS levels were remarkably increased. The activities of ACP, AKP, and lysozyme were obviously decreased, while the activities of MPO and NF-κB were significantly increased. The expressions of immune-related mRNA were significantly affected. After co-treatment with PFOS and PDTC, the growth inhibition, the morphological damage, the ROS induction, and the expressions of immune-related mRNA were reversed. Taken together, the results indicated that PFOS can significantly inhibit the growth, disturb the immune system by changing the normal structure of liver, the activities of immune-related enzymes, and a series of gene transcriptions involved in immune regulation in liver of male zebrafish. PFOS-induced pro-inflammatory effect of hepatocytes was observed, and the involvement of NF-κB signaling pathway was participated in its action mechanism. These findings provide further evidence that PFOS interferes with the immune regulation of liver of male zebrafish under in vivo conditions.

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.

It has been shown that piRNA is the largest class of small non-coding RNA in germline cells of animals which plays key roles in transposons regulation and transcriptional activities. In the present study, piRNAs from two small RNA libraries including ovary and testis of Nile tilapia were identified and characterized. By length and k-mer based small RNA prediction algorithm, 279,059 and 583,230 small RNA reads were confirmed as piRNA from ovary and testis, respectively. The identified piRNAs showed evolutionarily conserved characterization, such as uridine bias in the 5′ ends. The 142,961 and 296,775 piRNAs from ovary and testis were mapped to the draft assembly of the tilapia genome, respectively. Both ovary and testis piRNAs were enriched from linkage (LG)6 and LG7. Meanwhile, the even distribution of +strand and −strand suggested the Ping–pong pathway (a double-displacement reaction of +strand and −strand) hypothesis. These piRNAs were derived from the upstream −2 kb and downstream +2 kb as well as gene regions which suggested a regulatory function on transcription activities. In gene regions, abundant piRNAs were derived from 5′UTR, 3′UTR and CDS. Furthermore, we characterized the differentially expressed piRNAs between ovary and testis. In total, 1979 and 2453 piRNAs were significantly higher and lower expressed in ovary compared to that in testis, respectively. Thereinto, the most concentrated up-regulate and down-regulate piRNAs were both from serine/threonine–protein kinase PIM genes of different transcripts. These findings will be helpful to facilitate studies on the piRNAs regulation on genes during gonad development of teleosts.

In this study, the tilapia was orally vaccinated by the attenuated Streptococcus agalactiae(S. agalactiae) strain YM001, and the distribution and the pathological effect of strain YM001 in different intestinal segments of tilapia were evaluated by real-time PCR(qPCR), immunohistochemistry(IHC) and histomorphology. The qPCR results showed that the number of bacteria was the highest in the intestinal tracts at 12 h post oral gavage in the YM001 group, then began to decrease sharply and eliminated at 7 d. And the number of bacteria was highest in the foregut, hindgut, and rectum at 12 h, 24 h, and 3 d, respectively. IHC indicated that bacteria mainly distributed in the margin epithelium and the goblet cells at 12 h - 24 h, and in the submucosa and muscle layer in the YM001 group in 3 d post gavage, then almost disappeared at 7 d. Histological examination of intestines post gavage displayed that an inflammation was observed at 7 d in the YM001 group and the intestinal structure was fully recovered at 15 d. and the intestinal structure was fully recovered at 15 d. Conclusion: The attenuated S. agalactiae vaccine strain YM001 could enter the intestinal tissue after oral gavage and had a strong spatial and temporal selectivity in the intestinal tract, which could cause obvious mucosal immune response and mild pathological reaction, but the pathological change could be gradually repaired with the extinction of bacteria in the body.

Selenium (Se) might be protective against oxidative stress at nutritional levels, but elevated Se concentrations in the diet has been revealed as the main culprit for the extinction of natural fish populations in Se-contaminated lakes. Though Se predominate as waterborne selenite (IV) and selenate (VI) in the water, the differences in bioaccumulation, effects (e.g., oxidative stress, antioxidants etc.) and molecular mechanisms between Se(IV) and Se(VI) have been relatively understudied in wild fish. In this study, the P. parva were exposed to waterborne Se (10, 200 and 1000 μg/L of Se(IV) or Se(VI)) and sampled at 4, 14 and 28 days. Bioaccumulation, tissue distributions of Se and following effects in different tissues were evaluated. The results showed that the levels of Se in the gills and intestine were significantly elevated with a seemingly concentration-dependent pattern in the Se(IV) treatment, with respectively 173.3% and 57.2% increase after 28 days of exposure, relative to that of Se(VI) treatment. Additionally, significant accumulation of Se was also observed in the muscle of Se(IV) treated fish. Se exposure increased the MDA levels in the brain and gills in the Se(IV) treatment, but less apparent in the Se(VI) treatment. Meanwhile, Se exposure lowered (at least 56%) the activity of GST in the gills, but increased the activity of AChE in the muscle (~69%) and brain (~50%) after 28 d. Most importantly, after 28 d of exposure, Se exposure caused significant decrease in GSH levels in the gills (at least 35%) and in all tissues examined at the highest test concentration. In general, the results showed that Se(IV) led to faster accumulation of Se than Se(VI) in P. parva, and the resulted lipid peroxidation was closely related to the levels of antioxidants, especially GSH. Our results suggest that the ecotoxicological effects of waterborne selenite and selenate differ in this freshwater species in the field.

One of the highest priority areas for improvement is the development of effective strategies for decreasing disease mortality levels in aquaculture production, a better understanding of the components of the fish immune system and their functions in the context of pathogen invasion is needed. Tilapia is the most common fish in South China, and Streptococcus agalactiae has become the most serious disease problem for tilapia industry in China. Here, we profiled gene expression differences between tilapia differing in their susceptibility to S. agalactiae both basally (before infection) and at three early timepoints post-infection (5 h, 50 h, and 7 d). Between group comparisons revealed 5756 unique genes differentially expressed greater than 2-fold at one or more timepoints. And the resistant fish showed much more strong ability in pathogen recognition, antigen presentation, immune activation, while the susceptible fish showed fast activation of apoptosis. Taken together, the immune profiles expand our knowledge for molecular mechanisms for disease resistance, as well as provide solid molecular resources for further identification of the candidate markers for disease-resistant selection and evaluation of disease prevention and treatment options for tilapia industry.

Cadmium (Cd) is one of the most toxic heavy metals in aquatic ecosystem which affects fish health and aquaculture. In the present study, we examined the bioaccumulation of Cd in the gonads of tilapia via dissolved and dietary routes. We evaluated the subchronic effects of Cd on the histology of gonads, steroid hormone levels and sex-related gene expressions in tilapia. In addition, we also studied maternal transfer of Cd. Our results indicated that Cd was accumulated significantly in both ovary and testis from both exposure routes. Histopathological analysis showed that Cd induced ovary and testis injuries. Estradiol levels were significantly increased in dissolved Cd exposed female fish. In addition, the Cd exposure displayed different effects on gene expressions in gonads. In females, the estrogen receptor (ERα) was stimulated in dissolved Cd-exposed fish at 70.32 and 143.78 μg/L for 30 days and in fish at 143.78 μg/L for 60 days. Vitellogenin expression was significantly down-regulated in the ovary of dissolved Cd-exposed fish. In testis, GR expression was elevated after 60 days of dissolved Cd and dietary exposure. Furthermore, Cd level was significantly higher in the eggs than that in the fry. Our results demonstrated that both dissolved and dietary Cd exposures affected gonad development by altering steroid hormone levels and sex-related gene expressions in tilapia.

Streptococcus iniae is the most significant bacterial disease of tilapia throughout the world, and commonly leads to tremendous economic losses. In contrast to other important fish species, our knowledge about the molecular mechanisms of tilapia in response to bacterial infection is still limited. Here, therefore, we utilized RNA-seq to first profiling of host responses in tilapia spleen following S. iniae infection at transcriptome level. A total of 223 million reads were obtained and assembled into 192,884 contigs with average length 844 bp. Gene expression analysis between control and infected samples at 5 h, 50 h, and 7 d revealed 1475 differentially expressed genes. In particular, the differentially expressed gene set was dramatically induced as early as 5 h, and rapidly declined to basal levels at 50 h. Enrichment and pathway analysis of the differentially expressed genes revealed the centrality of the pathogen attachment and recognition, cytoskeletal rearrangement and immune activation/inflammation in the pathogen entry and host inflammatory responses. Understanding of these responses can highlight mechanisms of tilapia host defense, and expand our knowledge of teleost immunology. Our findings will set a foundation of valuable biomarkers for future individual, strain, and family-level studies to evaluate immune effect of vaccine and individual response in host defense mechanisms to S. iniae infection, to select disease resistant families and strains.

Risk assessing newly synthesized chemicals prior to their applications is extremely important, if we want to ensure substitution of risky chemicals with more benign ones. During the past two decades, many analogs of bisphenol A (BPA) have been manufactured, while their toxicity remains less studied. The aim of this study was to compare the acute toxicity of a synthesized lignin-derived BPA (LD-BP) with that of BPA in representative aquatic organisms including two algal species (Chlorella pyrenoidosa and Scenedesmus obliquus), a cladoceran species (Daphnia magna), and the Japanese medaka (Oryzias latipes). The results revealed that the two algal species showed different responses to the two chemicals. For C. pyrenoidosa, both BPA and LD-BP stimulated growth within 48 h of exposure, except for the 50 mg L−1 of LD-BP treatment. After 96 and 144 h of exposures, BPA stimulated the growth of C. pyrenoidosa at low-exposure concentrations but inhibited its growth at high concentrations, while LD-BP caused a concentration-dependent response in C. pyrenoidosa. S. obliquus exhibited a monotonic concentration-response curve for both BPA and LD-BP exposures. For both D. magna and O. latipes, concentration-responses were monotonic with 96 h–LC50 of BPA and LD-BP of 11.7 and 5.0 mg L−1 and 9.4 and 4.1 mg L−1, respectively. Our results demonstrate that LD-BP is more toxic than BPA in the representative aquatic organisms, and it can pose higher ecological risk to the aquatic ecosystem than BPA.

Fluoride is ubiquitously distributed in natural waters. Elevated fluoride may cause histopathological changes and induce oxidative stress in the gills of the common carp (Cyprinus carpio). The present study further evaluates the effects of fluoride on growth performance, body composition, and biochemical measurements of C. carpio. The results showed that food intake, growth, serum osmolality, body composition, and biochemical measures in the blood were affected by fluoride. Weight gain rate and specific growth rate in the exposed fish decreased compared with those of the control fish. Levels of crude protein and crude lipids were reduced in the fluoride-exposed fish. The major ion levels in the sera of fluoride-exposed fish were severely disturbed, resulting in a lower osmolality. All the biochemical parameters measured in the blood were affected by the exposure to fluoride. Total protein, albumin, globulin, and glucose in fish exposed to 63.6 mg/L, 77.7 mg/L, and 124.4 mg/L were lower than those in the control fish. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were markedly increased in exposed fish compared with control. Taken together, the data showed that exposure to fluoride caused a suite of detrimental effects in C. carpio, which might lead to a decrease in growth and food utilization efficiency. The results of the present study indicated that high levels of fluoride could pose a threat to carp in the field.

Piwi proteins as constituent factors of the piRNA pathway are required for germline maintenance, meiosis and gonad development. Previous study showed hCG could regulate the Piwi expression in ovary of teleosts. In this study, we revealed effects of LHRH-A and hCG on Piwi expression in testis of tilapia using Real-time PCR and Western blot. Both in vivo and in vitro study suggest that LHRH-A and hCG significantly down-regulated Piwil-1 and Piwil-2 in mRNA or protein levels compared with controls. Meanwhile, tissue and cell distribution showed that Piwi proteins were mainly expressed in spermatocytes rather than mature sperms. These results indicated that HPG suppresses Piwis which may play a crucial role in testis differentiation and development.

Genetic diversity plays a vital role for maintainingeconomically important traits in fish populations.The goal of this study was to assess the geneticvariability among four tilapia populations in China.Two populations named XA (Oreochromis aureus,30 samples) and AN (Egyptian Oreochromis niloticus,24 samples) were gathered from Freshwater Fish-eries Research Center (FFRC), and the other twonamed AL (O. aureus, 30 samples) and AJ (Egyp-tian O. niloticus, 30 samples) were from GuangxiFisheries Research Institute (GFRI). GFRI intro-duced AL and AJ from XA and AN, and selectedthem for generations. We want to know if AL andAJ have genetic changes after selection and gener-ations. Fifteen polymorphic microsatellite markerswere used. Our results indicated that the four tila-pia populations showed moderate genetic variabil-ity. The polymorphism information content (PIC)of the four populations was from 0.28 to 0.39; A e was from 1.67 to 2.15; the H e was from 0.35 to0.46; the genetic similarity indexes were from 0.94to 0.69 and also the clustering result showed thatthese four populations were highly related. Therewere no significant changes in genetic variationbetween late-introduced population and early-introduced population of O. aureus. Our resultshelped to understand the genetic variability of tila-pia populations in China and for future marker-assisted selection for important economic traits.Keywords: microsatellite, Nile tilapia, Blue tila-pia, heterozygosity, genetic diversity, selection,loss of genetic variationIntroductionTilapia belongs to Perciformes Cichlida and it isnative to Africa and Middle East. Tilapia (Oreochr-omis mossambicus) was introduced to China in theearly 1950s. Later, many species and strains (Or-eochromis niloticus, Oreochromis aureus, etc.) wereintroduced to China too (Li 2001; Li & Li 2001;Ye 2008). After their introduction to China, tila-pia has been undergone artificial selection forvarious economically important traits, such asfast growth, resistance to environmental stressorsand high percentage of males. Since the expandedproduction of tilapia started, China has been thelargest supplier of tilapia in the world till now(Li, Li, Tang & Cai 2009).Fresh Water Fisheries Research Center (FFRC) isthe most famous genetic and breeding resourcesCenter of Tilapia in China well known as the bestquality of purebred Tilapia parents. And now,FFRC was the head of China Agriculture Techni-que System-tilapia (CARS). The Blue tilapia(O. aureus, XA) was introduced from Auburn Uni-versity by FFRC in 1983, and they have selectedand bred a new Blue tilapia strain, named “Xia’ao1”. This strain is widely used for cross-breedingwith Nile tilapia to get all male tilapia fry inChina. The Nile tilapia samples (AN) used in thisstudy were introduced from Central laboratory forAquaculture Research, Abbassa, Ministry of Agri-culture and Land Reclamation of Egypt by FFRC in1999. Guangxi Fisheries Research Institute (GFRI)was the most famous tilapia fry productive unit inChina as the high male rate of cross-bred tilapiafry. GFRI introduced two strains of tilapia: XA(O. aureus) and AN (Egyptian O. niloticus) from