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BACKGROUND: Early blight caused by Alternaria spp. is amongst the most important diseases in potato. Demethylation inhibitor (DMI) fungicides are widely used to control the disease but long‐term use may decrease its control efficacy due to fungicide resistance. This study investigated the occurrence of difenoconazole resistance in Alternaria spp. and molecular resistant mechanisms. RESULTS: EC₅₀ values of 160 isolates to difenoconazole ranged from 0.026 μg mL⁻¹ to 15.506 μg mL⁻¹ and the frequency of difenoconazole sensitivity formed a non‐normal distribution curve with a major and a minor peak. Isolates with EC₅₀ values of 4.121 and 5.461 μg mL⁻¹ were not controlled effectively at fungicide doses of 50 and 100 μg mL⁻¹. Cross‐resistance was observed between DMI fungicides difenoconazole and propiconazole, but not between difenoconazole and other fungicide groups, including boscalid, iprodione, or carbendazim. The CYP51gene was 1673 bp encoding 525 amino acids in length and contained two introns. All sensitive and resistant isolates had the identical amino acid sequence of CYP51, with the exception of one resistant isolate carrying a mutation of R511W. A 6 bp insertion in the upstream region was observed in half of the resistant isolates. In the absence of propiconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. In the presence of difenoconazole, expression of CYP51 gene was induced significantly in the DMI‐resistant isolates but not in the sensitive ones. CONCLUSION: Induced expression of CYP51 in resistant isolates exposed to difenoconazole is an important determinant for DMI resistance in potato pathogens Alternaria sect. © 2019 Society of Chemical Industry

The endocrine regulatory roles of the hypothalamic–pituitary–adrenocortical axis on anxiety-like behavior and metabolic status have been found throughout animal taxa. However, the precise effects of the balancing adrenal corticosteroid biosynthesis under the influence of adrenocorticotrophic hormone (ACTH), a pro-opiomelanocortin (POMC)-derived peptide, on animal energy expenditure and somatic growth remain unknown. POMC has also been identified as one of the candidate loci for polycystic ovary syndrome, which features hyperandrogenism and some prevalence of obesity in patients. Here we show that zebrafish lacking functional POMCa exhibit similar phenotypes of stress response and body weight gain but not obesity as observed in mammalian models. In contrast with the impaired anorexigenic signaling cascade of melanocyte-stimulating hormones and leptin, which are responsible for their obesity-prone weight gain observed in various pomc mutant mammals, analyses with our pomca mutant series indicate that ACTH is the key regulator for the phenotype with enhanced somatic growth without obesity in pomca-deficient zebrafish. Hypocortisolism associated with hyperandrogenism has been observed in the pomca-deficient zebrafish, with enhanced activation of mammalian target of rapamycin complex 1; reutilization of amino acids and fatty acid β-oxidation are observed in the muscle tissue of the pomca-deficient fish. After reducing hyperandrogenism by crossing our pomca mutant fish with a cyp17a1-deficient background, the phenotype of enhanced somatic growth in pomca-deficient fish was no longer observed. Thus, our work also demonstrated that the role of POMCa in stress response seems to be conserved in vertebrates, whereas its effect on adipostasis is unique to teleosts.

Zika virus (ZIKV), a mosquito-borne flavivirus, poses a serious threat to public health worldwide, and Aedes albopictus is one of its vectors. To evaluate the potential of the entomopathogenic fungus Beauveria bassiana for ZIKV vector control, we compared the vectorial capacity of Ae. albopictus females blood-fed with ZIKV with or without exposure to Beauveria bassiana. We found that fungal infection significantly decreased the amount of ZIKV by 3.6-, 12.3- and 7.8-fold in mosquito midguts, heads and salivary glands, respectively. Similarly, fungal infection also reduced the rates of ZIKV dissemination, potential transmission and potential population transmission for mosquitoes by 26.8%, 38.4% and 35.2%, respectively. On the other hand, the median survival time and fecundity of fungus-infected mosquitoes were reduced by 84.2% and 39.8% in comparison with those of the non-fungus-infected mosquitoes. The first gonotrophic cycle length was increased by 15.3% because of fungal infection. This study revealed that B. bassiana infection significantly reduced the vectorial capacity of A. albopictus for ZIKV, which suggested that B. bassiana could be broadly and efficiently used in the field for the control of Zika vectors.

Key points In a cold environment, mammals increase their food intake while fish decrease or stop feeding. However, the physiological value of fasting during cold resistance in fish is currently unknown. Fasting for more than 48 h enhanced acute cold resistance in zebrafish, which correlated with lipid catabolism and cell damage attenuation. Lipid catabolism and autophagy were necessary for cold resistance in fish and the inhibition of mitochondrial fatty acid β-oxidation or autophagy weakened the fasting-induced cold resistance. Repression of mechanistic target of rapamycin (mTOR) signalling pathway by rapamycin largely mimicked the beneficial effects of fasting in promoting cold resistance, suggesting mTOR signalling may be involved in the fasting-induced cold resistance in fish. Our study demonstrates that fasting may be a protective strategy for fish to survive under cold stress. Abstract In cold environments, most homeothermic animals increase their food intake to supply more energy to maintain body temperature, whereas most poikilothermic animals such as fishes decrease or even stop feeding under cold stress. However, the physiological value of fasting during cold resistance in poikilotherms has not been explained. Here, we show that moderate fasting largely enhanced cold resistance in fish. By using pharmacological (fenofibrate, mildronate, chloroquine and rapamycin) and nutritional approaches (fatty acids diets and amino acids diets) in wild-type or specific gene knock-out zebrafish models (carnitine palmitoyltransferase-1b-deficient strain, CPT1b-/- , or autophagy-related protein 12-deficient strain, ATG12-/- ), we verified that fasting-stimulated lipid catabolism and autophagy played essential roles in the improved cold resistance. Moreover, suppression of the mechanistic target of rapamycin (mTOR) pathway by using rapamycin mostly mimicked the beneficial effects of fasting in promoting cold resistance as either the physiological phenotype or transcriptomic pattern. However, these beneficial effects were largely reduced when the mTOR pathway was activated through high dietary leucine supplementation. We conclude that fasting helps fish to resist cold stress by modulating lipid catabolism and autophagy, which correlates with the mTOR signalling pathway. Therefore, fasting can act as a protective strategy of fish in resisting coldness.

The essential oil extracted from roots and rhizomes of Ligusticum jeholense Nakai et Kitagawa was investigated for its chemical composition by GC-MS analysis, and evaluated for its contact toxicity and repellency against Tribolium castaneum and Lasioderma serricorne, along with some of its individual components. The essential oil was rich in aromatics (65.34%) with low molecular weight. Major components included sedanolide (33.95%), 3-butylidenephthalide (18.76%), spathulenol (8.90%) and myristicin (6.76%). The results of bioassays indicated that the essential oil of L. jeholense and 3-butylidenephthalide possessed significant repellent activities against T. castaneum at 2 and 4 h post-exposure. Meanwhile, 3-butylidenephthalide had potent contact toxicity against L. serricorne (LD50 = 13.64 µg/adult). The minor component n-butylbenzene in the oil was highly toxic to T. castaneum (LD50 = 23.99 µg/adult) and L. serricorne (LD50 = 7.86 µg/adult) in contact assays, but failed to repel these beetles at all testing concentrations. Spathulenol and myristicin exerted good insecticidal and repellent effects on the two target insects. This work suggests that the essential oil of L. jeholense has promising potential for development as natural insecticide or repellent to control pest damage in warehouses.

Dietary usage of microencapsulated oils has been proved to promote growth and gut health in land animals, however, these beneficial effects have not been intensively investigated in aquatic animals. The present study hypothesized that dietary microencapsulated oils could alleviate negative effects caused by high-fat diet (HFD) in fish, thus we fed juvenile Nile tilapia with HFD (15% fat) by using normal or microencapsulated oils as lipid sources for 8 weeks. To compare the effects between different microencapsulated oils with different fatty acid composition, two oil mixtures (PL: 60% palm oil +40% linseed oil; FL: 60% fish oil +40% linseed oil) were used. After feeding trial, the growth, body composition, fatty acid composition, resistance to pathogenic bacteria challenge, activities of immune enzymes, expressions of inflammation genes, intestinal structure and microbiota were measured, respectively. The results indicated that microencapsulated oils didn’t affect fish growth, body composition, and muscle fatty acid composition, but improved the intestinal activities of lysozyme (LZM) and resistance against Aeromonas hydrophila. Further tests showed that microencapsulated oils not only improved the structure of intestine, but also increased the number of beneficial bacteria (Bacillus and Paenibacillus) and decreased the number of harmful bacteria (Pseudoalteromonas and Roseovarius) in the intestine. Those beneficial effects are more significant between normal PL oil and microencapsulated PL oil, but no significant difference in FL oil groups. Taken together, these results suggested that microencapsulated oil might enhance immune activities in the Nile tilapia fed with HFD by improving intestinal structure and microflora. Moreover, the beneficial effects of the microencapsulated oil are correlated to the oil composition. To the best of our knowledge, this is the first study illustrating the beneficial effects of dietary microencapsulated oil in the fish fed with HFD.

Toxoplasma gondii is an intracellular parasite that infects humans and other warm-blooded animals. Exosomes are endocytic-derived vesicles released by cells, representing an important mode of intercellular communication. In exosomes, specific molecules of proteins, lipids, and mRNAs or miRNAs have been detected, some of which are capable of transferring biologically active molecules to recipient cells. Dendritic cells (DCs) are the only antigen-presenting cells (APCs) that activate the initial immune response. In this study, high-throughput sequencing was used to analyze the exosomal miRNA profile of DC2.4 cells infected with Toxoplasma gondii for 28 h, compared with those of uninfected DC2.4 cells. Differential exosomal miRNAs (DEmiRs) from these two cell groups were analyzed. Through high-throughput sequencing, 3434 DEmiRs were obtained, and 12 stably enriched DEmiRNAs were verified by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) and selected for further analysis. The target genes of these 12 miRNAs were predicted with online analysis software and subjected to bioinformatics analyses including protein&ndash, protein interaction (PPI) network analysis, key driver analysis (KDA), gene ontology (GO) enrichment, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. These DEmiRs were found to be associated with a variety of biological processes and signaling pathways involved in host ubiquitin system, innate immunity, biosynthesis, and transferase activity and could be potential biomarkers for T. gondii infection.

Background Metalaxyl is a phenylamide fungicide, introduced for oomycete management in the 1970s. RPA190 gene has been reported to be associated with metalaxyl resistance, although other genes may also be involved in the process. This study assessed the evolutionary association of amino acid (AA) mutations in RPA190 with metalaxyl resistance in Phytophthora infestans. Results All isolates from Yunnan were resistant to metalaxyl with multiple AA mutations in RPA190. Two main evolutionary pathways were found in metalaxyl resistance: in one pathway, RPA190 from sensitive isolates (SAA) underwent a single type AA mutation at position 1476, changing from valine to glycine (V1476G); the second pathway involved multiple steps of point mutations. In a phylogenetic tree, nucleotide sequences of the gene clustered into six clades largely corresponding to AA isoform groups. Among the clusters, sequences carrying mutations V1476G and P980S formed two independent clades; other clades shared a common mutation of F382Y. All three mutations together with another two were shown to be under positive selection and the number of AA mutations in an isolate was positively correlated with EC50 values. Conclusion Multiple mutations such as V1476G, P980S, and F382Y in RPA190 contribute to metalaxyl resistance, and resistance to metalaxyl can emerge in at least two independent pathways in P. infestans. © 2018 Society of Chemical Industry.

Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SCAT) have different structures and metabolic functions and play different roles in the regulation of the mammal endocrine system. However, little is known about morphology and physiological and metabolic functions between VAT and SCAT in fish. We compared the morphological, physiological, and biochemical characteristics of VAT and SCAT in Nile tilapia and measured their functions in energy intake flux, lipolytic ability, and gene expression patterns. SCAT contained more large adipocytes and nonadipocytes than VAT in Nile tilapia. VAT had higher lipid content and was the primary site for lipid deposition. Conversely, SCAT had higher hormone-induced lipolytic activity. Furthermore, SCAT had a higher percentage of monounsaturated and lower polyunsaturated fatty acids than VAT. SCAT had higher mitochondrial DNA, gene expression for fatty acid β-oxidation, adipogenesis, and brown adipose tissue characteristics, but it also had a lower gene expression for inflammation and adipocyte differentiation than VAT. SCAT and VAT have different morphological structures, as well as physiological and metabolic functions in fish. VAT is the preferable lipid deposition tissue, whereas SCAT exhibits higher lipid catabolic activity than VAT. The physiological functions of SCAT in fish are commonly overlooked. The present study indicates that SCAT has specific metabolic characteristics that differ from VAT. The differences between VAT and SCAT should be considered in future metabolism studies using fish as models, either in biomedical or aquaculture studies.

Energy metabolism plays important roles in stress resistance and immunity in mammals, however, such functions have not been established in fish. In the present study, Nile tilapia (Oreochromis niloticus) was fed with mildronate, an inhibitor of mitochondrial fatty acid (FA) β-oxidation, for six weeks subsequently challenged with Aeromonas hydrophila and ammonia nitrogen exposure. Mildronate treatment reduced significantly l-carnitine concentration and mitochondrial FA β-oxidation efficiency, while it increased lipid accumulation in liver. The fish with inhibited hepatic FA catabolism had lower survival rate when exposed to Aeromonas hydrophila and ammonia nitrogen. Moreover, fish fed mildronate supplemented diet had lower immune enzymes activities and anti-inflammatory cytokine genes expressions, but had higher pro-inflammatory cytokine genes expressions. However, the oxidative stress-related biochemical indexes were not significantly affected by mildronate treatment. Taken together, inhibited mitochondrial FA β-oxidation impaired stress resistance ability in Nile tilapia mainly through inhibiting immune functions and triggering inflammation. This is the first study showing the regulatory effects of lipid catabolism on stress resistance and immune functions in fish.

1α,25(OH)2D3 (vitamin D3) is crucial for mineral homeostasis in mammals, but the precise effects of 1α,25(OH)2D3 in adipose tissue remain to be clarified in vivo. The initial 25-hydroxylation is catalyzed by liver microsomal cytochrome P450 2R1 (CYP2R1), which is conserved in vertebrates. To probe the physiological function(s) of 1α,25(OH)2D3 in teleosts, we generated two independent cyp2r1-deficient zebrafish lines. These mutants exhibit retarded growth and increased obesity, especially in the visceral adipose tissue (VAT). These defects could be rescued with 25(OH)D3 treatments. ChIP-PCR analyses demonstrated that pgc1a is the target of the vitamin D receptor in the liver and VAT of zebrafish. Significantly decreased protein levels of Pgc1a, impaired mitochondrial biogenesis, and free fatty acid oxidation are also observed in the cyp2r1 mutant VAT. Our results demonstrate that regulation of 1α,25(OH)2D3 during lipid metabolism occurs through the regulation of Pgc1a for mitochondrial biogenesis and oxidative metabolism within zebrafish VAT.

Dexamethasone, a known immunosuppressant, can inhibit the immune response and increase the amount of pathogen in body, but the role of dexamethasone affecting susceptibility of crucian carp (Carassius auratus) to pathogen is unclear. The effects of dexamethasone on susceptibility of crucian carp to Aeromonas hydrophila were investigated in this study. The fish were divided into four groups randomly and injected intraperitoneally by dexamethasone for 0 day (group D), 3 days (group C), 6 days (group B), and 9 days (group A), respectively. The serum lysozyme activity was significantly declined in group A, B and C. Relative immune gene expression such as il-1β, cxcl-8, tnfα and crp in kidney were down-regulation compared to group D. After that crucian carp were infected with A. hydrophila, crucian carp treated by dexamethasone had higher mortality (group A 95%, group B 76%, group C 31%) when compared to group D (4% mortality); the amount of pathogen in was significantly increased (P

Background The entomopathogenic fungus Beauveria bassiana has been widely used to kill mosquito larvae and adults in the laboratory and field. However, its slow action of killing has hampered its widespread application. In our study, the B. bassiana fungus was genetically modified to express the Bacillus thuringiensis (Bt) toxin Cyt2Ba to improve its efficacy in killing mosquitoes. Methodology/Principal findings The efficacy of the wild type (WT) of B. bassiana and a transgenic strain expressing Cyt2Ba toxin (Bb-Cyt2Ba) was evaluated against larval and adult Aedes mosquitoes (Aedes aegypti and Aedes albopictus) using insect bioassays. The Bb-Cyt2Ba displayed increased virulence against larval and adult Aedes mosquitoes compared with the WT: for Ae. aegypti adults, the median lethal time (LT50) was decreased by 33% at the concentration of 1× 108 conidia/ml, 19% at 1× 107 conidia/ml and 47% at 1× 106 conidia/ml. The LT50 for Ae. albopictus adults was reduced by 20%, 23% and 29% at the same concentrations, respectively. The LT50 for Ae. aegypti larvae was decreased by 42% at 1× 107 conidia/ml and 25% at 1× 106 conidia/ml, and that for Ae. albopictus larvae was reduced by 33% and 31% at the same concentrations, respectively. In addition, infection with Bb-Cyt2Ba resulted in a dramatic reduction in the fecundity of Aedes mosquitoes. Conclusions/Significance In conclusion, our study demonstrated that the virulence of B. bassiana against mosquitoes can be significantly improved by introducing the Bt toxin gene Cyt2Ba into the genome to express the exogenous toxin in the fungus. The transgenic strain Bb-Cyt2Ba significantly reduced the survival and fecundity of Ae. aegypti and Ae. albopictus compared with the WT strain, which suggested that this recombinant B. bassiana has great potential for use in mosquito control., Author summary Mosquito vectors transmit many diseases to humans and animals, causing illness and death and resulting in substantial socio-economic burdens in endemic countries. The control of mosquitoes has almost exclusively relied on the use of chemical insecticides, which has recently led to the broad resistance of important mosquito vectors worldwide. Entomopathogenic fungi, such as Beauveria bassiana, are an important alternative or complement to chemical insecticides. However, the relatively slow action of fungal pathogens in killing mosquitoes, compared with chemical insecticides, has hampered their widespread application. To improve the insecticidal efficacy of the entomopathogen B. bassiana, the fungus was genetically modified to express the Bacillus thuringiensis toxin Cyt2Ba. The mitotically stable transformant (Bb-Cyt2Ba) successfully expressed Cyt2Ba toxin, and the virulence of this strain against adults and larvae of Aedes aegypti and Aedes albopictus mosquitoes was significantly improved. In addition, egg laying was significantly affected by Bb-Cyt2Ba infection. Infection with this fungus resulted in a dramatic reduction in fecundity of the target mosquitoes. Therefore, this recombinant B. bassiana has great potential for use in mosquito control.

Leptin is known to inhibit appetite and promote energy metabolism in vertebrates. Leptin resistance (LR) commonly occurs in diet-induced obesity (DIO) in mammals. However, the roles of leptin in the energy homeostasis in DIO animals with LR remain unclear. Here we first verified the high expression of leptin in subcutaneous adipose tissue (SCAT) as in liver in Nile tilapia. Furthermore, we produced two types of DIO Nile tilapia by using a high-carbohydrate diet (HCD) or a high-fat diet (HFD), and confirmed the existence of LR in both models. Notably, we found that HCD-DIO fish retained leptin action in the activation of lipid metabolism and showed LR in glucose metabolism regulation, while this selective leptin action between lipid and glucose metabolism was reversed in HFD-DIO fish. Fasting the fish for 1 week completely recovered leptin actions in the regulation of lipid and glucose metabolism. Therefore, leptin may retain more of its activities in animals with LR than previously believed. Evolutionally, this selective regulation of leptin in nutrients metabolism could be an adaptive mechanism in animals to store surplus calories when different types of food are abundant.

Lipophagy degrades lipid droplets (LDs) through the lysosomal degradative pathway, thus plays important roles in regulating lipid metabolism in mammals. However, information on the existence and functions of lipophagy in fish lipid metabolism is still limited. In the present study, we confirmed the existence of lipophagy by observing the structures of LDs sequestered in autophagic vacuoles in the zebrafish liver cell line (ZFL) via electronic microscopy. Moreover, starved cells increased the mRNA expression of the microtubule-associated protein 1A/1B light chain 3 beta (LC3), which is a marker protein for autophagy and protein conversion from LC3-I to LC3-II. Inhibiting autophagy with chloroquine increased significantly the LDs content and decreased fatty acid β-oxidation and esterification activities in the ZFL cells cultured in the fed state. Furthermore, inhibiting autophagy function downregulated the mRNA expression of the genes and their proteins related to lipid metabolism. Altogether, the present study verified the existence of lipophagy and its essential regulatory roles in lipid metabolism in fish cells.

The monogenean Dactylogyrus intermedius and the bacterium Flavobacterium columnare are 2 common pathogens in aquaculture. The objective of the present study was to examine the effect of prior parasitism by D. intermedius on the susceptibility of goldfish to F. columnare and to explore the potential immune mechanisms related to the parasite infection. A F. columnare challenge trial was conducted between D. intermedius-parasitized and non-parasitized goldfish. The F. columnare load in gill, kidney, spleen and liver were compared. The expression of immune-related genes (IL-1β2, TNF-α1, TGF-β, iNOS-a, C3 and Lyz) in gill and kidney of D. intermedius-only infected and uninfected control fish were evaluated. D. intermedius-parasitized goldfish exhibited higher mortality and significantly higher loads (3051 to 537,379 genome equivalents [GEs] mg(-1)) of F. columnare, which were 1.13 to 50.82-fold higher than non-parasitized fish (389 to 17,829 GEs mg(-1)). Furthermore, the immune genes IL-1β2, TNF-α1, iNOS-a and Lyz were up-regulated while the TGF-β and C3 were down-regulated in the gill and kidney of parasite-infected fish compared to the non-parasitized controls. The down-regulation TGF-β and C3 was especially noteworthy, as this might indicate the suppression of the host immune functions due to the parasitism by D. intermedius. Taken together, these data demonstrate that parasite infection can enhance bacterial invasion and presents a hypothesis, based on gene expression data, that modulation of host immune response could play a role.

Scientific Reports 7: Article number: 40815; published online: 19 January 2017; updated: 23 March 2017 The original version of this Article contained an error in the spelling of the author Xuan Qin, which was incorrectly given as Xun Qin. This has now been corrected in the PDF and HTML versions of the Article.

Gut microbiota of grass carp plays an important role in host. However, detailed information regarding the changes of microbiota after probiotics administration in relation to the gastrointestinal microbiota is absent. In the present study, dietary administration of putative probiotics Shewanella xiamenensis A-1, Aeromonas veronii A-7, and Bacillus subtilisstrains was conducted in grass carp to investigate if there is a discernible alteration in intestinal microbiota and whether the alteration is associated with previous study about the immunity regulation in grass carp. Bacterial 16S rRNA gene sequence-based comparisons of the bacterial communities in the grass carp intestine were detected after 28 days feeding by five diets, and results demonstrated the changes of microbial community composition at genus level. The abundance of Cetobacterium genus with potential immunity function increased. Potential pathogens and probiotics are important constitutions of the intestinal microbiota. Orally taken probiotics considerably reduced the abundance of the potential pathogenic bacteria (e.g., Pseudomonas and Flavobacterium genus) in the intestine. Meanwhile, putative probiotics used in this study were favorable to the reproduction of potential probiotics in THE intestine of grass carp (e.g., Vibrio, Streptococcus, and Enterococcus genus). Moreover, modulation of intestinal environment by the probiotics could impact the abundance of cellulose-degrading bacteria (e.g., Citrobacter genus). Those results suggested that oral probiotics administration can positively improve the composition of intestinal microbial community in grass carp, and this was associated with regulation of immunity in grass carp. Probiotics-induced alteration of microbiota may potentially lower the risk of disease outbreaks during cultivation stage of grass carp.

Peroxisome proliferation activated receptor α (PPARα) is an important transcriptional regulator of lipid metabolism and is activated by high-fat diet (HFD) and fibrates in mammals. However, whether nutritional background affects PPARα activation and the hypolipidemic effects of PPARα ligands have not been investigated in fish. In the present two-phase study of Nile tilapia (Oreochromis niloticus), fish were first fed a HFD (13% fat) or low-fat diet (LFD; 1% fat) diet for 10 weeks, and then fish from the first phase were fed the HFD or LFD supplemented with 200 mg/kg body weight fenofibrate for 4 weeks. The results indicated that the HFD did not activate PPARα or other lipid catabolism-related genes. Hepatic fatty acid β-oxidation increased significantly in the HFD and LFD groups after the fenofibrate treatment, when exogenous substrates were sufficiently provided. Only in the HFD group, fenofibrate significantly increased hepatic PPARα mRNA and protein expression, and decreased liver and plasma triglyceride concentrations. This is the first study to show that body fat deposition and dietary lipid content affects PPARα activation and the hypolipidemic effects of fenofibrate in fish, and this could be due to differences in substrate availability for lipid catabolism in fish fed with different diets.

Excess fat accumulation has been observed widely in farmed fish; therefore, efficient lipid-lowering factors have obtained high attention in the current fish nutrition studies. Dietary L-carnitine can increase fatty acid β-oxidation in mammals, but has produced contradictory results in different fish species. To date, the mechanisms of metabolic regulation of L-carnitine in fish have not been fully determined. The present study used zebrafish to investigate the systemic regulation of nutrient metabolism by dietary L-carnitine supplementation. L-carnitine significantly decreased the lipid content in liver and muscle, accompanied by increased concentrations of total and free carnitine in tissues. Meanwhile, L-carnitine enhanced mitochondrial β-oxidation activities and the expression of carnitine palmitoyltransferase 1 mRNA significantly, whereas it depressed the mRNA expression of adipogenesis-related genes. In addition, L-carnitine caused higher glycogen deposition in the fasting state, and increased and decreased the mRNA expressions of gluconeogenesis-related and glycolysis-related genes, respectively. L-carnitine also increased the hepatic expression of mTOR in the feeding state. Taken together, dietary L-carnitine supplementation decreased lipid deposition by increasing mitochondrial fatty acid β-oxidation, and is likely to promote protein synthesis. However, the L-carnitine-enhanced lipid catabolism would cause a decrease in glucose utilization. Therefore, L-carnitine has comprehensive effects on nutrient metabolism in fish.

Using rare minnow (Gobiocypris rarus) embryos as experimental model, the developmental toxicity of Cu, Zn and Cd were investigated following exposure to 0.001-1.000mg/L for 72h, and the toxicity effects were evaluated by larval malformation rate, heart rate, pericardial area, spontaneous movements, tail length, enzyme activities and biomarker genes. Our results revealed that increased malformation rate provide a gradual dose-response relationship, and the most pronounced morphological alteration was heart and body malformations. Values of 72h EC50 with their 95 percent confidence intervals on G. rarus embryos were 0.103 (0.072-0.149)mg/L for Cu, 0.531 (0.330-1.060)mg/L for Zn, 0.219 (0.147-0.351)mg/L for Cd. Enzyme activities can be regard as a type of low-dose stimulation and high-dose inhibition. Stress and metabolism-related genes (hsp70, cyp1a and mt) were significantly up-regulated, development-related genes (wnt8a, vezf1 and mstn) were significantly down-regulated after the treatment by Cu, Zn and Cd. Overall, the present study points out Cu, Zn and Cd are highly toxic to G. rarus embryos. The information presented in this study will be helpful in fully understanding the toxicity induced by Cu, Zn and Cd in fish embryos.

The zebrafish skeletal muscle-specific promoter mylz2 was used to cause crucian carp overexpression of the zebrafish IGF-1 cDNA. In stable transgenic germline F1 progenies, a 5-fold increase in the level of IGF-1 in skeletal muscle was observed. Evident skeletal muscle hyperplasia was observed in the transgenic fish through histologic analysis. By analyzing the RNA sequencing transcriptome of the skeletal muscle of IGF-1 transgenic fish and nontransgenic control fish at 15 months of age, 10 966 transcripts with significant expression levels were identified with definite gene descriptions based on the corresponding zebrafish genome information. Based on the results of our RNA sequencing transcriptome profiling analysis and the results of the real-time quantitative PCR analysis performed to confirm the skeletal muscle transcriptomics analysis, several pathways, including IGF-1 signaling, aerobic metabolism, and protein degradation, were found to be activated in the IGF-1-overexpressing transgenic fish. Intriguingly, our transcriptional expression and protein assays indicated that the overexpression of IGF-1 stimulated a significant shift in the myofiber type toward a more oxidative slow muscle type. Although the body weight was surprisingly decreased by IGF-1 transgenic expression, significantly higher oxygen consumption rates were measured in IGF-1-overexpressing transgenic fish compared with their nontransgenic control fish. These results indicate that the sustained overexpression of IGF-1 in crucian carp skeletal muscle promotes myofiber hyperplasia and cellularity changes, which elicit alterations in the body energy metabolism and skeletal muscle growth.

Natural selection endows animals with the abilities to store lipid when food is abundant and to synthesize lipid when it is limited. However, the relevant adaptive strategy of lipid metabolism has not been clearly elucidated in fish. This study examined the systemic metabolic strategies of Nile tilapia to maintain lipid homeostasis when fed with low- or high-fat diets. Three diets with different lipid contents (1%, 7%, and 13%) were formulated and fed to tilapias for 10 weeks. At the end of the feeding trial, the growth rate, hepatic somatic index, and the triglyceride (TG) contents of serum, liver, muscle, and adipose tissue were comparable among three groups, whereas the total body lipid contents and the mass of adipose tissue increased with the increased dietary lipid levels. Overall quantitative PCR, western blotting and transcriptomic assays indicated that the liver was the primary responding organ to low-fat (LF) diet feeding, and the elevated glycolysis and accelerated biosynthesis of fatty acids (FA) in the liver is likely to be the main strategies of tilapia toward LF intake. In contrast, excess ingested lipid was preferentially stored in adipose tissue through increasing the capability of FA uptake and TG synthesis. Increasing numbers, but not enlarging size, of adipocytes may be the main strategy of Nile tilapia responding to continuous high-fat (HF) diet feeding. This is the first study illuminating the systemic adaptation of lipid metabolism responding to LF or HF diet in fish, and our results shed new light on fish physiology.

To reduce the lethal hemorrhagic disease caused by grass carp reovirus (GCRV) and improve the production of grass carp, efficient and economic prophylactic measure against GCRV is the most pressing desired for the grass carp farming industry. In this work, a novel SWCNTs-pEGFP-vp5 DNA vaccine linked vp5 recombinant in the form of plasmid pEGFP-vp5 and ammonium-functionalized SWCNTs by a chemical modification method was prepared to enhance the efficacy of a vp5 DNA vaccine against GCRV in juvenile grass carp. After intramuscular injection (1, 2.5 and 5 μg) and bath administration (1, 10, and 20 mg/L), the ability of the different immune treatments to induce transgene expression was analyzed. The results showed that higher levels of transcription and expression of vp5 gene could be detected in muscle tissues of grass carp in SWCNTs-pEGFP-vp5 treatment groups compare with naked pEGFP-vp5 treatment groups. Moreover, antibody levels, immune-related genes, and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pEGFP-vp5 vaccine. In addition, we found that a good immune protective effect was observed in bath immunization group; which at a concentration of 20 mg/L could reach the similar relative percentage survival (approximately 100%) in injection group at a dose of 5 μg. All these results indicated that ammonium-functionalized SWCNTs could provide extensive application prospect to aquatic vaccine and might be used to vaccinate fish by intramuscular injection or bath administration method.

The Metronidazole (MTZ), a widely used antibiotic for treating variations of infections, recently is applied in a powerful tool for specifically ablating cells or tissues when combined with E. coli nitroreductase (NTR). Although some undesired biological effects on eukaryote cells have been reported previously, the toxicological mechanism of MTZ has not been uncovered yet. In current study, we found that MTZ can induce proopiomelanocortin a (pomca) expression in zebrafish larvae. The effect of MTZ is in stage-sensitive and dose-dependent manner. A pro-proliferation activity of MTZ on pomca-expressing cells in the pituitary at larval stage was also observed. Furthermore, up-regulated levels of prolactin (prl) and glycoprotein hormone subunit α (gsuα) were also observed after the MTZ treatment. Therefore, utilizing our zebrafish as in vivo model, we concluded that MTZ can interfere the endocrine signals in the pituitary hormone genes expression. Our current results raised the cautions to the intensively application of MTZ in clinical practices and biomedical researches.

Triazophos (TAP) has been widely used in agriculture for controlling insect pests and is a known organo-phosphorus pesticide. Due to TAP characteristics, such as high chemical and photochemical stability, itspotential toxicity to aquatic organisms has gained great interest. To explore the potential developmentaltoxicity of TAP, Gobiocypris rarus embryos and larvae were exposed to various concentrations of TAP (0.1–15 mg L 1 ) until 72 h. Results showed that values of 72 h LC 50 and EC 50 were 7.44 and 5.60 mg L 1 forembryos, 2.52 and 1.37 mg L 1 for larvae. Increased malformation, decreased heart rate and body lengthprovide a gradual concentration-dependent pattern. Enzyme activities and mRNA levels were signifi-cantly changed even at low concentration (0.05 mg L 1 for embryos and 0.01 mg L 1 for larvae). Overall,the present study points out that TAP is likely a risk to the early development of G. rarus. The informationpresented in this study will be helpful in better understanding the toxicity induced by TAP in fishembryos and larvae.2014 Elsevier Ltd. All rights reserved.

Human respiratory syncytial virus (RSV) is a major viral pathogen of the lower respiratory tract of infants and young children worldwide. No effective prevention measure is available. Attenuated Salmonella strains expressing heterologous antigens can be delivered by the oral route, triggering efficient antigen-specific humoral, cellular, and mucosal immunity. In this study, we orally administered attenuated Salmonella strain SL7207, carrying the plasmid pcDNA3.1/F expressing the RSV F gene, to BALB/c mice and showed significant elevations of serum anti-RSV IgG and bronchoalveolar lavage secretory IgA as compared with the control group carrying empty plasmid (p0.001). The ratio of IgG1 and IgG2a was 0.96. The experimental group also showed a stronger cytotoxic T cell response (p0.01 at effector:target ratios of 100:1 and 50:1) and a higher stimulation index value of T cell proliferation (p0.05) than the respective control group. RSV titers in the lung homogenates of the experimental group on day 3 and day 5 postchallenge were lower than in the control group (p0.05). Histopathological analysis showed obvious differences in infiltration of inflammatory cells and pulmonary alveolar wall thickness (p0.01) between the two groups. In summary, our results demonstrate the potential of orally administered SL7207-based DNA vaccines against RSV infection.