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As one of the most serious pathogens in the freshwater aquatic environment, spring viraemia of carp virus (SVCV) induces a high mortality rate in several cyprinid fishes. In this study, we designed and synthesized a total of 44 coumarin derivatives to evaluate the anti-SVCV activity. By comparing the inhibitory concentration at half-maximal activity (IC 50 ), two imidazole coumarins (B4 and C2) were selected, with maximum inhibitory rates on SVCV more than 90%. Mechanistically, B4 or C2 did not affect viral adhesion and delivery from endosomes to the cytosol. Further, B4 and C2 could decline the apoptosis in SVCV-infected cells and the viral activated caspase-3, 8, 9 activities. Other results showed that SVCV induced the cytoskeletal structure to be a circumferential ring of microtubules near the nucleus, with occurring a disrupted microfilament organization. In comparison, cytoskeleton structure in drug-treated cells kept complete. In addition, the cellular microstructure in drug treatments showed no significant change; while SVCV-infected cells were seriously shrunk, and observed typical apoptotic features including cell shrinkage, volume reduction and cell blebbing. More importantly, B4 and C2 enhanced anti-oxidative enzyme gene expression and triggered the Nrf-2 pathway to keep balance of intracellular redox state. Therefore, the use of two imidazole coumarins (B4 and C2) could be a viable way of preventing and controlling SVCV infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Moroxydine hydrochloride (Mor) is known to have multi-antiviral activities against DNA and RNA viruses but very little information exists on its pharmacology. The paper was undertaken to explore the antiviral response and antiapoptotic mechanism of Mor against grass carp reovirus (GCRV) in Ctenopharyngodon idella kidney (CIK) cells. The results showed that exposing GCRV-infected cell to 6.3 μg mL(-1) of Mor for 96 h avoid ca. 50% apoptosis. Meanwhile, Mor had lower cytotoxicity than ribavirin (Rib) as the value of safe concentration was threefold higher than effective concentration and the compound could ensure sufficient into and out of cells within 4 h when tested at the maximal safe concentration. Mor blocked the GCRV-induced cytopathic effects and eliminated nucleocapsids in CIK cells to keep the normal morphological structure. Moreover, the expressions of viral protein genes were significantly inhibited especially the guanylyl transferase and RNA-dependent RNA polymerase related expression. Furthermore, GCRV caused Bcl-2 down-regulation and Bax mitochondrial translocation was prevented by treatment of CIK cells with Mor. The downstream effector, caspase activity was also significantly inhibited in Mor treated cells. The potential mechanism might be that mitochondrial apoptotic signals were not activated by the intervention of Mor for targeting viral gene expression. Taken together, Mor showed high anti-GCRV activity and had been proved as a secure and promising agent in viral controlling in aquaculture industry., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Infectious diseases of viral origin cause major aquatic production losses in different parts of the world. Because of formidable barriers for gastrointestinal tract, skin and cell, large amounts of antiviral drugs have limited therapeutic effect. In the current study, functionalized single-walled carbon nanotubes (SWCNTs) were selected as a drug carrier to carry antiviral drug for the treatment of viral diseases on fish. The results show that increasing antiviral drug (ribavirin) intake was observed by SWCNTs carrier and therapeutic dosage to kill grass carp reovirus is significantly reduced. At 12d post infection, survival rate and infection rate were 29.7% and 50.4% for naked ribavirin treatment group exposed to the highest concentration (20 mg/L); however, survival rate of 96.6% and infection rate of 9.4% were observed in 5 mg/L ribavirin-SWCNTs treatment group. In addition, the drug detention time in different organs and tissues (blood, gill, liver, muscle, kidney and intestine) was also significantly extended (about 72 h) compared with the same dosage in naked ribavirin treatment group. Moreover, the toxicity of functionalized SWCNTs in grass carp can be minimal, and physiological markers (some antioxidant enzymes activities, apoptotic factors activities and their corresponding genes expression) remained within normal ranges following treatment. Our results indicated that drug delivery with functionalized SWCNTs can improve the antiviral effect on grass carp and has a potential application value to control fish viral diseases in aquaculture., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Due to the high chemical and photochemical stability, an organophosphorus pesticide triazophos might enter aquatic ecosystems and impose negative effect on aquatic organisms. In order to investigate short-term toxicity of triazophos on goldfish (Carassius auratus), antioxidant response in brain, spleen, kidney and liver was tested in this study. As a confirmation, the impact of triazophos on acetyl cholinesterase (AChE) activity was found a reduction in all studied tissues, especially in brain. In addition, 0.1 and 0.5 mg L(-1) triazophos induced MDA level increased, while glutathione content (GSH), superoxide dismutase (SOD), catalase (CAT) and lactate dehydrogenase (LDH) activities decreased. Of note, more prominent oxidative stress was provoked in kidney and liver, but weaker in brain and spleen. These results revealed that triazophos could cause a generalized oxidative stress and tissue specific antioxidant response in goldfish. Furthermore, neuroendocrine-growth-related gene expression (growth hormone (GH), luteinizing hormone (LH) and peptide YY) in brain was also changed by exposed to triazophos during 4 and 7d exposure periods. Linked with the above results, the present study pointed out that triazophos might induce a neurotoxic effect and oxidative damage in goldfish, and the goldfish brain should be a critical target for triazophos-induced damage., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Strain A2 with algicidal activity against Microcystis aeruginosa was isolated and identified with the genus Acinetobacter on the basis of phenotypic tests and 16S rRNA gene analysis. It was identified with the species Acinetobactor guillouiae by partial rpoB sequence analysis. When 10% (v/v) of the bacterial culture was co-incubated with M. aeruginosa culture, algicidal efficiency reached 91.6% after 7 days. Supernatant of A2 culture showed similar algicidal activity, while the cell pellet had little activity, suggesting that Acinetobacter guillouiae A2 indirectly attacked M. aeruginosa cells by secreting an extracellular algicidal compound, which was characterized as heat-stable. A significant decrease in the microcystin (microcystin-LR) concentration was observed after 10% (v/v) addition of A2 culture. Transcription of three microcystin-related genes (mcyA, mcyD and mcyH) was also found to be inhibited. The algicidal compound 4-hydroxyphenethylamine was obtained by further isolation and purification using various chromatographic techniques. The EC50, 3d and EC50, 7d values of 4-hydroxyphenethylamine against M. aeruginosa were 22.5 and 10.3 mgL(-1), respectively. These results indicate that A. guillouiae strain A2 inhibits growth of M. aeruginosa and degrades microcystin production. The identified compound, 4-hydroxyphenethylamine, has potential for development as a new algicidal formulation or product., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The strobilurins are used widely in the world as effective fungicidal agents to control Asian soybean rust. In this study, the early life stage of grass carp (Ctenopharyngodon idella), which is one of the most important aquaculture species in China, was chosen to measure the acute toxicity of three common strobilurin-derived fungicides (trifloxystrobin (TFS), azoxystrobin (AZ) and kresoxim-methyl (KM)). As endpoints, normal developmental parameters (lethal concentration (LC₅₀) and average heart rate), expression of relative genes, and three antioxidant enzyme activities in the developing juveniles were recorded during a 48 h exposure. The results revealed that values of LC₅₀ were TFS 0.051 (0.046-0.058) mg L⁻¹, AZ 0.549 (0.419-0.771) mg L⁻¹ and KM 0.338 (0.284-0.407) mg L⁻¹ for juveniles. For the potential toxicity mechanisms, these three fungicides increased catalase (CAT) and peroxidase (POD) activity and decreased superoxide dismutase (SOD) activity, significantly inhibited expressions of three growth-related genes (IGF-1, IGF-2 and GHR) and two energy-related-genes (CCK and PYY), and caused pronounced up-regulation a stress-gene (HSP70). The present study demonstrated potential toxic effects of TFS, AZ and KM on the early development of C. idella. Overall, three strobilurins (TFS, AZ and KM) might cause serious damages to the aquatic species; therefore, their pollution supervision in water ecological environment should be strengthened.

The present study was undertaken to isolate the active compounds responsible for the anthelmintic activity of methanol extract of Semen pharbitidis against Dactylogyrus intermedius in goldfish (Carassius auratus). The active methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation, eventually yielding two bioactive compounds: palmitic acid and pharnilatin A by comparing spectral data (NMR and ESI-MS) with literature values. According to in vivo anthelmintic assays, they were found to be 50% effective at the concentrations (EC(50)) of 5.3 and 1.4 mg L(-1), respectively. The promising palmitic acid and pharnilatin A from S. pharbitidis were also subjected to acute toxicity tests for the evaluation of their safety to the host (goldfish). After 48h exposure, the mortalities of goldfish were recorded, and the established LC(50) values were 2.45- and 5.29-fold higher than the corresponding EC(50), demonstrating that pharnilatins A may have better application potential than palmitic acid. The present results provide evidence that pharnilatins A might be potential source of new anti-parasitic drug for the control of Dactylogyrus., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this work, acute toxicities of heavy metals (Cu, Zn and Cd) were evaluated singly or in mixtures on Chinese rare minnow (Gobiocypris rarus) in its early stages of development (embryos, larvae). Normal embryos not later than 3 h post-fertilization and newly hatched larvae were selected for the tests. The embryos were exposed to the metal solutions studied until developing to the stages of somite formation (15 h), tail detachment (25 h 10 min), heart-beat visible (34 h 10 min), pectoral fin bud appearance (47 h 40 min) and hatching (75 h), respectively. Exposures of the larvae were continued for 24 h. Results from the single toxicity tests revealed that the mortality of embryos increases obviously with increasing exposure duration. G. rarus appears to be more sensitive to heavy metal exposures at larval stage than at embryonic stage. The toxicity order of the tested metals was Cu>Cd>Zn for the embryos and Cu>Zn>Cd for the larvae with LC₅₀ as the toxicity criterion. These data suggest that the sensitivity of G. rarus to heavy metals (Cu, Zn and Cd) is different and it depends on the exposition duration and the stages of fish development. Among the metals tested, Cu is the one most toxic to G. rarus at its early development stages. In addition, results of the mixture experiments showed that binary combinations of Cu-Zn and Cu-Cd had strictly synergistic lethal effects on the larvae. The enhanced toxicity suggested inclusion of mixture considerations in the risk assessment of heavy metals might be recommended., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The present study was designated to ascertain the anthelmintic activity of the dried fruits of Brucea javanica and to isolate and characterise the active constituents. The methanol extract from the fruits of B. javanica showed significant anthelmintic activity against Dactylogyrus intermedius (EC(50) (median effective concentration) value=49.96 mg l(-1)). Based on this finding, the methanol extract was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording two known quassinoids showing potent activity, bruceine A and bruceine D. Both bruceine A and D exhibited significant activity against D. intermedius with EC(50) values of 0.49 mg l(-1) and 0.57 mg l(-1), respectively, which were more effective than the positive control, mebendazole (EC(50) value=1.25 mg l(-1)). In addition, the 48-h median lethal concentration (LC(50)) for bruceine A and D against the host (Carassius auratus) was 10.6-fold and 9.7-fold higher than the EC(50) for D. intermedius. These results provide evidence that the isolated compounds might be potential sources of new anti-parasitic drugs for the control of Dactylogyrus. This is the first report on an in vivo anthelmintic investigation for B. javanica against D. intermedius., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The present study aims to evaluate the anthelmintic properties of aerial part of Macleaya microcarpa (Maxim) Fedde. Bioassay-guided fractionation and isolation of the compounds with anthelmintic activity were performed on the ethanolic extract of M. microcarpa yielding five bioactive alkaloids namely: sanguinarine, cryptopine, beta-allocryptopine, protopine and 6-methoxyl-dihydro-chelerythrine by comparing spectral data (UV, NMR, and EI-MS) with literature values. According to in vivo anthelmintic assays, they were found to be 100% effective at the concentrations of 0.7, 8.0, 8.0, 16.0 and 7.0 mgl(-1), and the median effective concentration (EC(50)) values for the five compounds were 0.37, 3.31, 4.64, 8.13 and 3.63 mgl(-1), respectively. Additionally, the acute toxicity on goldfish for the five active compounds was also investigated with median lethal concentrations (LC(50)) values of 1.13, 16.12, 15.88, 21.69 and 10.91 mgl(-1), respectively. The resulting therapeutic indices for sanguinarine, cryptopine, beta-allocryptopine, protopine and 6-methoxyl-dihydro-chelerythrine were 3.03, 4.82, 3.40, 2.66 and 2.99 correspondingly. Correlations analysis between the logP and EC(50), LC(50) of the five alkaloids revealed that the activity of the five alkaloids was well correlated with their hydrophobicity and r(2)=0.45 is for anthelmintic activity while r(2)=0.47 is for acute toxicity for goldfish, respectively. These results provided evidence that the studied plant extract, as well as the isolated compounds, especially sanguinarine, might be potential plant-based medicines for the treatment of D. intermedius infection., (Copyright 2010 Elsevier B.V. All rights reserved.)