Gao-Xue Wang

In vivo anthelmintic activity of five alkaloids from Macleaya microcarpa (Maxim) Fedde against Dactylogyrus intermedius in Carassius auratus

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.

Osthol and isopimpinellin from Fructus cnidii for the control of Dactylogyrus intermedius in Carassius auratus

The efficacy of two active compounds, isolated from the fruit of Fructus cnidii, was determined against Dactylogyrus intermedius in goldfish (Carassius auratus). The chemical structure of the two compounds was identified by spectral analysis. Designated Compounds I and II, they were isolated by successive silica gel column chromatography and used, in combination, in an in vivo anthelmintic efficacy assay. At dose rates of 1.6 and 9.5 mg l(-1), respectively, the two compounds were 100% effective against D. intermedius compared to mebendazole (positive control), used at a concentration of 1.5 mg l(-1), which is 100% efficacious. Compounds I and II were shown to be safe for goldfish at dose rates of up to 6.4 and 12.5 mg l(-1), respectively. The median effective dose (ED(50)) of Compounds I and II for D. intermedius-infested goldfish after 48 h exposure was 0.807 and 8.050 mg l(-1), respectively. If the median lethal dose (LD(50)) values for goldfish of the two compounds are 6.749 and 12.759 mg l(-1), respectively, the ED(50) values for D. intermedius yielded a therapeutic index of 8.14 and 1.52 for Compounds I and II, respectively, at 48 h. Compounds I and II were identified as osthol and isopimpinellin by physico-chemical detection.

Single and joint action toxicity of heavy metals on early developmental stages of Chinese rare minnow (Gobiocypris rarus)

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.

Protective immunity of grass carp immunized with DNA vaccine encoding the vp7 gene of grass carp reovirus using carbon nanotubes as a carrier molecule

The uses of single walled carbon nanotubes (SWCNTs) as carriers for DNA delivery have received considerable attention in cell studies. DNA vaccination of fish has been shown to elicit durable transgene expression, but no reports exist on intramuscular administration of SWCNTs-DNA vaccine electrostatic complexes which prepared through non-covalent conjugation. In this study, we injected grass carp intramuscularly with a plasmid vector containing a major capsid protein gene (vp7) of grass carp reovirus as a) naked pcDNA-vp7, b) SWCNTs-pcDNA-vp7, c) empty plasmid vector, or phosphate buffered saline. After intramuscular administration, the ability of the different immune treatments to induce transgene expression was analyzed. The results indicated that higher levels of transcription and expression of the vp7 gene could be detected in muscle tissues of grass carp 28 days intramuscular injection in SWCNTs-pcDNA-vp7 treatment groups compare with naked pcDNA-vp7 treatment groups. Moreover, the serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, immune-related genes, antibody levels and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pcDNA-vp7 vaccine. The data in this study suggested that SWCNTs were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by intramuscular approach.

Effects of dietary supplementation of probiotic Shewanella colwelliana WA64, Shewanella olleyana WA65 on the innate immunity and disease resistance of abalone, Haliotis discus hannai Ino

The effects of dietary administration of two probiotics, Shewanella colwelliana WA64 and Shewanella olleyana WA65, on the innate immunity of abalone (Haliotis discus hannai Ino), and survival of juvenile abalone challenged with Vibrio harveyi have been studied. Two groups of abalone were fed with three different diets: one control, and two diets supplemented with 10(9) cell g(-1) of probiotic WA64 (WA64 diet) and WA65 (WA65 diet) for up to four weeks. Results showed that abalone fed diets containing S. colwelliana WA64 and S. olleyana WA65 had led to an enhanced cellular and humoral immune response, notably higher haemocytes, respiratory burst activity, serum lysozyme activity and total protein levels were recorded after one week of probiotic administration. On the other hand, mortality after the challenges with V. harveyi in the group fed with control diet ranged from 77 to 80%, while mortality rates observed in the groups fed with diets supplemented with WA64 and WA65 ranged from 27 to 50% and 30-43%, respectively. The results demonstrated potential for S. colwelliana WA64 and S. olleyana WA65 to improve innate immunity and disease resistance in H. discus hannai.

Algicidal activity of Salvia miltiorrhiza Bung on Microcystis aeruginosa—Towards identification of algicidal substance and determination of inhibition mechanism

The present study was to isolate and identify a potent algicidal compound from extract of Salvia miltiorrhiza and study the potential inhibition mechanism on Microcystis aeruginosa. Column chromatography and bioassay-guided fractionation methods were carried out to yield neo-przewaquinone A, which was identified by spectral analysis. The EC50 of neo-przewaquinone A on M. aeruginosa were 4.68 mg L(-1). In addition, neo-przewaquinone A showed relatively higher security on Chlorella pyrenoidosa and Scenedesmus obliquus, with the EC50 values of 14.78 and 10.37 mg L(-1), respectively. For the potential inhibition mechanisms, neo-przewaquinone A caused M. aeruginosa cells morphologic damage or lysis, increased malondialdehyde content and decreased the soluble protein content, total antioxidant and superoxide dismutase activity, and significantly inhibited three photosynthesis-related genes (psaB, psbD, and rbcL). The results demonstrated the algicidal effect of neo-przewaquinone A on M. aeruginosa and provided the possible inhibition mechanisms.

Bioassay-guided isolation and identification of active compounds from Semen pharbitidis against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

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.

Developmental toxicity of 3,4-dichloroaniline on rare minnow (Gobiocypris rarus) embryos and larvae

Gobiocypris rarus is a freshwater cyprinid, which possesses lots of attractive features (short life cycle, high fecundity, and especially the transparent trait during early life stage) that make it a suitable model in aquatic toxicity tests. In this study, the effects of 3,4-dichloroaniline (3,4-DCA) on the early life stages of G. rarus were measured. As endpoints, normal developmental parameters (survival rate, malformation rate, total body length and average heart rate) as well as biomarker genes (stress response (hsp70), organizer function and axis formation (wnt8a), vascular system development (vezf1), detoxification (cyp1a) and endocrine disruption (erα)) in the developing embryos and larvae were recorded during a 72 h exposure. The results revealed that reduced survival rate, increased malformation, changes in heart rate and total body length provide a gradual dose-response relationship, values of 72 h LC(50) were 4.146 (3.665-4.713) mg L(-1) for embryos and 1.088 (0.832-1.432) mg L(-1) for larvae. The developmental biochemical biomarkers are very promising tools to determine the severity of toxicants in the growing G. rarus embryos and larvae, even at a concentration of 1% for LC(50). Gene expressions of wnt8a and cyp1a in embryos were highly up-regulated (more than 100-fold) after exposure to 3,4-DCA. Overall, the present study points out that 3,4-DCA is high toxic to the early development of G. rarus, and offers a practicable and highly sensitive bioassay for the general assessment of chemical toxicity.

Effect of a new recombinant Aeromonas hydrophila vaccine on the grass carp intestinal microbiota and correlations with immunological responses

Intestinal microbiota has become an integral component of the fish, and plays a key role in host metabolism, immunity and health maintenance. However, information on the immune responses after vaccine administration in relation to the intestinal microbiota is absent in fish. The present study focused on the effect of a new recombinant Aeromonas hydrophila vaccine (Aera) by using a novel functionalized, single-walled carbon nanotubes (SWCNTs) as a delivery vehicle on the intestinal microbiota of grass carp (Ctenopharyngodon idella) through the bath immunization, and further explored the immunological responses in intestine, kidney and spleen. By performing deep sequencing, a total of 81,979 valid reads and 609 OTUs obtained from 4 intestine samples were analyzed. We detected 141 genuses, most of which belonged to Firmicutes, Fusobacteria and Proteobacteria. Of note, the quantity of Aeromonas in library Aera (after 6 h Aera vaccine pretreatment, fish were transferred to tanks without SWCNTs-Aera for 28 d) and Aera-GD (6 h Aera vaccine pretreated prior to the group injected by A. hydrophila) was declined 6.5% and 14.6% compared with the control, respectively. Moreover, the expression of seven immune-related genes (IFN-I, TNF-α, CRP, IL-8, IgM, MHC I and CD8α) in the intestine, kidney and spleen of Aera treated fish was significantly enhanced, which indicated that a better tissue immune response in grass carp was induced by the SWCNTs-Aera vaccine. Therefore, a new recombinant SWCNT-Aera vaccine may represent potentially efficient and immunological role in grass carp intestine to resist A. hydrophila infection.

Single-walled carbon nanotubes as candidate recombinant subunit vaccine carrier for immunization of grass carp against grass carp reovirus

Grass carp reovirus (GCRV), the most pathogenic aquareovirus, can cause fatal hemorrhagic disease in fingerling and yearling grass carp. Vaccination by injection is by far the most effective method of combating disease. However it is labor intensive, costly and not feasible to vaccinate large numbers of the fish. Thus, an efficient and economic strategy for the prevention of GCRV infection becomes urgent. Here, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture SWCNTs-VP7 subunit vaccine with chemical modification. Different developmental stages of grass carps were immunized by VP7/SWCNTs-VP7 subunit vaccine against GCRV by intramuscular injection and bath immunization. The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP7 subunit vaccine were induced in comparison with VP7 subunit vaccine alone. Immunization doses/concentrations are significantly reduced (about 5-8 times) to prevent GCRV infection in different developmental stages of grass carp with injection or bath treatment when SWCNTs carrier was used. A good immune protective effect (relative percentage survival greater than 95%) is observed in smaller size fish (0.2 g) with SWCNTs-VP7 bath immunization. In addition, serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes and antibody levels were significantly enhanced in fish immunized with vaccine. This study suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate fish by bath approach.