[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: As one of the most serious pathogens in the aquatic environment, Saprolegnia can only in freshwater induce a high mortality rate during the fish egg incubation period. This study investigated anti-Saprolegnia activity of a total of 108 plants on Saprolegnia parasitica in vitro, and Dioscorea collettii was selected for further studies. By loaded on an open silica gel column and eluted with petroleum ether-ethyl acetate-methanol, dioscin (C45H72O16) was isolated from D. collettii. S. parasitica growth was inhibited significantly when dioscin concentration was more than 2.0 mg L(-1). Compared with formalin and hydrogen peroxide, dioscin showed relatively higher inhibitory effect. For the potential inhibition mechanisms, dioscin could cause the S. parasitica mycelium morphologic damage, dense folds, or disheveled protuberances by the observation of field emission scanning electron microscopy and the influx of PI. The structural changes in the treated mycelium were indicative of an efficient anti-Saprolegnia activity of dioscin. The oxidative stress result showed that dioscin also accumulated the reactive oxygen species (ROS) excessively, and increased total antioxidant and superoxide dismutase activity. These situations could render S. parasitica more vulnerable to oxidative damage. Additionally, when dioscin concentration was less than 2.0 mg L(-1), the survival rate of embryo was more than 70%. Therefore, the use of dioscin could be a viable way for the prevention and control of saprolegniasis.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Intestinal microorganisms of aquatic animals have a crucial impact on the host and some may have the potential to be used as probiotics. In this work, three strains of autochthonous bacteria were isolated from the intestinal tract of the healthy grass carp (Ctenopharyngodon idellus). The effects of single or combined use of dietary Shewanella xiamenensis A-1, S. xiamenensis A-2, and Aeromonas veronii A-7 were assessed on innate immunity and disease resistance of grass carp. The grass carp were fed 28 days by five experimental diets: i) one control diet with no probiotics, ii) three diets supplemented with 1 × 108 cell g− 1 of intestinal bacteria A-1 (group A1), A-2 (group A2) and A-7 (group B), and iii) mixed diet (group MIX) comprising of bacteria A 1, A-2 and A 7 dosed at 1 × 108 cell g− 1, at a ratio of 4:2:1, which was set according to their quantity ratio in our study. At the end of the 28-day feeding experiment, the survival of grass carp against Aeromonas hydrophila was challenged for 14 days. Results showed that responses of several non-specific immune parameters of groups fed probiotics were enhanced; i.e. all probiotic groups led to significant enhancement of both respiratory burst, phagocytic and lysozyme activities than that of control activity. The complement C3, total serum proteins, albumin and globulin levels were also significantly improved on day 14 of feeding regime with probiotics compared to the control. Moreover, the expression of four immune-related genes (IL-8, IL-1β, lysozyme-C, and TNF-α genes) was significantly up-regulated in grass carp fed probiotic diets compared to control group on days 7, 14, and 28. The cumulative mortality of grass carp, experimentally challenged with A. hydrophila, was reduced in groups fed probiotics, i.e. A-1 = 46.67%, A-2 = 33.33%, B = 53.33% and mixed = 26.67%, respectively, than that of controls (80%) after 14 days of post-infection. Overall, the results of this study indicated the potential of using the three isolated autochthonous bacteria (single or combined) to improve immunity and disease resistance of grass carp.
[Show abstract][Hide abstract] ABSTRACT: This study investigated the short-term toxicity of azoxystrobin (AZ), one of strobilurins used as an effective fungicidal agent to control the Asian soybean rust, on aquatic unicellular algae Chlorella vulgaris. The median percentile inhibition concentration (IC50) of AZ for C. vulgaris was found to be 510 μg L(-1). We showed that the algal cells were obviously depressed or shrunk in 300 and 600 μg L(-1) AZ treatments by using the electron microscopy. Furthermore, 19, 75, and 300 μg L(-1) AZ treatments decreased the soluble protein content and chlorophyll concentrations in C. vulgaris and altered the energy-photosynthesis-related mRNA expression levels in 48- and 96-h exposure periods. Simultaneously, our results showed that AZ could increase the total antioxidant capacity (T-AOC) level and compromise superoxide dismutase (SOD), peroxidase (POD), glutathione S transferase (GST), glutathione peroxidase (GPx) activities, and glutathione (GSH) content. These situations might render C. vulgaris more vulnerable to oxidative damage. Overall, the present study indicated that AZ might be toxic to the growth of C. vulgaris, affect energy-photosynthesis-related mRNA expressions, and induce reactive oxygen species (ROS) overproduction in C. vulgaris.
Environmental Science and Pollution Research 02/2015; 22(10). DOI:10.1007/s11356-015-4121-7 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 A. 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.
Research in Microbiology 01/2015; 166(2). DOI:10.1016/j.resmic.2014.12.013 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for recombinant Aeromonas hydrophila vaccine administration via bath or injection in juvenile grass carp were studied. The results showed that SWCNT as a vector for the recombinant protein aerA, augmented the production of specific antibodies, apparently stimulated the induction of immune-related genes, and induced higher level of survival rate compared with free aerA subunit vaccine. Furthermore, we compared the routes of bath and intramuscular injection immunization by SWCNTs-aerA vaccine, and found that similar antibody levels induced by SWCNTs-aerA were observed in both immunization routes. Meanwhile, a similar relative percentage survival (approximately 80%) was found in both a 40 mg/L bath immunization group, and a 20 μg injection group. The results indicate that functionalized SWCNTs could be a promising delivery vehicle to potentiate the immune response of recombinant vaccines, and might be used to vaccinate juvenile fish by bath administration method.
[Show abstract][Hide abstract] ABSTRACT: The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1β, TNFα, MyD88 and IgM) in C. idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1β, TNFα, and MyD88 in C. idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L−1). According to in vivo insecticidal assays against Dactylogyrus intermedius, compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L−1 and safe to goldfish (Carassius auratus). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C. idella kidney cells and to some extent, eliminate the virus and parasitic infections.
[Show abstract][Hide abstract] ABSTRACT: Trifloxystrobin (TFS) is the widely used strobilurin fungicide. However, little information is so far available regarding the uptake kinetics and developmental toxicity of TFS to fish. The present study was conducted to investigate the uptake kinetics, potential environment risk and toxicity of TFS on Gobiocypris rarus embryos. Results revealed that increased malformation, decreased body length and heart rate, affected spontaneous movement and swimming speed provide a gradual concentration-dependent manner; values of 144 h LC50 (median lethal concentration) and EC50 (median effective concentration) were 1.11 and 0.86 μg/L. Continuous exposure to TFS resulted in a steady accumulation with no evidence of elimination. Enzyme activities were significantly changed; reactive oxygen species and DNA damage were significantly induced after TFS treatment. Certain genes related to cell apoptosis (p53), metabolism (cyp1a), stress response (hsp70) and blood vessels (vezf1) development were all significantly up-regulated. This is the first study to define uptake kinetics and to focus on behavioral consequences, physiological changes and mRNA expression following TFS exposure in the early life stages of fish. Our results suggest that TFS is highly toxic to fish embryos.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Abstract Carbon nanotubes (CNTs) are widely used in industrial and commercial applications, but few studies systematically evaluate their developmental toxicity on aquatic organism. Using rare minnow (Gobiocypris rarus) at early life stages as experimental models, developmental toxicity of functionalized single-walled CNTs (SWCNTs) was investigated following exposure to 0-320 mg/L for 144 h. Results revealed that significantly increased in mortality and malformation was only observed after hatching. Decreased body length, heart rate and swimming speed provide a concentration-dependent manner on larvae; values of 144 h LC50 and EC50 were 140.8 and 109.8 mg/L, respectively. Antioxidant enzyme activities (superoxide dismutase, catalase and glutathione S-transferase) and antioxidant enzyme related mRNA expressions were significant changed; cell apoptosis activities (caspase-3, -8, -9) and cell apoptosis related mRNA expressions were significant up-regulated; reactive oxygen species and DNA damage were significantly induced when the concentration of SWCNTs above 100 mg/L. Fluorescence and electron microscopy sliced observation show that SWCNTs were well dispersed in larvae within 0.5 h, eventually cleared from the larvae at 144 h. This is the first study to define uptake kinetics and to focus on behavioral consequences, physiological changes and mRNA expression following SWCNTs exposure in the early life stages of fish. The results obtained in the present study demonstrated that functionalized SWCNTs have the potential to affect aquatic life when released into the aquatic environment and reached high concentration. In the increasing economical context of SWCNTs, complementary studies must be undertaken, especially including mechanistic and environmental investigations.
[Show abstract][Hide abstract] ABSTRACT: Current chemotherapy of mono-component for Dactylogyrus is unsatisfactory due to its limited efficacy with side effects including pesticide resistance and drug residue. The present study is conducted to investigate the effects of herbal ingredients combination against Dactylogyrus on Carassius auratus under in vivo conditions. The ingredients of Dioscorea zingiberensis (D. zingiberensis) and Ginkgo biloba (G. biloba) are administered individually and in combination in an experimental fish infection model in this study. Finally, strong synergy actions are observed in both the methanol extract combinations and the active compound combinations between D. zingiberensis and G. biloba. The anthelmintic effects of the combination are expressed in terms of the values of EC50, EC75, EC90, and EC99 to compare with the phytomedicine alone. The combination index (CI) is also calculated by using the combination index algorithm. Both the values of effective concentration and combination index indicate a highly synergistic anthelmintic effect against Dactylogyrus. Our study provides a powerful means by which anthelmintic combination therapies can be examined and demonstrates that the combinations of herbal ingredients have excellent properties and are predicted to give improved cure rates in monogenean management of aquaculture.
[Show abstract][Hide abstract] ABSTRACT: Cyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, fungi, bacteria and cell lines. Recently the main methods of controlling cyanobacteria are using chemicals, medicinal plants and microorganism but fewer involved the safety research in hydrophytic ecosystems. In search of an environmentally safe compound, 53 chemicals were screened against the developed heavy cyanobacteria bloom Microcystis aeruginosa using coexistence culture system assay. The results of the coexistence assay showed that 9 chemicals inhibited M. aeruginosa effectively at 20 mg L(-1) after 7 days of exposure. Among them dimethomorph, propineb, and paraquat were identified that they are safe for Chlorella vulgaris, Scenedesmus obliquus, Carassius auratus (Goldfish) and Bacillus subtilis within half maximal effective concentration (EC50) values 5.2, 4.2 and 0.06 mg L(-1) after 7 days, respectively. Paraquat as the positive control observed to be more efficient than the other compounds with the inhibitory rate (IR) of 92 % at 0.5 mg L(-1). For the potential inhibition mechanism, the chemicals could destroy the cell ultrastructure in different speed. The safety assay proved dimethomorph, propineb and paraquat as harmless formulations or products having potential value in M. aeruginosa controlling, with the advantage of its cell morphology degrading ability.
[Show abstract][Hide abstract] ABSTRACT: Triazophos (TAP) has been widely used in agriculture for controlling insect pests and is a known organophosphorus pesticide. Due to TAP characteristics, such as high chemical and photochemical stability, its potential toxicity to aquatic organisms has gained great interest. To explore the potential developmental toxicity 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 LC50 and EC50 were 7.44 and 5.60 mg L−1 for embryos, 2.52 and 1.37 mg L−1 for larvae. Increased malformation, decreased heart rate and body length provide a gradual concentration-dependent pattern. Enzyme activities and mRNA levels were significantly 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 information presented in this study will be helpful in better understanding the toxicity induced by TAP in fish embryos and larvae.
[Show abstract][Hide abstract] ABSTRACT: Using rare minnow (Gobiocypris rarus) at early-life stages as experimental models, the developmental toxicity of five widely used triazole fungicides (myclobutanil, fluconazole, flusilazole, triflumizole, and epoxiconazole) were investigated following exposure to 1-15 mg/L for 72 h. Meanwhile, morphological parameters (body length, body weight, and heart rate), enzyme activities (superoxide dismutase (SOD), glutathione S-transferase (GST), adenosine triphosphatase (ATPase), and acetyl cholinesterase (AChE)), and mRNA levels (hsp70, mstn, mt, apaf1, vezf1, and cyp1a) were also recorded following exposure to 0.2, 1.0, and 5.0 mg/L for 72 h. Results indicated that increased malformation and mortality, decreased body length, body weight, and heart rate provide a concentration-dependent pattern; values of 72 h LC50 (median lethal concentration) and EC50 (median effective concentration) ranged from 3 to 12 mg/L. Most importantly, the results of the present study suggest that even at the lowest concentration, 0.2 mg/L, five triazole fungicides also caused notable changes in enzyme activities and mRNA levels. Overall, the present study points out that those five triazole fungicides are highly toxic to the early development of G. rarus embryos. The information presented in this study will be helpful in better understanding the toxicity induced by triazole fungicides in fish embryos.
Environmental Science and Pollution Research 07/2014; 21(23). DOI:10.1007/s11356-014-3317-6 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of single or combined use of dietary Shewanella haliotis D4, Bacillus cereus D7 and Aeromonas bivalvium D15, isolated from the gut of shrimp, were assessed on the growth, innate immunity and disease resistance of Pacific white shrimp (Litopenaeus vannamei). Shrimp was fed 28 days by five experimental diets — i) control diet with no probiotics, ii) three probiotic diets, i.e. feed supplemented with 107 cell g− 1 of D4 (D4 diet), D7 (D7 diet) and D15 (D15 diet), and iii) mixed diet comprising of strains D4, D7 and D15 at a ratio of 2:1:1, dosed at 107 cell g− 1. Results showed that growth of shrimp, i.e. final weight, weight gain, percent weight gain and specific growth rate, was improved after 28-days of feeding regime with probiotics compared to the control. Respond of several non-specific immune parameters of groups fed probiotics was also enhanced; i.e. diet D4 and D15 led to significant enhancement of both respiratory burst and superoxide dismutase activities than that of control. Moreover, the respiratory burst activity was also enhanced by mixed diet at the end of 28 days. All probiotic groups were able to increase acid phosphatase activity of shrimp between days 7 and 21. Moreover, the expression of prophenoloxidase and < beta>-1, 3-glucan-binding protein was significantly up regulated in shrimp fed D4 and D15 diets compared to control group, but penaeidin 3 expression up regulated significantly only with mixed diet after 28 days. The cumulative mortality of shrimp, experimentally challenged with Vibrio harveyi, was reduced in groups fed probiotics, i.e. D4 = 36.67%, D7 = 41.67%, D15 = 13.33% and mixed = 11.67%, respectively, than that of controls (43.33%) after 10 days of post-infection. Overall, the results of this study indicated the potential of using probiotics to improve growth, immunity and disease resistance of white shrimp.
Aquaculture 05/2014; s 428–429:141–149. DOI:10.1016/j.aquaculture.2014.03.016 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study investigated the effects of trifloxystrobin that one strobilurin used widely in the world as an effective fungicidal agent to control Asian soybean rust on aquatic unicellular algae Chlorella vulgaris. We determined the potential toxic effect of trifloxystrobin on C. vulgaris, and found median inhibition concentration (IC50) value 255.58 (95% confidence interval, 207.81-330.29)μgL(-1). In addition, the algal cells were obviously depressed or shrunk at different concentrations by electron microscopy. In the study, a real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL, and one energy gene, ATPs. The results showed that trifloxystrobin reduced the transcript abundances of the three genes and enhanced expression of ATPs after 48 and 96h. The lowest abundances of psaB, psbC and rbcL transcripts in response to trifloxystrobin exposure were 58%, 79% and 60% of those of the control, respectively. For the potential toxic influences, trifloxystrobin could decrease the soluble protein and total antioxidant contents (T-AOC), and increase superoxide dismutase (SOD) and peroxidase (POD) activity with a gradual concentration-response relationship. Overall, the present study demonstrated that trifloxystrobin could affect the activities of antioxidant enzymes, disrupts photosynthesis in C. vulgaris, and damage cellular structure.