Ammonia exposure alters the expression of immune-related and antioxidant enzymes-related genes and the gut microbial community of crucian carp (Carassius auratus).

Chronic exposure of ammonia in fish can affect the activities of antioxidant enzymes but few studies investigate the influence of ammonia exposure on the expression of immune-related and antioxidant enzymes-related genes. Also, there is no study demonstrates the effect of ammonia exposure on gut microbial community of fish. In this study, crucian carp (Carassius auratus) were exposed to the ammonia concentrations, 0 (control), 10 mg L-1 (low) or 50 mg L-1 (high) for consecutive 30 days at 25 ± 1 °C temperature, respectively, and after that, the fish from all exposure groups were maintained in control conditions for another 15 days. The results showed that low concentration ammonia increased the expression of immune-related genes and antioxidant enzymes-related genes, but high concentration ammonia inhibited the expression of immune-related genes and antioxidant enzymes-related genes. After a 15-day treatment without ammonia, the expression of antioxidant enzymes-related genes and immune-related genes showed no significant changes compared with control. The results of high-throughput sequencing showed that gut microbial communities were significantly differentiated following ammonia exposure. The abundance of Bacteroides and Cetobacterium (two kinds of potential probiotics) increased while fish exposed to 10 mg L-1 ammonia. The Flavobacterium (a potential fish pathogen) showed increasing trends when the exposure dose reached 50 mg L-1, while the Bacteroides and Cetobacterium showed almost no abundance. The results also revealed that ammonia exposure concentration or time can alter the intestinal microbial community. In conclusion, ammonia exposure could induce the immune response in crucian carp, and alter the gut microbial community. The results may help us understand the correlations of gut microbial community shift and ammonia exposure and extend our knowledge to comprehend the effects of environmental factors on intestinal microbial community.

Potential application of antiviral coumarin in aquaculture against IHNV infection by reducing viral adhesion to the epithelial cell surface.

Due to the lack of relevant therapies for infectious haematopoietic necrosis virus (IHNV) infection, the viral outbreak invariably causes serious economic losses in salmonid species. In this study, we evaluated the anti-IHNV effects of 7-(6-benzimidazole) coumarin (C10) and 4-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[4,3-d]pyrimidin-5-one (S5) in vitro and in vivo. The results revealed that C10 at 12.5 mg/L and S5 at 25 mg/L significantly inhibited IHNV replication in epithelioma papulosum cyprini (EPC) cells with a maximum inhibitory rate >90%, showing that IHNV-induced cytopathic effect (CPE) was alleviated by C10 and S5. There are two complementary effects on antiviral mechanism: 1. C10 completely inhibited IHNV infectivity when the virus was preincubated with C10 at 12.5 mg/L, determining that C10 may have a negative impact on IHNV binding to the cell; 2. C10 also up-regulated the gene expression of extracellular proto type galectin-1 (Gal1-L2) and a chimera galectin-3 (Gal3-L1) of EPC cells to inhibit IHNV adhesion. For the in vivo study, injection and immersion of the coumarins enhanced the survival rate of rainbow trout (Oncorhynchus mykiss) juveniles by 25% (at least) at 12 dpi. IHNV loads in the kidney and spleen were also obviously decreased at 96 h, and thus we considered that they had a delaying effect on IHNV replication in vivo. Meanwhile, C10 with a high stability in aquacultural water in immersion suppressed IHNV horizontal transmission by decreasing the viral loads in recipient fish. Overall, our data suggest that there is a positive effect of C10 and S5 against IHNV infection in aquaculture, and C10 had the potential to be a broad-spectrum antiviral against fish rhabdoviruses.

In vitro and in vivo evaluation of antiviral activity of a phenylpropanoid derivative against spring viraemia of carp virus.

Phenylpropanoids, common natural compounds, possess many different biological activities such as antioxidant, anti-inflammatory and antiviral. Spring viraemia of carp virus (SVCV) can cause a high mortality in common carp (Cyprinus carpio). However, there are currently no licenced drugs that effectively cure this disease. In this study, we designed and synthesized a phenylpropanoid derivative 4-(4-methoxyphenyl)-3,4-dihydro-2H-chromeno[4,3-d]pyrimidine-2,5(1 H)-dione (E2), and explored the antiviral effect against SVCV in vitro and in vivo. Up to 25 mg/L of E2 significantly inhibited the expression levels of SVCV protein genes in the epithelioma papulosum cyprini (EPC) cell line by a maximum inhibitory rate of >90%. As expected, E2 remarkably declined the apoptotic of SVCV-infected cells and suppressed potential enhancement of the mitochondrial membrane potential (ΔΨm), these data implied that E2 could protect mitochondria from structural damage in response to SVCV. Meanwhile, E2 was added to EPC cells under four different conditions: time-of-addition, time-of-removal, pre-treatment of viruses and pre-treatment of cells indicated that E2 may block the post-entry transport process of the virus. Additionally, the up-regulation of six interferon (IFN)-related genes also demonstrated that E2 indirectly activated IFNs for the clearance of SVCV in common carp. Drug cure effect showed that treatment with E2 at 0.5 d post infection (dpi) is more effective than at 0, 1 or 2 dpi. Most importantly, intraperitoneal therapy of E2 markedly improved common carp survival rate and reduced virus copies in body. Therefore, the E2 has potential to be developed into a novel anti-SVCV agent.

Developmental toxicity of Fe3O4 nanoparticles on cysts and three larval stages of Artemia salina.

Using Artemia salina cysts (capsulated and decapsulated) and larvae (instar I, II and III) as experimental models, the potential effects of Fe3O4 nanoparticles (Fe3O4-NPs) on marine ecosystems were investigated. Hatchability, mortality and a number of ethological, morphological and biochemical parameters were selected as end-points to define the toxic responses. Data showed that the hatching rates of capsulated and decapsulated cysts were significantly decreased (p < 0.01) following exposure to 600 mg/L for 24 and 36 h. The LC50 values for instar II and III were 482 and 561 mg/L (could not be measured for instar I), and the EC50 values for swimming inhibition of instar I, II and III were 474, 365 and 421 mg/L, respectively. Effects on hatchability, mortality and swimming were accounted for Fe3O4-NPs rather than iron ion released from the NPs. Instar II larvae showed the greatest sensitivity to Fe3O4-NPs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Body lengths of instar I, II and III larvae were decreased in dose-dependent manners. Fe3O4-NPs attached onto the gills and body surface, resulting in irreversible damages. Reactive oxygen species, malondialdehyde content, total antioxidant capacity and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities were substantially increased following exposure, indicating that toxic effects were related to oxidative stress. Mitochondrial malformation, cristae rupturing and membranous structure disruption were clearly observed after Fe3O4-NPs exposure. Fe3O4-NPs were ingested and well distributed in the gut, yolk and primary body cavity. Uptake kinetics data showed that the maximum Fe3O4-NPs content (16.4 mg/g) was reached at 30 h. The combined results so far indicate that Fe3O4-NPs have the potential to affect aquatic organisms when released into the marine ecosystems.