Responses of pro- and anti-inflammatory cytokines in zebrafish liver exposed to sublethal doses of Aphanizomenon flosaquae DC-1 aphantoxins.

Blooms of the dominant cyanobacterium Aphanizomenon flosaquae are frequently encountered in natural waters, and their secretion of neurotoxic paralytic shellfish toxins called aphantoxins threatens environmental safety and human health worldwide. The liver is the primary detoxification organ in animals, and its pro- and anti-inflammatory responses are important functions in the detoxification of toxins. Therefore, we investigated the response of these inflammatory factors to aphantoxins in the liver of zebrafish (Danio rerio). A. flosaquae DC-1 was sampled during blooms in Dianchi Lake, China and cultured, and the toxin was extracted and analyzed using high performance liquid chromatography. The primary constituents were gonyautoxins 1 (34.04%) and 5 (21.28%) and neosaxitoxin (12.77%). Zebrafish were injected intraperitoneally with 5.3 µg (low dose) or 7.61 µg (high dose) of saxitoxin equivalents [equivalents (eq.)]/kg body weight of A. flosaquae DC-1 aphantoxins. Hyperemia, the hepatosomatic index (HSI), and physiological and molecular responses of pro- and anti-inflammatory cytokines in the zebrafish liver were investigated at different time points 1-24 h post-exposure. Aphantoxins significantly enhanced hepatic hyperemia and altered the HSI 3-24 h post-exposure, suggesting that inflammation caused morphological changes. Subsequent investigations using the enzyme-linked immunosorbent assay showed that the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1ß (IL-1ß), IL-6, and IL-8 and anti-inflammatory cytokines IL-10 and transforming growth factor ß were higher in the liver of zebrafish exposed to aphantoxins, which indicated physiological inflammatory responses. Further analysis by real-time fluorescence quantitative polymerase chain reaction demonstrated upregulated mRNA expression of these cytokines, suggesting molecular inflammatory responses in the zebrafish liver. These changes showed dose- and time-dependent patterns. These results indicated that aphantoxins induced hyperemia and altered the HSI, and subsequently increased the levels of proinflammatory cytokines TNF-α, IL-1ß, IL-6 and IL-8 to induce physiological inflammatory responses. These changes activated the anti-inflammatory cytokines IL-10 and TGF-ß to suppress inflammatory damage. The induced changes were the result of upregulated mRNA expression of these inflammatory cytokines caused by aphantoxins. Aphantoxins resulted in hepatic immunotoxicity and response by inducing pro-inflammatory cytokines. Zebrafish liver in turn suppressed the inflammatory damage by upregulating the activities of anti-inflammatory cytokines. In the future, these pro- and anti-inflammatory cytokines in the zebrafish liver may be prove to be useful biomarkers of aphantoxins and blooms in nature.

Respiratory toxicity of cyanobacterial aphantoxins from Aphanizomenon flos-aquae DC-1 in the zebrafish gill.

Aphantoxins from Aphanizomenon flos-aquae are frequently identified in eutrophic waterbodies worldwide. These toxins severely endanger environmental safety and human health due to the production of paralytic shellfish poisons (PSPs). Although the molecular mechanisms of aphantoxin neurotoxicity have been studied, many questions remain to be resolved such as in vivo alterations in branchial histology and neurotransmitter inactivation induced by these neurotoxins. Aphantoxins extracted from a naturally isolated strain of A. flos-aquae DC-1 were determined by high performance liquid chromatography. The basic components of the isolated aphantoxins identified were gonyautoxin 1 (GTX1), gonyautoxin 5 (GTX5), and neosaxitoxin (neoSTX), which comprised 34.04, 21.28, and 12.77% of the total, respectively. Zebrafish (Danio rerio) was administrated 5.3 or 7.61mg STX equivalents (eq)/kg (low and high doses, respectively) of the A. flos-aquae DC-1 aphantoxins by intraperitoneal injection. Histological alterations and changes in neurotransmitter inactivation in the gills of zebrafish were investigated for 24h following exposure. Aphantoxin exposure significantly increased the activities of gill alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and resulted in histological alterations in the gills during the first 12h of exposure, indicating the induction of functional and structural damage. Gill acetylcholinesterase (AChE) and monoamine oxidase (MAO) activities were inhibited significantly, suggesting an alteration of neurotransmitter inactivation in zebrafish gills. The observed alterations in gill structure and function followed a time- and dose-dependent pattern. The results demonstrate that aphantoxins or PSPs lead to structural damage and altered function in the gills of zebrafish, including changes in histological structure and increases in the activities of AST and ALT. The inhibition of the activities of AChE and MAO suggest that aphantoxins or PSPs could induce respiratory toxicity in the zebrafish gill. Furthermore, these parameters may be used as bioindicators for investigating aphantoxin exposure and cyanobacterial blooms in nature.

Antioxidative responses in zebrafish liver exposed to sublethal doses Aphanizomenon flos-aquae DC-1 aphantoxins.

Aphanizomenon flos-aquae secretes paralytic shellfish poisons (PSPs), termed aphantoxins, and endangers environmental and human health via eutrophication of water worldwide. Although the molecular mechanism of neuronal PSP toxicity has been well studied, several issues remain unresolved, notably the in vivo hepatic antioxidative responses to this neurotoxin. Aphantoxins extracted from a natural isolate of A. flos-aquae DC-1 were resolved by high performance liquid chromatography. The primary components were gonyautoxins 1 and 5 and neosaxitoxin. Zebrafish (Danio rerio) were treated intraperitoneally with either 5.3 or 7.61 (low and high doses, respectively) µg saxitoxin (STX) equivalents (eq)/kg of A. flos-aquae DC-1 aphantoxins. Antioxidative responses in zebrafish liver were examined at different timepoints 1-24h post-exposure. Aphantoxin administration significantly enhanced hepatic malondialdehyde (MDA) content 1-12h post-exposure, indicative of oxidative stress and lipid peroxidation. By contrast, levels of reduced glutathione (GSH) in zebrafish liver declined significantly after 3-24h exposure, suggesting that GSH participates in MDA metabolism. A significant upregulation of the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) was observed, suggesting that aphantoxins induce lipid peroxidation in zebrafish liver and are likely to be hepatotoxic. Hepatic levels of MDA and GSH, and of the three enzymes (SOD, CAT, and GPx), therefore provide potential biomarkers for studying environmental exposure to aphantoxins/PSPs from cyanobacterial blooms.

Morphological alterations and acetylcholinesterase and monoamine oxidase inhibition in liver of zebrafish exposed to Aphanizomenon flos-aquae DC-1 aphantoxins.

Aphanizomenon flos-aquae is a cyanobacterium that produces neurotoxins or paralytic shellfish poisons (PSPs) called aphantoxins, which present threats to environmental safety and human health via eutrophication of water bodies worldwide. Although the molecular mechanisms of this neurotoxin have been studied, many questions remain unsolved, including those relating to in vivo hepatic neurotransmitter inactivation, physiological detoxification and histological and ultrastructural alterations. Aphantoxins extracted from the natural strain of A. flos-aquae DC-1 were analyzed by high-performance liquid chromatography. The main components were gonyautoxins 1 and 5 (GTX1, GTX5) and neosaxitoxin (neoSTX), which comprised 34.04%, 21.28%, and 12.77% respectively. Zebrafish (Danio rerio) were exposed intraperitoneally to 5.3 or 7.61 µg STX equivalents (eq)/kg (low and high doses, respectively) of A. flos-aquae DC-1 aphantoxins. Morphological alterations and changes in neurotransmitter conduction functions of acetylcholinesterase (AChE) and monoamine oxidase (MAO) in zebrafish liver were detected at different time points 1-24h post-exposure. Aphantoxin significantly enhanced hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and histological and ultrastructural damage in zebrafish liver at 3-12 h post-exposure. Toxin exposure increased the reactive oxygen species content and reduced total antioxidative capacity in zebrafish liver, suggesting oxidative stress. AChE and MAO activities were significantly inhibited, suggesting neurotransmitter inactivation/conduction function abnormalities in zebrafish liver. All alterations were dose- and time-dependent. Overall, the results indicate that aphantoxins/PSPs induce oxidative stress through inhibition of AChE and MAO activities, leading to neurotoxicity in zebrafish liver. The above parameters may be useful as bioindicators for investigating aphantoxins/PSPs and cyanobacterial blooms in nature.

[Development and succession of biological soil crusts and the changes of microbial biomasses].

Biological soil crusts (BSCs) play important ecological roles in vegetation and ecological restoration in desert regions, and different crust developmental and successional stages have different ecological functions. In this experiment, the BSCs in Shapotou region (at southeast edge of Tengger Desert) were investigated to study crust development and succession through field investigation, microscopic observation combined with quantitative analysis of microbial biomasses. The results showed that BSCs in this region generally developed and succeeded from algal crusts, lichen crusts to moss crusts. With the development and succession of BSCs, crust photosynthetic biomass gradually increased, while microalgal biomass showed a first increasing and then decreasing trend. Among the crust algae (cyanobacteia), Microcoleus vaginatus, as the first dominant species, occupied the most algal biomass and reached a maximum of 0.33 mm3 x g(-1) crusts in algal crusts; while Scytonema javanicum and Nostoc sp. have their maximal biomasses in the later lichen crusts. In addition, it was found that the heterotrophic microbial biomass began to increase in algal crusts, and then decreased in lichen crusts; followed by another increase and the increase achieved the maximum at last in moss crusts. Through the correlation analysis, it was found that bacterial biomass significantly positively correlated with crust organic carbon and Na+ content, while fungal biomass positively correlated with K+ and Na+ content (P < 0.05). In conclusion, this study investigated the developmental and successional patterns of BSCs in Shapotou region, and discussed the effects of crust development and succession on several microbial biomasses from the point of view of environmental adaptation and functional requirement, which may be helpful for us to understand crust development and succession, and provide theoretical and practical significances for crust maintenance and management in ecological restoration of desertification regions.

[Development and succession of artificial biological soil crusts and water holding characteristics of topsoil].

In order to understand the improving effects of cyanobacterial inoculation on water retention of topsoil in desert regions, this work focused on the development and succession of biological soil crusts and water holding characteristics of topsoil after cyanobacterial inoculation in Qubqi Desert. The results showed that after the artificial inoculation of desert cyanobacteria, algal crusts were quickly formed, and in some microenvironments direct succession of the algal crusts to moss crusts occurred after 2-3 years. With the development and succession of biological soil crusts, the topsoil biomass, polysaccharides content, crust thickness and porosity increased, while the soil bulk density decreased. At the same time, with crust development and succession, the topsoil texture became finer and the percents of fine soil particles including silt and clay contents increased, while the percents of coarse soil particles (sand content) decreased proportionately. In addition, it was found that with crust development and succession, the water holding capacity and water content of topsoil showed an increasing trend, namely: moss crust > algal crusts > shifting sand. The water content (or water holding capacity) in algal and moss crusts were 1.1-1.3 and 1.8-2.2 times of those in shifting sand, respectively. Correlation analysis showed that the water holding capacity and water content of topsoil were positively correlated with the crust biomass, polysaccharides content, thickness, bulk density, silt and clay content; while negatively correlated with the porosity and sand content. Furthermore, stepwise regression analysis showed that the main factor affecting water content was the clay content, while that affecting water holding capacity was the porosity.

Lipid peroxidation and antioxidant responses in zebrafish brain induced by Aphanizomenon flos-aquae DC-1 aphantoxins.

Aphanizomenon flos-aquae is a cyanobacterium that is frequently encountered in eutrophic waters worldwide. It is source of neurotoxins known as aphantoxins or paralytic shellfish poisons (PSPs), which present a major threat to the environment and human health. The molecular mechanism of PSP action is known, however the in vivo effects of this neurotoxin on oxidative stress, lipid peroxidation and the antioxidant defense responses in zebrafish brain remain to be understood. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed using high performance liquid chromatography. The major components of the toxins were gonyautoxins 1 and 5 (GTX1 and GTX5, 34.04% and 21.28%, respectively) and neosaxitoxin (neoSTX, 12.77%). Zebrafish (Danio rerio) were injected intraperitoneally with 7.73 µg/kg (low dose) and 11.13 µg/kg (high dose) of A. flos-aquae DC-1 aphantoxins. Oxidative stress, lipid peroxidation and antioxidant defense responses in the zebrafish brain were investigated at various timepoints at 1-24h post-exposure. Aphantoxin exposure was associated with significantly increased (>1-2 times) reactive oxygen species (ROS) and malondialdehyde (MDA) in zebrafish brain compared with the controls at 1-12h postexposure, suggestive of oxidative stress and lipid peroxidation. In contrast, reduced glutathione (GSH) levels in the zebrafish brain exposed to high or low doses of aphantoxins decreased by 44.88% and 41.33%, respectively, after 1-12h compared with the controls, suggesting that GSH participated in detoxification to ROS and MDA. Further analysis showed a significant increase in the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) compared with the controls, suggesting elimination of oxidative stress by the antioxidant response in zebrafish brain. All these changes were dose and time dependent. These results suggested that aphantoxins or PSPs increased ROS and MDA and decreased GSH in zebrafish brain, and these changes induced oxidative stress. The increased activity of SOD, CAT and GPx demonstrated that these antioxidant enzymes could play important roles in eliminating excess ROS and MDA. These results also suggest that MDA, ROS, GSH and these three antioxidant enzymes in the brain of zebrafish may act as bioindicators for investigating A. flos-aquae DC-1 aphantoxins or PSPs and algal blooms in nature.

Zebrafish locomotor capacity and brain acetylcholinesterase activity is altered by Aphanizomenon flos-aquae DC-1 aphantoxins.

Aphanizomenon flos-aquae (A. flos-aquae) is a source of neurotoxins known as aphantoxins or paralytic shellfish poisons (PSPs) that present a major threat to the environment and to human health. Generally, altered neurological function is reflected in behavior. Although the molecular mechanism of action of PSPs is well known, its neurobehavioral effects on adult zebrafish and its relationship with altered neurological functions are poorly understood. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed by HPLC. The major analogs found in the toxins were the gonyautoxins 1 and 5 (GTX1 and GTX5; 34.04% and 21.28%, respectively) and the neosaxitoxin (neoSTX, 12.77%). Zebrafish (Danio rerio) were intraperitoneally injected with 5.3 and 7.61 µg STXeq/kg (low and high dose, respectively) of A. flos-aquae DC-1 aphantoxins. The swimming activity was investigated by observation combined with video at 6 timepoints from 1 to 24 h post-exposure. Both aphantoxin doses were associated with delayed touch responses, reduced head-tail locomotory abilities, inflexible turning of head, and a tailward-shifted center of gravity. The normal S-pattern (or undulating) locomotor trajectory was replaced by a mechanical motor pattern of swinging the head after wagging the tail. Finally, these fish principally distributed at the top and/or bottom water of the aquarium, and showed a clear polarized distribution pattern at 12 h post-exposure. Further analysis of neurological function demonstrated that both aphantoxin doses inhibited brain acetylcholinesterase activity. All these changes were dose- and time-dependent. These results demonstrate that aphantoxins can alter locomotor capacity, touch responses and distribution patterns by damaging the cholinergic system of zebrafish, and suggest that zebrafish locomotor behavior and acetylcholinesterase can be used as indicators for investigating aphantoxins and blooms in nature.

The genetic basis of white tigers.

The white tiger, an elusive Bengal tiger (Panthera tigris tigris) variant with white fur and dark stripes, has fascinated humans for centuries ever since its discovery in the jungles of India. Many white tigers in captivity are inbred in order to maintain this autosomal recessive trait and consequently suffer some health problems, leading to the controversial speculation that the white tiger mutation is perhaps a genetic defect. However, the genetic basis of this phenotype remains unknown. Here, we conducted genome-wide association mapping with restriction-site-associated DNA sequencing (RAD-seq) in a pedigree of 16 captive tigers segregating at the putative white locus, followed by whole-genome sequencing (WGS) of the three parents. Validation in 130 unrelated tigers identified the causative mutation to be an amino acid change (A477V) in the transporter protein SLC45A2. Three-dimensional homology modeling suggests that the substitution may partially block the transporter channel cavity and thus affect melanogenesis. We demonstrate the feasibility of combining RAD-seq and WGS to rapidly map exotic variants in nonmodel organisms. Our results identify the basis of the longstanding white tiger mystery as the same gene underlying color variation in human, horse, and chicken and highlight its significance as part of the species' natural polymorphism that is viable in the wild.