Yongding Liu

Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR

The freshwater, bloom-forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin, which causes the damage of animal liver. Recently, toxic Microcystis blooms frequently occur in the eutrophic Dianchi Lake (300 km2 and located in the South-Western of China). Microcystin-LR from Microcystis in Dianchi was isolated and purified by high performance liquid chromatography (HPLC) and its toxicity to mouse and fish liver was studied (Li et al., 2001). In this study, six biochemical parameters (reactive oxygen species, glutathione, superoxide dismutase, catalase, glutathione peroxide and glutathione S-transferase) were determined in common carp hepatocytes when the cells were exposed to 10 microg microcystin-LR per litre. The results showed that reactive oxygen species (ROS) contents increased by more than one-time compared with the control after 6 h exposure to the toxin. In contrast, glutathione (GSH) levels in the hepatocytes exposed to microcystin-LR decreased by 47% compared with the control. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GSH-Px) increased significantly after 6 h exposure to microcystin-LR, but glutathione S-transferase (GST) activity showed no difference from the control. These results suggested that the toxicity of microcystin-LR caused the increase of ROS contents and the depletion of GSH in hepatocytes exposed to the toxin and these changes led to oxidant shock in hepatocytes. Increases of SOD, CAT and GSH-Px activities revealed that these three kinds of antioxidant enzymes might play important roles in eliminating the excessive ROS. This paper also examined the possible toxicity mechanism of microcystin-LR on the fish hepatocytes and the results were similar to those with mouse hepatocytes.

FIRST REPORT OF THE CYANOTOXINS CYLINDROSPERMOPSIN AND DEOXYCYLINDROSPERMOPSIN FROM RAPHIDIOPSIS CURVATA (CYANOBACTERIA)

A strain of Raphidiopsis (Cyanobacteria) isolated from a fish pond in Wuhan, P. R. China was examined for its taxonomy and production of the alkaloidal hepatotoxins cylindrospermopsin (CYN) and deoxy‐cylindrospermopsin (deoxy‐CYN). Strain HB1 was identified as R. curvata Fritsch et Rich based on morphological examination of the laboratory culture. HB1 produced mainly deoxy‐CYN at a concentration of 1.3 mg·g−1 (dry wt cells) by HPLC and HPLC‐MS/MS. CYN was also detected in trace amounts (0.56 μg·g−1). A mouse bioassay did not show lethal toxicity when tested at doses up to 1500 mg dry weight cells·kg−1 body weight within 96 h, demonstrating that production of primarily deoxy‐CYN does not lead to significant mouse toxicity by strain HB1. The presence of deoxy‐CYN and CYN in R. curvata suggests that Raphidiopsis belongs to the Nostocaceae, but this requires confirmation by molecular systematic studies. Production of these cyanotoxins by Raphidiopsis adds another genus, in addition to Cylindrospermopsis, Aphanizomenon, and Umezakia, now known to produce this group of hepatotoxic cyanotoxins. This is also the first report from China of a CYN and deoxy‐CYN producing cyanobacterium.

Effect of desert soil algae on the stabilization of fine sands

Four filamentous cyanobacteria, Microcoleusvaginatus, Phormidium tenue,Scytonemajavanicum (Kutz.) and Nostoc sp., and asingle-celled green alga, Desmococcus olivaceus, allisolated from Shapotou (Ningxia Hui Autonomous Region of China), were batchcultured and inoculated onto unconsolidated sand in greenhouse and fieldexperiments. Their ability to reduce wind erosion in sands was quantified byusing a wind tunnel laboratory. The major factors related to cohesion of algalcrusts, such as biomass, species, species combinations, bioactivity, niche,growth phase of algae, moisture, thickness of the crusts, dust accretion(including dust content and manner of dust added) and other cryptogams(lichens,fungi and mosses) were studied. The best of the five species were M.vaginatus and P. tenue, while the best mix wasablend of 80% M. vaginatus and 5% each of P.tenue,S. javanicum,Nostocsp. and D. olivaceus. The threshold friction velocity wassignificantly increased by the presence of all of the cyanobacterial species,while the threshold impact velocity was notably increased only by thefilamentous species. Thick crusts were less easily eroded than thin crusts,while biomass was more effective than thickness. Dust was incorporated bestintoMicrocoleus crust when added in small amounts over time,and appeared to increase growth of the cyanobacterium as well as strengthen thecohesion of the crust. Microbial crust cohesion was mainly attributed to algalaggregation, while lichens, fungi and mosses affected more the soil structureand physico-chemical properties.

The vertical microdistribution of cyanobacteria and green algae within desert crusts and the development of the algal crusts

Substantial amounts of algal crusts were collected from five different desert experimental sites aged 42, 34, 17, 8 and 4 years, respectively, at Shapotou (China) and analyzed at a 0.1 mm microscale of depth. It was found that the vertical distribution of cyanobacteria and microalgae in the crusts was distinctly laminated into an inorganic-layer (ca.0.00–0.02 mm, with few algae), an algae-dense-layer (ca.0.02–1.0 mm) and an algae-sparse-layer (ca.1.0–5.0 mm). It was interesting to note that in all crusts Scytonema javanicum Born et Flah (or Nostoc sp., cyanobacterium), Desmococcus olivaceus (Pers ex Ach., green alga) Laundon and Microcoleus vaginatus Gom. (cyanobacterium) dominated at the depth of 0.02–0.05, 0.05–0.1 and 0.1–1.0 mm, respectively, from the surface. Phormidium tenue Gom. (or Lyngbya cryptovaginatus Schk., cyanobacterium) and Navicula cryptocephala Kutz.(or Hantzschia amphioxys (Ehr.) Grun. and N. cryptocephala together, diatom) dominated at the depth of 1.0–3.0 and 3.5–4.0 mm, respectively, of the crusts from the 42 and 34 year old sites. It was apparent that in more developed crusts there were more green algae and the niches of Nostoc sp., Chlorella vulgaris Beij., M. vaginatus, N. cryptocephala and fungi were nearer to the surface. If lichens and mosses accounted for less than 41.5% of the crust surface, algal biovolume was bigger when the crust was older, but the opposite was true when the cryptogams other than algae covered more than 70%. In addition to detailed species composition and biovolume, analyses of soil physicochemical properties, micromorphologies and mineral components were also performed. It was found that the concentration of organic matter and nutrients, electric conductivity, silt, clay, secondary minerals were higher and there were more micro-beddings in the older crusts than the less developed ones. Possible mechanisms for the algal vertical microdistribtion at different stages and the impact of soil topography on crust development are discussed. It is concluded that biomethods (such as fine species distribution and biovolume) were more precise than mineralogical approaches in judging algal crust development and thus could be a better means to measure the potentiality of algal crusts in desert amelioration.

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects on loach embryo-larval and juvenile development. The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development. Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption, small head, curved body and tail, and abnormal hatching. Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. In addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC(50)=593.3 microg/l).

Microcystin production of Microcystis viridis (cyanobacteria) under different culture conditions

Toxic cyanobacterium Microcystis viridis was isolated from the eutrophic Dianchi Lake of south‐western China. Three microcystins were detected from this strain by high‐pressure liquid chromatography. Among them, [Dha7] microcystin‐RR is the major component and represents about 70–80% of the total toxins of this strain. We examined its microcystin production pattern in relation to culture conditions such as light intensity, nutrition, temperature, pH and growth phase. Of all the factors examined, light intensity and pH influenced the toxin production much more than other factors. At 25°C, a high production of [Dha7] microcystin‐RR was obtained at light intensity 15 μE s‐1 m2, and both low and high pH (pH 7.0 and pH 9.2) enhanced the yield of microcystin‐RR. No strong relationships were found between total toxin and temperature ranging from 15°C. The highest production of total toxins was measured in the mid‐exponential growth phase and lowest production towards the declining growth phase. Remarkable changes in toxin production and morphology emerged coincidentally following the transfer of nitrogen‐deficient cells into fresh MA medium during the time course. These findings may shed light on the correlation between toxin production and morphogenesis of this strain.

Toxicological effects of nanometer titanium dioxide (nano-TiO2) on Chlamydomonas reinhardtii.

The toxicological effects of nanometer titanium dioxide (nano-TiO2) on a unicellular green alga Chlamydomonas reinhardtii were assessed by investigating the changes of the physiology and cyto-ultrastructure of this species under treatment. We found that nano-TiO2 inhibited photosynthetic efficiency and cell growth, but the content of chlorophyll a content in algae did not change, while carotenoid and chlorophyll b contents increased. Malondialdehyde (MDA) content reached maximum values after 8h exposure and then decreased to a moderately low level at 72 h. Electron microscopy images indicated that as concentrations of nano-TiO2 increased, a large number of C. reinhardtii cells were noted to be damaged: the number of chloroplasts declined, various other organelles were degraded, plasmolysis occurred, and TiO2 nanoparticles were found to be located inside cell wall and membrane. It was also noted that cell surface was surrounded by TiO2 particles, which could present an obstacle to the exchange of substances between the cell and its surrounding environment. To sum up, the effect of nano-TiO2 on C. reinhardtii included cell surface aggregation, photosynthesis inhibition, lipid peroxidation and new protein synthesis, while the response of C. reinhardtii to nano-TiO2 was a rapid process which occurs during 24 h after exposing and may relate to physiological stress system to mitigate damage.

UV‐B‐induced Oxidative Damage and Protective Role of Exopolysaccharides in Desert Cyanobacterium Microcoleus vaginatus

UV-B-induced oxidative damage and the protective effect of exopolysaccharides (EPS) in Microcoleus vaginatus, a cyanobacterium isolated from desert crust, were investigated. After being irradiated with UV-B radiation, photosynthetic activity (Fv/Fm), cellular total carbohydrates, EPS and sucrose production of irradiated cells decreased, while reducing sugars, reactive oxygen species (ROS) generation, malondialdehyde (MDA) production and DNA strand breaks increased significantly. However, when pretreated with 100 mg/L exogenous EPS, EPS production in the culture medium of UV-B stressed cells decreased significantly; Fv/Fm, cellular total carbohydrates, reducing sugars and sucrose synthase (SS) activity of irradiated cells increased significantly, while ROS generation, MDA production and DNA strand breaks of irradiated cells decreased significantly. The results suggested that EPS exhibited a significant protective effect on DNA strand breaks and lipid peroxidation by effectively eliminating ROS induced by UV-B radiation in M. vaginatus.

Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences

The taxonomy of Aphanizomenon flos-aquae strain NH-5, a producer of cyanotoxins, was re-evaluated by comparison with six other Aphanizomenon strains using morphological characteristics and 16S rRNA gene sequences. Strain NH-5 was concluded to be improperly identified as Aph. flos-aquae based upon (1) lack of bundle formation in the trichomes, (2) location of akinetes next to heterocytes, (3) lower similarities (less than 97.5%) in the 16S rRNA gene sequences relative to Aph. flos-aquae strains, and (4) comparison within a phylogenetic tree constructed from 16S rRNA gene sequences. The Aphanizomenon strains investigated in this study are classified to four morphological groups as described by the classical taxonomy of Komárek & Kovácik (1989). This classification was supported from the phylogenetic results of 16S rRNA gene sequences. This study also discusses the generic boundaries between Aphanizomenon and Anabaena.

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 μg−1 microcystin-RR. The results showed that the growth of Synechococcus elongatus (expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Significant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.