Effects of heavy metals （Pb^2＋ and Cd^2＋） on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb²⁺ and Cd²⁺ (0, 0.5, 1, 2, 4, 6 and 8 mg/L). The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased (>4 mg/L Pb²⁺) metal concentration. The photosynthetic apparatus (thylakoid membranes) were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration (8 mg/L Pb²⁺), the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations (0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L Pb²⁺), both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents (chlorophyll α, β carotene and phycocyanin) were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb²⁺, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll α, β carotene and phycocyanin respectively over the control. Corresponding reductions for the same Cd²⁺concentrations were 57.83, 48.94 and 56.90%. Lethal concentration (96 h LC₅₀) values (3.47 mg/L Cd²⁺ and 12.11 mg/L Pb²⁺) indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd²⁺ than Pb²⁺. Supported by the Chinese Scholarship Council     

Studies on hemolysis of hemolysin produced by Synechocystis sp. PCC 6803

Hemolysin produced by various bacteria,may destroy erythrocyte membranes via a pore-forming mechanism,a deter-gent action,or a lipase activity.Previous to this experiment,the mode of action used by cyanobacterial hemolysin had not been re-ported.To characterize the action mode of hemolysin produced by the wild-type strain of Synechocystis sp.PCC6803,hemolysis of erythrocytes originating from human,mouse,sheep,rabbit and goldfish was studied.The erythrocytes of mouse,sheep and rabbit were sensitive,while those of human and fish were resistant,to this hemolysin.Using rabbit erythrocytes,it was shown that hemoly-sis occurred in two steps:a binding step within the first 10 min of treatment and a lytic step after 30 min.Both binding and lysis were highly temperature-dependent.Effects of erythrocyte density on hemolysis suggest that the hemolysin might target erythrocytes via a multiple-hit mechanism.In the osmotic protection experiment,all tested osmotic protectants,with molecular diameters ranging from 0.9 ?5.66 nm,failed to effectively inhibit hemolysis.Scanning electron micrographs showed that the hemolysin caused protuberances or echinocytes in rabbit erythrocytes,and then disrupted and ruptured the erythrocytes.Characteristics of hemolysis showed distinct differences from other pore-forming mechanisms,suggesting that this hemolysin might act through a detergent-like or lipase mecha-nism,rather than a pore-forming mechanism.     

Characterization of calcium deposition induced by Synechocystis sp. PCC6803 in BG11 culture medium

Calcium carbonate （CaCO3） crystals in their preferred orientation were obtained in BG11 culture media inoculated with Synechocystis sp. PCC6803 （inoculated BG11）. In this study, the features of calcium carbonate deposition were investigated. Inoculated BGll in different calcium ion concentrations was used for the experimental group, while the BGll culture medium was used for the control group. The surface morphologies of the calcium carbonate deposits in the experimental and control groups were determined by scanning and transmission electron microscopy. The deposits were analyzed by electronic probe micro-analysis, Fourier transform infrared spectrum, X-ray diffraction, thermal gravimetric analysis and differential scanning calorimetry. The results show that the surfaces of the crystals in the experimental group were hexahedral in a scaly pattern. The particle sizes were micrometer-sized and larger than those in the control group. The deposits of the control group contained calcium （Ca）, carbon （C）, oxygen （O）, phosphorus （P）, iron （Fe）, copper （Cu）, zinc （Zn）, and other elements. The deposits in the experimental group contained Ca, C, and O only. The deposits of both groups contained calcite. The thermal decomposition temperature of the deposits in the control group was lower than those in the experimental group. It showed that the CaCO3 deposits of the experimental group had higher thermal stability than those of the control group. This may be due to the secondary metabolites produced by the algae cells, which affect the carbonate crystal structure and result in a close-packed structure. The algae cells that remained after thermal weight loss were heavier in higher calcium concentrations in BGll culture media. There may be more calcium- containing crystals inside and outside of these cells. These results shall be beneficial for understanding the formation mechanism of carbonate minerals.     

Insertal orientation has no influence on the expression ofgfp gene and the growth of the hostSynechococcus sp. PCC7942

In transgenic process, a foreign gene can be integrated in the host genome in two directions, which may influence its expression. In order to study the effects of insertal orientation, the gfp reporter gene was inserted in the isiAB locus of Synechococcus sp.PCC7942 in different directions, and the GFP expression levels and the growth of the transgenic algae were compared. It was showed that the gfp gene could express in each direction, and no significant difference was detected on algal growth and GFP expression levels between the two recombinant algae.     

Effects of heavy metal ions (Cu2+, Pb2+ and Cd2+) on DNA damage of the gills, hemocytes and hepatopancreas of marine crab, Charybdis japonica

There are rising concerns about the hazardous effects of heavy metals on the environment. In this study, comet assay and DNA alkaline unwinding assay were conducted on the tissues (gills, hepatopancreas, and hemocytes) of Charybdis japonica in order to illustrate genotoxicity of three heavy metal ions (Cu2+, Pb2+, and Cd2+) on the marine crabs C. japonica. The crabs were exposed to Cu2+ (10, 50, and 100 ?g.L?1), Pb2+ (50, 250, and 500 ?g L?1) and Cd2+ (5, 25, and 50 ?g L?1), and the tissues were sampled at days 0.5, 1, 3, 6, 9, and 15. DNA alkaline unwinding assay was used for testing the DNA single strand break in gills and hepatopancreas and comet assay was employed for testing the DNA damage in hemocytes. The results showed that the DNA damage (F-value) of gills in the crabs exposed to the three heavy metals was decreased gradually during the exposure periods and there was a dose-time response relationship in certain time, suggesting that the levels of DNA single strand break in all the experimental groups increased significantly compared to the controls. Changes of F-value in hepatopancreas of the crabs exposed to the three heavy metals were similar to those in gills except that the peak values were found in the 500 ?g L?1 Pb2+ treatment group at day 3 and the 50 ?g L?1 Cd2+ treatment group at day 9. The ranks of DNA damage in gills and hepatopancreas induced by the three heavy metal ions (50 ?g L?1, day 15) were Cd2+ >Pb2+ >Cu2+ and Pb2+ >Cu2+ >Cd2+. The levels of DNA damage in gills were higher than those in hepatopancreas in the same experimental group. It can be concluded that indices of DNA damage can be used as the potential biomarkers of heavy metal pollution in marine environment.     

Bioaccumulation of Pb^2＋ and Its Effects on Growth, Morphology and Pigment Contents of Spirulina （Arthrospira）platensis

A laboratory experiment was conducted to assess the bioaccumulation of Pb2 and its effects on growth,morphology andpigment contents of Spirulina (Arthrospira) platensis.The specimen cultured in Zarrouk liquid medium was treated with variousinitial metal concentrations (0,5,10,30,50 and 100 μgmL-1).The growth of S.platensis was adversely affected by Pb2 at high con-centrations (30,50 and 100 μg mL-1).However,at low concentrations (5 μg mL-1),Pb2 could stimulate its growth slightly.The pig-ment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner.The highest reductions (67% and 53%respectively in chlorophyll α and β carotene) were observed in 100 μg mL-1 treatment group.The LC50 (96 h) of Pb2 was measured as75.34μg mL-1.Apart from a few cases of filament breakages at elevated concentrations (50 and 100μg mL-1),morphological abnor-malities are not specific.Metal bioaccumulation increased with Pb2 concentrations,but decreased with exposure time.The maxi-mum accumulated amount was 188 mgg-1 dry weight.The bioconcentration factor (BCF) reached to a peak at day 2,followed by agradual reduction for all the exposure concentrations.S.platensis is able to tolerate considerably high Pb2 concentrations.Conse-quently it can be used as a potential species to remove heavy metal from contaminated waters.     

Combined effects of light intensity and NH^＋4-enrichment on growth,pigmentation, and photosynthetic performance of Ulva prolifera （Chlorophyta）

The aim of this study was to investigate the effects of light intensity and enhanced nitrogen supply on the growth and photosynthesis of the green-tide macroalga, Ulva prolifera. Thalli of U. prolifera were grown in natural or NH 4 +-enriched seawater under two different light intensities for 7 days, and then the growth rate, pigmentation, and photosynthetic performance of the thalli were evaluated. The results show that the relative growth rate(RGR) was markedly higher under the high light level than under the low light level. Enrichment with NH 4 + enhanced the RGR under high light intensity, but did not affect RGR under low light intensity. In low light conditions, NH 4 +-enrichment resulted in a marked decrease in the maximal photosynthetic rate( P m) and the maximum carbon fixation rate( V max), but it did not affect the half saturation constant for carbon( K 0.5) or the ratio of V max to K 0.5, which reflects the carbon acquisition efficiency. In high light conditions, P m, K 0.5, and the dark respiration rate( R d) increased under NH 4 + enrichment, but V max and the V max / K 0.5 ratio decreased. Regardless of the light intensity, NH 4 +-enrichment did not affect the apparent photosynthetic efficiency( α), which refl ects the ability of the alga to use light energy at low light levels. Under both low and high light intensities, the chlorophyll a(Chl a), chlorophyll b(Chl b), and carotenoids(Car) contents in thalli were higher in NH 4 +-enriched than in natural seawater, except that there was a decrease in the Chl b content of thalli in NH 4 +-enriched seawater under low light intensity. Therefore, NH 4 + enrichment improved the growth and photosynthetic performance of U. prolifera under high light intensity, but not under low light intensity. We discuss the possible mechanisms underlying these physiological responses.     

Removal of eutrophication factors and heavy metal from a closed cultivation system using the macroalgae, Gracilaria sp. (Rhodophyta)

In this study,the ability of macroalgae Gracilaria sp.of removing eutrophication factors and toxic heavy metals Al,Cr,and Zn in a closed cultivation system is reported.The results show that the concentration of the three heavy metals decreased significantly during the experimental period in an algal biomass dependent manner.The biofiltration capacity of the alga for Al,Cr,and Zn is 10.1%-72.6%,52.5%-83.4% and 36.5%-91.7%,respectively.Using more materials resulted in stronger heavy metal removal.Additionally...     

Effects of Pseudoalteromonas sp. BC228 on digestive enzyme activity and immune response of juvenile sea cucumber (Apostichopus japonicus)

A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0(control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control(P 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet(P 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets(P 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.     

Effects of salinity on survival,feeding behavior and growth of the juvenile swimming crab,Portunus trituberculatus (Miers, 1876)

Effect of salinity on survival,feeding behavior and growth of juvenile swimming crab P ortunus trituberculatus was investigated under 5 salinity levels of 5,10,20,30 and 40. The results show that the crab juveniles fed 2 or 3 times at the salinity 20 and 30,each lasted for about 25 minutes,for a total feeding time of 73.2±22.65 minutes per day. At these salinities,there were significantly higher in the frequency of feeding and in total feeding time than those at lower salinities of 5 and 10. All crab juveniles moulted when reared at a salinity of 20 during the 5 days duration of the experiment,which is significantly higher than those at other salinities. All juveniles survived at salinity 20,and the survivorship was not significantly different from that at 30,but was signif icantly higher than those at other salinities. The crab juveniles reared at a salinity of 20 had the highest value of food ration of 0.190 8±0.011 3 g/g BW,average body weight gain of 0.796±0.128 g,gain rate of 87%–96%,and food conversion ratio of 1.20±0.09. There was no significant difference in the values found between 20 and 30 but these values were significantly lower than that at the other salinities( P 0.05). Highest activities of digestive enzymes(Amylase,Protease,Lipase) and lowest activities of protective enzymes(SOD,PO,CAT) were also obtained on crab juveniles reared at salinity of 20.     

STUDY ON EMBRYONIC DEVELOPMENT AND EARLY GROWTH OF TRIPLOID AND GYNOGENETIC DIPLOID LEFI—EYED FLOUNDER, PARALICHTHYS OLIVACEUS （T. et S. ）

The early effects of chromosomal manipulation of eggs and sperm on the yields of trip-loid and gynogenefic diploid larvae of Paralichthys olivaceus were investigated. Triploidy was achieved by cold shocking fen.ilized eggs at 0 - 2℃ for 45 minutes duration 5 minutes after fen.ilization, and the in-duced triploidy rates were 31.2% - 50% and the relative hatching rates were 53.3% - 99%. Gynnge-aetic diploids were obtained when eggs were inseminated with irradiated sperm and cold shocked at 0 -2℃ for 45 minutes duration 5 minutes after fertihzation. The induced gynogenetic diploid rates and the relative hatching rates were 94 % - 96 % and 48.5 % - 68.5 % respectively. The embryonic development of the triploid experimental group and of the gynogenetic diploid experimental group was delayed at first compared with the control group. But from the gastrula stage, it was not delayed anymore. There were no significant differences in the growth of the triploid experimental group larvae and the control group larvae, and in the growth of the gynogenetic diploid experimental group larvae and the control group larvae ac-cording to Student‘s t-test (α = 0.05). The relationship between the early growth of the triploid experi-mental group larvae and that of gynogenetic diploid experimental group larvae was also studied.     

Study on the distribution,abundance and growth condition of Antarctic Krill(Euphausia superba)in the Prydz Bay region,Antarctica during the Austral summer of 1999/2000

Antarctic krill (Euphausia superba) were collected from the Prydz Bay region during the austral summer of 1999/2000. The sample - collection was made at 15 sites in 3 longitudinal transects. Although krill were encountered at 14 sites, the abundance was comparatively low in general. The main component of the krill population was of post -larval stages. The mean numerical and weight density of krill integrated for all sampling stations in the survey area were 16. 17 ind. 1000 m -3and 12.02 g 1000 m -3 , respectively. In the survey area, stations with larger krill density are mainly located in the slope zone, except that the largest sample was collected in the open sea zone. No krill occurred in the only station of the shelf zone during our investigation. Krill collected from the slope zone were under better growth condition than those from the open sea zone, but when the survey area is considered as a whole, the growth condition is normal. The results of the comprehensive analysis of the environmental fa     

Effects of Na+/K+ ratio of groundwaters on the gill ion-transport enzyme activity, plasma osmolality and growth of Cynoglossus semilaevis juveniles

The effects of environmental Na⁺/K⁺ ratio on the gill ion-transport enzyme activity, plasma osmolality and growth of Cynoglossus semilaevis juveniles were investigated. The results showed that, plasma osmolality was similar among flounder adapted to different Na⁺/K⁺ ratios of saline groundwaters (P>0.05), while the growth, gill Na⁺, K⁺-ATPase and HCO₃ ⁻-ATPase activities were affected by Na⁺/K⁺ ratio significantly (P<0.05). The gill Na⁺, K⁺-ATPase activity reached its maximum on day 3, then decreased gradually from day 3 to day 9 and remained constant from day 9 to day 15. The peak values of gill Na⁺, K⁺-ATPase activity were detected on day 3 for all Na⁺/K⁺ ratios of saline groundwaters, then the enzyme activities descended, and on day 9 the enzyme activities achieved steady state, and the gill HCO₃ ⁻-ATPase activity increased rapidly and achieved steady state after one day. During steady state, the gill Na⁺, K⁺-ATPase and HCO₃ ⁻-ATPase activity of Na⁺/K⁺ ratios 20 and 40 treatments were significantly higher than those in the control group (Na⁺/K⁺ ratio 27.5), while there were no significant differences between the Na⁺/K⁺ ratio 30 treatment and the control group; the gill Na⁺, K⁺-ATPase activity of Na⁺/K⁺ ratio 20 and 40 treatments were significantly higher than that for ratio 30 treatment, but there were no significant differences of gill HCO₃ ⁻-ATPase activity among these treatments. At the end of the 15-day experiment, the weight gain (%) and specific growth rate (SGR) of flounders maintained in seawater were significantly higher than those in groundwaters; significant differences also occurred among the treatments; Na⁺/K⁺ ratio 30 treatment had the highest values (33.7% and 1.94 respectively), which were significantly higher than those under Na⁺/K⁺ ratios 20 and 40 treatments. Therefore, for the saline groundwater used in this experiment, it is suggested that the Na⁺/K⁺ ratio be adjusted to approximately 30, i.e., as close to that of natural seawater as possible in the culture of flounder.     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.     

Effects of algal concentration and initial density on the population growth of Diaphanosoma celebensis Stingelin （Crustacea, Cladocera）

The effects of algal concentration and initial density on the population growth of the estuarine cladocera,Diaphanosoma celebensis Stingelin,were evaluated in an indoor experiment.A 2 × 4 layout that included two algal concentrations(Chlorella pyrenoidosa,1 × 106 and 3 × 106 cell/mL) and four inoculation densities(100,200,300 and 400 ind./L) were established.Diaphanosoma celebensis were reared in 150 mL flasks containing 50 mL of algal medium at 22°C,under salinity of 10 and a photoperiod of 12 h L:12 h D.T...     

Effect of temperature and irradiance on the growth and reproduction of Enteromorpha prolifera J. Ag. （Chlorophycophyta,Chlorophyceae）

Effect of temperature and irradiance on growth and reproduction of Enteromorpha prolifera that bloomed offshore along the Qingdao coast in summer 2008, was studied. It was showed that E. prolifera propagated mainly asexually with specific growth rate （SGR） of 10.47 at 25℃/40 μmol m^-2s^-1. Under this condition, gametes with two flagellate formed and released in 5 days. At the beginning of the development, the unicell gamete divided into two cells with heteropolarity, and then the apical cell developed into thalli primordial cells, whereas the basal cell developed into rhizoid primordial cells. In 8-day culture, the monoplast gamete developed into juvenile germling of 240 μm in length. Unreleased gametes can develop directly within the alga body. E. prolifera could either reproduce through lateral branching or fragmenting except apomixis revealed by Microscopic observation. On aged tissue of E. prolifera, although the degraded pigments partially remained in faded algal filaments, numerous vegetative cells could still divide actively in the algal tissues.