The microcystins-induced DNA damage in the liver and the heart of zebrafish,Danio rerio

Microcystins (MCYST) are the freshwater cyanobacterial toxins, known to induce hepatocellular carcinoma, necrosis, intrahepatic bleeding, as well as human and livestock mortality. Within hepatocytes, MCYST selectively bind to protein phosphatases 1 and 2A, resulting in severe liver damage. The toxicology of MCYST in mice and rats has been well studied, but little is known regarding genotoxicity in aquatic animals. In this study, the zebrafish, Danio rerio was exposed to crude extract of Microcystis aeruginosa bloom. Liver and heart were examined for MCYST-induced toxicity. Light microscopy at 36?h revealed severe, widespread apoptotic necrosis of the majority of hepatocytes, and cytoskeletal deformation in myocardiocytes. Hepatocytes were dissociated with cell shrinkage and margination of nuclear chromatin. Laddering of genomic DNA from the liver and heart of the exposed fish in an increment of 180–200?bp was consistent with apoptosis. Fluorimetric analysis of DNA unwinding was carried out to determine the DNA strand breakage. After 36?h exposure, the % double-stranded DNA was significantly reduced in hepatocytes and myocardiocytes. In conclusion, the results obtained in this study indicate that, the extract of M. aeruginosa bloom is genotoxic to fish. The DNA damage observed in this study may be attributed to the activation of DNA endonucleases. This model of DNA damage may contribute for identifying novel molecular mechanisms of interest for therapeutic application.     

RAPD markers suggest genotypic effects on forager specialization in a eusocial wasp

Genetic variability within insect societies may provide a mechanism for increasing behavioral diversity among workers, thereby augmenting colony efficiency or flexibility. In order to assess the possibility that division of labor has a genetic component in the eusocial wasp Polybia aequatorialis, I asked whether the genotypes of workers within colonies correlated with behavioral specialization. Workers specialized by foraging for one of the four materials (wood pulp, insect prey, nectar, or water) gathered by their colonies. I collected foragers on 2 days from each of three colonies and identified the material the foragers were carrying when collected. I produced random amplified polymorphic DNA (RAPD) markers from the genomic DNA of these foragers and estimated genotypic similarity of foragers based on sharing of variable RAPD marker bands. Contingency tests on 20 variable loci per colony showed statistically significant (P <0.05) biases in RAPD marker frequencies among forager types in the three colonies. Patterns of association of RAPD marker bands with specializations were constant in two colonies, but changed between collection days in one colony. RAPD marker biases suggest that division of labor among workers includes a genetic component in P. aequatorialis. Colony-level selection on variation in division of labor is a possible factor favoring the evolutionary maintenance of high genotypic variability (low relatedness) in epiponine wasp colonies and in other eusocial insects. Received: 18 July 1995/Accepted after revision: 1 October 1995     

Effect of fomesafen on glutathione S-transferase and cellulase activity and DNA damage in the earthworm (Eisenia fetida)

Biochemical responses and DNA damage of earthworm (Eisenia fetida) exposed to various concentrations of fomesafen (0, 10, 100, and 500 μg kg^?1) for 3, 7, 14, 21, and 28 days were investigated in vivo. Glutathione S-transferase (GST) activity was significantly stimulated upon treatment with 500 μg kg^?1 fomesafen for 21 days, while the cellulase activity was markedly inhibited after 14 days of treatment. On day 28, the activities of GST and cellulase had recovered to the level similar to that of controls. Low or mild DNA damage in earthworm was induced within 14 days of exposure, and the damage was reduced or disappeared with the extension of exposure.     

石油烃对栉孔扇贝血淋巴细胞DNA损伤的初步研究

为研究石油烃对海洋生物的毒性效应,将栉孔扇贝(Chlamys farreri)暴露于0.08、0.21和0.88mg·L-1石油烃中,采用单细胞凝胶电泳实验(彗星实验)技术检测不同暴露时间扇贝血淋巴细胞的DNA损伤程度,对照组中石油烃背景浓度为0.04mg·L-1。结果显示,低浓度(0.08mg·L-1)的石油烃短期(<7d)内即可导致栉孔扇贝血淋巴细胞的DNA损伤,并且随石油烃浓度的增大和暴露时间的延长,DNA损伤程度增加,石油烃浓度达0.88mg·L-1时,DNA损伤程度已非常严重。3d恢复实验后,各浓度组DNA损伤又均有不同程度的恢复。研究表明,彗星实验是检测石油烃对海洋贝类DNA损伤的一种有效手段,贝类血淋巴细胞DNA损伤有望成为石油烃污染的一种生物标志物,用于海洋污染的早期预警监测。     

Growth and DNA damage in young Laminaria sporophytes exposed to ultraviolet radiation: implication for depth zonation of kelps on Helgoland (North Sea)

Growth as an integrative parameter of all physiological processes was measured in young sporophytes of temperate Laminaria digitata, Laminaria saccharina and Laminaria hyperborea exposed in the laboratory to irradiance consisting of either only photosynthetically active radiation (PAR) or to a spectrum including ultraviolet radiation (UVR) (PAR+UVA+UVB) by use of cut-off glass filters. Size increment was measured every 10 min over a period of 18–21 days using growth chambers with on-line video measuring technique. In the chamber, plants were grown at 10±2°C and 16:8 h light–dark cycles with 6 h additional UVR exposure in the middle of the light period. Tissue morphology and absorption spectra were measured in untreated young sporophytes while chlorophyll a content and DNA damage were measured in treated thalli at the end of the experiment. Sensitivity of growth under UVR was found to be related to the observed upper depth distribution limit of the upper sublittoral L. digitata, upper to mid sublittoral L. saccharina and lower sublittoral L. hyperborea. Tissue DNA damage is, however, dependent on thallus thickness which minimizes UVR effect where outer cell layers shade inner cells and provide longer pathlength for UVR. Exposure to UVR causes cellular, enzymatic and molecular damage. Presence of UV-absorbing compounds further reduces effective UVR from reaching physiological targets. The cost of producing higher amount of UV-absorbing compounds and effective DNA repair mechanism can, however, divert photosynthate at the expense of growth. Tissue chlorophyll a content was not significantly different between treatments suggesting a capacity for acclimation to moderate UVR fluence. Growth acclimation to repeated UVR exposure was observed within a period of 12 days while growth inhibition was observed after a longer UVR exposure period of 21 days. The results give further insight into the effects of UVR on the cellular level and show how ecological parameters such as the upper depth distribution limit are dependent on cellular processes.     

Tissue-specific stress and hepatic DNA damage in Pelteobagrus fulvidraco caused by low concentrations of cadmium

Physiological stress and DNA damage in Pelteobagrus fulvidraco induced by continuous exposure to cadmium at concentrations of 170 and 1700 µg/L for up to 28 days was evaluated. The activities of acetylcholinesterase and monoamine oxidase in brain tissue, Na⁺-K⁺-ATPase and glutathione in gill tissue, and superoxide dismutase and catalase in liver tissue were measured. In studying random amplified polymorphic DNA to evaluate cadmium-induced hepatic genotoxicity, both the appearance of new bands and the disappearance of existing bands were observed, as well as increased levels of monoamine oxidase and Na⁺-K⁺-ATPase and decreased activities of antioxidant enzymes. The results suggest that continuous exposure to cadmium at the studied levels can induce biochemical and physiological changes and DNA damage in P. fulvidraco.     

The rosette agent of chinook salmon (Oncorhynchus tshawytscha) is closely related to choanoflagellates,as determined by the phylogenetic analyses of its small ribosomal subunit RNA

The rosette agent of Chinook salmon (Oncorhynchus tshawytscha), initially described and characterized in the mid 1980s, is the cause of a serious infectious disease in the Pacific Northwest of North America. Previous work, utilizing rosette agent maintained by growth in embryo salmon cell-culture, has shown it to be a eukaryotic obligate intracellular parasite. However, its ultrastructural features do not suggest a relationship with any specific eukaryotic group. We have utilized a molecular approach to further investigate the phylogeny of rosette agent previously maintained in vitro during 1990 and 1991. We have amplified the genomic DNA encoding the small subunit ribosomal RNA (16S-like rRNA), and determined the complete DNA nucleotide sequence of this gene segment. Comparison with other eukaryotic 16S-like rRNA sequences suggests that the rosette agent shares a unique evolutionary history with choanoflagellates.     

Distribution and relative abundance of Pseudocalanus moultoni and P. newmani (Copepoda: Calanoida) on Georges Bank using molecular identification of sibling species

The sibling species, Pseudocalanus moultoni (Frost, 1989) and P. newmani (Frost, 1989), occur sympatrically on Georges Bank. Taxonomic discrimination of the species relies on subtle morphological characteristics, making routine identification of the species very difficult. DNA sequence variation of two mitochondrial genes, 16S rRNA and cytochrome oxidase I (COI), reliably discriminated P. moultoni and P.␣newmani. Levels of DNA sequence variation for both genes were consistent with those between species of calanoid copepods. A molecular systematic protocol (based on allele-specific PCR amplification) was designed from the COI sequences and used to discriminate females of the two species. The distributions and relative abundances of the two species were mapped for April 1996 based on samples of 15 to 30 females from 12 collections across Georges Bank. The results of this study indicated that P. moultoni females predominated along the northern flank of Georges Bank, while P. newmani females were common on the southern flank, deeper than the 60-m isobath. Received: 16 May 1997 / Accepted: 26 April 1998     

Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)

Quantification of feeding rates and selectivity of zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, methodological limitations have made many of these studies difficult. Recently, molecular based methods have demonstrated that DNA from prey species can be used to identify zooplankton gut contents, and further, quantitative gut content estimates by quantitative PCR (qPCR) assays targeted to the 18S rRNA gene have been used to estimate feeding rates in appendicularians and copepods. However, while standard single primer based qPCR assays were quantitative for the filter feeding appendicularian Oikopleura dioica, feeding rates were consistently underestimated in the copepod Calanus finmarchicus. In this study, we test the hypothesis that prey DNA is rapidly digested after ingestion by copepods and describe a qPCR-based assay, differential length amplification qPCR (dla-qPCR), to account for DNA digestion. The assay utilizes multiple primer sets that amplify different sized fragments of the prey 18S rRNA gene and, based on the differential amplification of these fragments, the degree of digestion is estimated and corrected for. Application of this approach to C. finmarchicus fed Rhodomonas marina significantly improved quantitative feeding estimates compared to standard qPCR. The development of dla-qPCR represents a significant advancement towards a quantitative method for assessing in situ copepod feeding rates without involving cultivation-based manipulation.     

朱■的随机扩增多态DNA分析与种内亲缘关系研究

利用随机扩增多态ＤＮＡ技术对朱（Ｎｉｐｐｏｎｉａｎｉｐｐｏｎ）８个个体进行了随机扩增多态ＤＮＡ分析．用２０个１０ｂｐ的随机引物对每只朱的基因组ＤＮＡ进行扩增,共得到１６８个扩增片段,其中共有片段为１０２个．根据聚类分析所得到的树状图确定了８只朱的亲缘关系,这为进一步构建全部朱个体的谱系关系图打下了基础,有利于制定更有效的朱保护计划     

Responses of the earthworm Eisenia andrei exposed to sublethal aluminium levels in an artificial soil substrate

Industrial discharges of Al are increasingly common. In this study, the activities of three antioxidases, malondialdehyde (MDA) content and changes in coelomocytes were investigated in earthworms (Eisenia andrei) exposed to different concentrations of aluminium chloride (range 0–200 mg Al kg^?1 dry soil) in artificial soils for 32 days. Within the first 16 days of Al exposure, catalase activity was elevated in most treatments, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were inhibited in all treatments. After 16 days exposure, SOD and GSH-Px activities returned to normal at lower Al concentrations, but were still inhibited at higher concentrations, hence the inhibition of SOD and GSH-Px showed some exposure-level dependence during the late exposure phase. MDA content was significantly elevated from 16 days after initial exposure. Neutral Red retention time of coelomocytes decreased at the highest exposure concentrations after 32 days, and an increased proportion of cells showing membrane damage was also observed at this time using flow cytometry. We conclude that excessive Al in soils can cause oxidative stress and cell damage in earthworms. Hence, Al accumulation in the environment may present an ecological hazard through suppression of the important functions of earthworms in soil ecosystems.     

2,4-D二甲胺盐对莲草直胸跳甲生存和繁殖的影响

为探究除草剂在防治喜旱莲子草(Alternanthera philoxeroides)的过程中对其天敌昆虫的影响,采用不同浓度的2,4-D二甲胺盐浸叶处理的喜旱莲子草饲喂莲草直胸跳甲,连续饲喂7d后,考察了莲草直胸跳甲的繁殖、存活和发育情况。结果表明,2,4-D二甲胺盐可显著降低莲草直胸跳甲成虫的取食量,且抑制作用随药剂浓度升高和取食时间延长而显著增强;较高浓度(1.72～3.44g·L~(-1))的2,4-D二甲胺盐还会导致莲草直胸跳甲雌成虫存活率和产卵量的明显降低,卵孵化率、幼虫存活率和蛹羽化率也显著降低。可见,高浓度(1.72～3.44g·L~(-1))的2,4-D二甲胺盐对莲草直胸跳甲的生长发育和繁殖有显著不利影响,在防治喜旱莲子草时,应将2,4-D二甲胺盐的施用浓度控制在0.22～0.86g·L~(-1)内。     

Bisphenol S induces oxidative stress and DNA damage in rat spermatozoa in vitro and disrupts daily sperm production in vivo

Bisphenol S has been introduced into some industrial applications and it may act as a xeno-estrogen that can alter endocrine functions and reproduction. The present study was carried out to examine the effect of bisphenol S exposure on oxidative stress, generation of reactive oxygen species, and DNA integrity in rat spermatozoa in vitro and daily sperm production and sperm DNA damage in vivo. Sperm were incubated with bisphenol S at concentrations of 0.5, 1, 10, and 100 µg/L. At the highest concentration, bisphenol S induced formation of reactive oxygen species, caused lipid peroxidation, affected superoxide dismutase levels, and increased DNA fragmentation. Adult rats were exposed to doses of 0.5, 5, 25, and 50 µg/kg/d for 28 days. Decrease in daily sperm production and an increase in sperm DNA damage was observed at the highest dose in the 50 µg/kg/d treated group, but sperm motility was not reduced.     

朱鹮的随机扩增多态DNA分析与种内亲缘关系研究

利用随机扩增多态DNA技术对朱鹮 ( Nipponia nippon ) 8个个体进行了随机扩增多态DNA分析. 用20个10bp的随机引物对每只朱鹮的基因组DNA进行扩增,共得到168个扩增片段,其中共有片段为102个. 根据聚类分析所得到的树状图确定了8只朱鹮的亲缘关系,这为进一步构建全部朱鹮个体的谱系关系图打下了基础,有利于制定更有效的朱鹮保护计划.     

Transmission,plasticity and the molecular identification of cyanobacterial symbionts in the Red Sea sponge <Emphasis Type="Italic">Diacarnus erythraenus</Emphasis>

Cyanobacterial symbionts in the sponge Diacarnus erythraenus from the Red Sea were identified in both adult sponges and their larvae by 16S rDNA sequencing. A single cyanobacterial type was found in all samples. This cyanobacterial type is closely related to other sponge cyanobacterial symbionts. The cyanobacterial rDNA, together with the morphological analysis by electron and fluorescence microscopy, provided evidence for vertical transmission of the symbionts in this sponge. In addition, we show phenotypic plasticity of the symbionts inside the sponge, probably as a result of variability in light availability inside the sponge tissue. Finally, the reproduction of Diacarnus erythraenus is also described.Matan Oren, Laura Steindler have contributed equally to the work.     

Mitochondrial and nuclear rRNA based copepod phylogeny with emphasis on the Euchaetidae (Calanoida)

Phylogenetic relationships within the copepod family Euchaetidae and between representatives of three copepod orders (Calanoida, Harpacticoida, and Poecilostomatoida) were investigated using partial nucleotide sequences of the mitochondrial 16S rRNA and the nuclear 28S rRNA genes. DNA isolation, polymerase chain reaction, cloning, and DNA sequencing techniques were customized for these crustaceans. Our results support the monophyly of each copepod order, but in contrast to traditional morphology-based phylogenies of copepod orders, the Poecilostomatoida are basal to the Calanoida and Harpacticoida on our DNA-based phylogenetic tree. Phylogenetic trees generated by maximum parsimony, neighbor-joining, and maximum-likelihood analyses support the classification of the genera Euchaeta and Paraeuchaeta in the family Euchaetidae; results, however, suggest that Euchaetaacuta Giesbrecht is more closely related to species of the genus Paraeuchaeta than to those of Euchaeta, although limited taxon sampling may be partially responsible for this result. Phylogenetic mapping using the most parsimonious 16S tree suggests that the morphological synapomorphies distinguishing the genus Euchaeta evolved independently twice during the history of the Euchaetidae. Further, phylogenetic mapping suggests that the most recent common ancestor of the Euchaetidae and the Aetideidae was a deep-living, vertically migrating copepod, and that a bathypelagic, vertically migrating lifestyle characteristic of Paraeuchaeta is an ancestral trait of the family Euchaetidae which was lost apomorphically by Euchaeta. The application of a molecular clock suggests that the sibling species Euchaeta rimana Bradford and Euchaeta marina (Prestandrea) diverged due to the emergence of the Panamanian land bridge. Received: 9 October 1997 / Accepted: 5 August 1998     

Performance evaluation of isoproturon-degrading indigenous bacterial isolates in soil microcosm

Isoproturon (IPU)-degrading soil bacteria were isolated from herbicide-applied wheat fields. These isolates were identified using cultural, morphological, biochemical and 16S rRNA sequencing methods. 16S rRNA sequences of both the bacterial isolates were compared with NCBI GenBank data base and identified as Bacillus pumilus and Pseudoxanthomonas sp. A soil microcosm study was carried out for 40 days in six different treatments. Experimental results revealed maximum 95.98% IPU degradation in treatment 6 where bacterial consortia were augmented in natural soil, followed by 91.53% in treatment 5 enriched with organic manure as an additional carbon source. However, only 14.03% IPU was degraded in treatment 1 (control) after 40 days. In treatments (2–4), 75.59%, 70.92% and 77.32% IPU degradation was recorded, respectively. IPU degradation in all the treatments varied significantly over the control. 4-Isopropylaniline was detected as IPU degradation by-product in the medium. The study confirmed that B. pumilus and Pseudoxanthomonas sp. performed effectively in soil microcosms and could be employed profitably for field-scale bioremediation experiments.     

低浓度阿特拉津长期暴露对鲫鱼DNA的影响

运用随机扩增多态性DNA(RAPD)技术在分子生物学水平上评价了低浓度阿特拉津长期暴露对鲫鱼DNA的影响.结果表明:0.006mg·L-1以上浓度(试验浓度系列为0.006、0.023、0.094、0.375、1.5、3mg·L-1)的阿特拉津持续暴露60d均会对鲫鱼的DNA产生影响;经筛选,确定有34条引物能够扩增出鲫鱼基因组DNA,其中有8条引物能检测出阿特拉津对鲫鱼基因组DNA影响的差异:各浓度组鲫鱼DNA的RAPD扩增产物条带均出现不同程度缺失;在较低浓度长期暴露下阿特拉津对鲫鱼基因组DNA的损伤无明显的剂量-效应关系,阿特拉津毒性在0.375mg·L-1时突然增强,但这一趋势并未向更高浓度组延续(1.5mg·L-1组表现出的毒性弱于0.375mg·L-1组和3mg·L-1组).     

S-phase,apoptosis and peroxisome proliferation in avian hepatocyte cultures following exposure to the phenoxy herbicide 2,4-D

Phenoxyherbicides induce peroxisome proliferation, as do other structurally related or unrelated chemicals which were found to be nongenotoxic carcinogens in rodents. The widespread use of 2,4-dichlorophenoxyacetic acid (2,4-D) raised concerns over the potential to produce cancer, yet results from epidemiological and experimental studies do not provide definitive evidence that 2,4-D is carcinogenic. The objective of the present study was to verify whether 2,4-D might interfere with cell growth similar to other peroxisome proliferators using cultured avian hepatocytes. Primary hepatocyte cultures from 18-day-old chick embryos were exposed to 2,4-D for up to 72 h followed by an assessment of replicative DNA synthesis and apoptosis. Further, peroxisome proliferation was evaluated. Results showed that 2,4-D stimulated DNA synthesis and suppressed spontaneous apoptosis, while the effects on peroxisome proliferation were less evident.