Effects of air pollutants on the growth and antioxidative system of Norway spruce exposed in open-top chambers

Grafted Norway spruce trees were subjected to exposure beginning in April 1988, to one of four different air treatments in open-top chambers: Charcoal filtered air (CF), non-filtered air (NF), non-filtered air with the addition of O(3) during summer (NFO), and SO(2) plus NO(2) during winter (NFOSN). CF trees were considered as the reference group. No effects on growth parameters were observed. Samples of the two youngest needle year classes were taken late in November 1989 for enzyme determinations. The activity of ascorbic acid peroxidase (A-POD) increased the same level in all treatments, and activities of catalase and dehydroascorbic acid reductase (DHA-R) increased only in NF and NFO treatments. A higher level of activity in the NFOSN treatment was observed only for glucose-6-phosphate-dehydrogenase (Glc-6-P-DH) and non-specific peroxidase (POD). Isoelectric focusing of POD showed a changed pattern in the NFOSN treatment. Neither activity nor isoelectric focusing of superoxidase dismutase (SOD) was changed in any of the treatments.     

Origin of Metazoa: Sponges as Living Fossils

Geodia cydonium  , which code for proteins. The analyses of their deduced amino acid sequences allowed a molecular biological approach to solve the problem of monophyly of Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin, and galectin as prominent examples, cell-surface receptors (tyrosine kinase receptor), elements of sensory systems (crystallin, metabotropic glutamate receptor), and homologs/modules of an immune system (immunoglobulin like molecules, scavenger receptor cysteine-rich, and short consensus repeats, rhesus system) classify the Porifera as true Metazoa. As living fossils, provided with simple, primordial molecules allowing cell-cell and cell-matrix adhesion as well as processes of signal transduction as known in a more complex manner from higher Metazoa, they also show peculiarities not known in other metazoan phyla. Tissues of sponges are rich in telomerase activity, suggesting a high plasticity in the determination of cell lineages. It is concluded that molecular biological studies with sponges as model will not only help to understand the evolution of Protoctista to Metazoa but also the complex, hierarchial regulatory network of cells in higher Metazoa.     

Ultrastructure of spermatozoa of <Emphasis Type="Italic">Onthophagus taurus</Emphasis> (Coleoptera,Scarabaeidae) exhibits heritable variation

Sperm competition is thought to be an important selective pressure shaping sperm form and function. However, few studies have moved beyond gross examinations of sperm morphology. Sperm length is subject to sexual selection via sperm competition in the scarab beetle Onthophagus taurus. Here, the structure and ultrastructure of spermatozoa in this species were investigated using light and electron microscopy. Spermatozoa were found to be filiform, measuring about 1,200 mm in length. The sperm head consists of a three-layered acrosome and a nuclear region bearing the anterior extension of the centriole adjunct. Acrosome and nuclear regions are bilaterally symmetric, with their axes of symmetry being orthogonal to each other. Head and flagellar structures are connected by a well-developed centriole adjunct. The sperm heads are asymmetrically surrounded by accessory material and embedded into the cytoplasm of the spermatocyst cell. The accessory material is produced inside the spermatids and then transferred to the outside due to a new membrane formed around the sperm’s organelles. The old spermatid membrane separates the accessory material from the cyst cell. The flagellum contains a 9+9+2 axoneme, two accessory bodies, and two mitochondrial derivatives of unequal size. The major mitochondrial derivative is significantly larger than the minor one. The axoneme is arranged in a sinusoidal manner parallel along the major mitochondrial derivative. The spermatozoa show no progressive motility when released in buffer solution which is likely to be the result of the flagellar arrangement and the structure of the major mitochondrial derivative. The cross-sectional area of the minor and the major mitochondrial derivatives show different patterns of genetic variation. The data provide the first estimates of genetic variation in sperm ultrastructure for any species, and give evidence for the persistence of genetic variation in ultrastructure required for the rapid and divergent evolution that characterizes spermatozoa generally.     

Reviving wood-pastures for biodiversity and people: A case study from western Estonia

Wood-pastures are associated with high cultural and biodiversity values in Europe. However, due to their relatively low productivity, large areas of wood-pastures have been lost over the last century. In some areas, incentive schemes have been developed to revive wood-pastures. We investigated the effects of one such scheme in western Estonia. We compared the structure of grazed wood-pastures (old and restored) to those of abandoned wood-pastures and ungrazed forest stands to explore the effects of management, and conducted interviews with 24 farmers to investigate their motivations to carry out the management. We found a positive influence of active management on the semi-open structure of wood-pastures. Financial support was vital for management, but personal values related to tradition also played an important role. The interviewees differed widely in their range of motivations, suggesting that other strategies in addition to financial incentives would further improve the management of wood-pastures in the region.

Electronic supplementary material

The online version of this article (doi:10.1007/s13280-015-0719-8) contains supplementary material, which is available to authorized users.     

Combined use of molecular biology taxonomy, Raman spectrometry, and ESEM imaging to study natural biofilms grown on filter materials at waterworks

DNA-based population analysis was applied in combination with Raman spectrometry and Environmental Scanning Electron Microscopy for the characterisation of natural biofilms from sand and activated carbon filters operated for a long term at a municipal waterworks. Whereas the molecular biology polymerase chain reaction combined with denaturing gradient gel electrophoresis approach provides a deeper insight into the bacterial biofilm diversities, Raman spectrometry analyses the chemical composition of the extracellular polymer substances (EPS), microorganisms embedded in EPS as well as other substances inside biofilm (inorganic compounds and humic substances). Microscopy images the spatial distribution of biofilms on the two different filter materials. In addition, bacterial bulk water populations were compared with biofilm consortia using the molecular fingerprint technique mentioned.Population analysis demonstrated the presence of more diverse bacterial species embedded in a matrix of EPS (polysaccharides, peptides, and nucleic acids) on the sand filter materials. In contrast to this, activated carbon granules were colonised by reduced numbers of bacterial species in biofilms. Besides α-, β-, and γ-Proteobacteria, a noticeable specific colonisation with Actinobacteria was found on activated carbon particles. Here, the reduced biofilm formation came along with a decreased EPS synthesis. The taxonomy profiles of the different biofilms revealed up to 60% similarity on the same filter materials and 32% similarity of different materials. Similarity of adherent communities from filter materials and bulk water populations from the filter effluent varied between 36% and 58% in sand filters and 6–40% in granular activated carbon filters.The biofilm investigation protocols are most crucial to subsequent acquisition of knowledge on biofilm dynamics and bacterial contributions to transformation or adsorption processes in waterworks facilities.     

Integral assessment of estrogenic potentials in sediment-associated samples

Background, aim, and scope  The enzyme-linked receptor assay (ELRA) detects estrogenic and anti-estrogenic effects at the molecular level of receptor binding and is a useful tool for the integrative assessment of ecotoxicological potentials caused by hormonally active agents (HAA) and endocrine disrupting compounds (EDC). The main advantage of the ELRA is its high sample throughput and its robustness against cytotoxicity and microbial contamination. After a methodological adaptation to salinity of the ELRA, according to the first part of this study, which increased its salinity tolerance and sensitivity for 17-β-estradiol, the optimised ELRA was used to investigate 13 native sediments characterised by different levels of salinity and chemical contamination. The applicability of the ELRA for routine analysis in environmental assessment was evaluated. Salinity is often a critical factor for bioassays in ecotoxicological sediment assessment. Therefore, salinity of the samples was additionally adjusted to different levels to characterise its influence on elution and binding processes of receptor-binding substances. Materials and methods  The ELRA was carried out with the human estrogen receptor α (ER) in a 96-well microplate format using the experimental setup known from the competitive immunoassay based on ligand–protein interaction. It is an important improvement that a physiologically relevant receptor was used as a linking protein instead of an antibody. The microplates were coated with a 17-β-estradiol-BSA conjugate, and dilution series of estradiol and of native sediment samples were added and incubated with the ER. After a washing step, a biotinylated mouse anti-ER antibody was added to each well. Receptor binding to estradiol, agonistic and antagonistic receptor binding, were determined by a streptavidin-POD-biotin complex with subsequent measurement of the peroxidase activity at the wavelength of 450 nm using a commercial ELISA multiplate reader. The sediment elutriates and pore water samples of sediments were tested in a dilution series to evaluate at which dilution step the receptor-binding potential ends. In the elution process (see Section 2.1 to 2.2), a method was developed to adjust the salinity to the levels of the reference testings, which offers an appropriate option to adjust the salinity in both directions. Statistical evaluation was made with a combination of the Mann–Whitney U test and the pT-method. Results  This part of the study characterised the environmental factor ‘salinity’ for prospective applications of the ELRA. Using reference substances such as 17-β-estradiol, the ELRA showed sigmoid concentration-effect relations over a broad range from 0.05 μg/l to 100 μg/l under physiological conditions. After methodological optimisation, both sensitivity and tolerance of the assay against salinity could be significantly raised, and the ELRA became applicable under salinity conditions up to concentrations of 20.5‰. The mean relative inter-test error (n = 3) was around 11% with reference substances and below 5% for single sediments elutriates in three replicates each. For sediment testings, the pore water and different salinity-adjusted elutriates of 13 sediments were used. A clear differentiation of the receptor-binding potential could be reached by application of the pT-method. Thereby, pT-values from one to six could be assigned to the sediments, and the deviation caused by the different salinity conditions was one pT-value. The mean standard deviation in the salinity adaptation procedure of the elutriates was below 5%. Discussion  Although the ELRA has already been used for assessments of wastewater, sludge and soil, its applicability for samples to different salinity levels has not been investigated so far. Even if the ELRA is not as sensitive as the E-screen or the YES-assay, with regard to reference substances like 17-β-estradiol, it is a very useful tool for pre-screening, because it is able to integrate both estrogenic as well as anti-estrogenic receptor-binding effects. According to the results of sediment testing, and given the integrative power to detect different directions of effects, the ELRA shows sufficient sensitivity and salinity tolerance to discriminate receptor-binding potentials in environmental samples. Conclusions  The optimised ELRA assay is a fast, cost-effective, reliable and highly reproducible tool that can be used for high-throughput screening in a microplate format in detecting both estrogenic and anti-estrogenic effects. Additionally, the ELRA is robust against microbial contaminations, and is not susceptible towards cytotoxic interferences like the common cell-culture methods. The general applicability and sufficient sensitivity of the ELRA was shown in freshwater environments. Marine and brackish samples can be measured up to salinity levels of 20.5‰. Recommendations and perspectives  In view of the proven sensitivity, functionality and the fastness of the ELRA, it is recommendable to standardise the test method. At the moment, no adequate in vitro test procedure exists which is standardised to DIN or ISO levels. The E-screen and the yeast estrogen/androgen screens (YES/YAS) sometimes underlie strong cytotoxic effects, as reported in the first part of this study. Further development of an ELRA assay using human androgen receptors appears to be very promising to gain information about androgenic and anti-androgenic effects, too. This would offer a possibility to use the ELRA as a fast and reliable pre-screening tool for the detection of endocrine potentials, thus minimising time and cost-expensive animal experiments.