Evaluation of confocal laser scanning microscopy for enumeration of virus-like particles in aquatic systems

Abundances of virus-like particles (VLPs, mostly bacteriophages) are high in aquatic environments; therefore, techniques for precise enumeration are essential in ecological monitoring. VLPs were determined after staining with SYBR Gold by conventional epifluorescence microscopy and compared to enumerations performed by confocal laser scanning microscopy (CLSM). In order to assess the potential of CLSM for viral direct counts (VDCs), we processed samples from different freshwater and marine systems. Optical sectioning by CLSM and production of an overlay picture of multiple scans enables the often uneven whole investigated filter area to be brought to the plane of focus. This allows for subsequent image analysis of digitally created high-quality images. Another advantage using the CLSM was that the short spot excitation of the stain via laser beam minimized fading of the stain. The VDC results show that there is no significant difference between the two methods. Regarding the known difficulties of viral abundance estimates on particulate material, CLSM was further applied to enumerate VLPs on a small set of marine transparent exopolymeric particles sampled from the Atlantic Ocean. Our data suggest that CLSM is a useful tool to count viruses in water samples as well as attached to certain types of aquatic aggregates.     

Biomarker responses associated with halogenated organic contaminants in northern fulmars (Fulmarus glacialis) breeding in the Canadian Arctic

We examined relationships between hepatic concentrations of halogenated organic contaminants and ethoxyresorufin O-deethylase (EROD) activity and retinoid (vitamin A) concentrations in livers, as well as retinol and thyroid hormone (TT₃, TT₄) levels in blood plasma, of northern fulmars at two breeding colonies in the Canadian High Arctic. Biomarker levels or responses did not differ significantly between males and females at either colony, nor was there any significant difference between colonies. No significant relationships were found between thyroid hormone or hepatic retinoid concentrations and any of the dioxin-like compounds or their Toxic Equivalents (TEQs) although significant positive correlations were found with plasma retinol (p < 0.03). EROD activity was significantly correlated with hepatic dioxin-like compounds and their TEQs (p < 0.001) as well as total PCBs (p < 0.01), which suggests that EROD induction occurs in northern fulmars at environmentally-relevant concentrations.     

Integral assessment of estrogenic potentials of sediment-associated samples

GOAL, SCOPE AND BACKGROUND: Exogenic endocrine-active substances are also called 'Endocrine Disrupting Chemicals' (EDC). They imitate or hinder the function of natural endogenic hormones or disturb the synthesis or the metabolism of hormones or of hormone receptors. The Enzyme-Linked Receptor Assay (ELRA) can detect estrogenic and anti-estrogenic effects at the level of receptor binding and is a useful tool for the integrative detection of contaminant effects. Although the test system has been used repeatedly in sediment assessments, the questions have remained concerning how it responds to variations in the physico-chemical matrix. For some bioassays, the salinity of the sample is a critical factor. This is especially relevant when testing wastewater samples or when sediment-associated samples in the tidal reaches of rivers are tested. Sediments in the tidal reaches of rivers change their salinity several times a day. Against this background, it would be beneficial to have a test procedure of known salinity tolerance. On account of this, the salinity tolerance of the ELRA was tested, assessed with reference substances at several salinity levels, and compared with the E-Screen method and a Yeast Estrogen Screen (YES), which are also frequently applied in environmental testing. The aim of this paper was to explore when the salinity limits within these test procedures are applicable. The trials should reveal the working range to be expected, characterize the salinity-dependent variations in sensitivity of the test, and provide options for methodological adjustments to improve the stability against increased salinity. METHODS: The ELRA was carried out with the human Estrogen Receptor alpha. (ER) using the same principle like a competitive immunoassay based on ligand-protein interaction. However, an essential difference is the use of a physiologically relevant receptor instead of an antibody as a linking protein. The ELRA measures the competition of sample estrogens and anti-estrogens against estradiol supplied as a BSA-coating conjugate for the binding site of dissolved ER. Estradiol or xeno-estrogen binding is quantified by a biotynilated anti-ER antibody and the subsequent measurement of peroxidase activity by a streptavidin-POD-biotin complex. The E-Screen was performed with the human breast cancer cell line MCF-7, which expresses the estrogen receptor constitutively. Cell proliferation depends on binding of estrogens or xeno-estrogens with the receptor. After incubation, estrogen-dependent cell growth was measured by sulforhodamin B staining. The YES was performed with a recombinant yeast strain, transfected with a receptor and a reporter plasmid bearing the estrogen receptor and a vitellogenin gene fused with the reporter gene lacZ. Estrogen or xeno-estrogen-dependent gene induction was measured indirectly by LacZ activity. The salinity levels were simulated in varying concentrations with NaCl from 0 to 40 per thousand or Artificial Sea Water (ASW) from 0 to 32 per thousand. RESULTS: The study characterized the factor 'salinity' for the prospective application fields of the ELRA. With reference substances such as 17-beta-estradiol, the ELRA showed classical sigmoidal concentration-effect relations in a range from 0.05 to 100 microg/l under physiological conditions. After a methodological adjustment to compensate decreasing receptor-binding affinity of estrogens and xeno-estrogens at higher salinity levels, the ELRA became applicable under salinity conditions up to concentrations of 20.5 per thousand. In tests, the ELRA reached under the influence of salinity a mean limit of detection of 0.062 microg/l 17-beta-estradiol. The mean relative inter-test error was around 11%. Above concentrations of 20.5 per thousand there is a risk of false negative assessment. Compared with the E-Screen method using the MCF7 cell line and the yeast estrogen test system (YES), the ELRA shows a lower sensitivity to 17-beta-estradiol. In the E-Screen, the cell proliferation was strongly reduced by sodium chloride induced cytotoxicity. In comparison with the E-Screen, the salinity tolerance of the YES and YAS methods is significantly higher. DISCUSSION: Despite adaption, total salinity tolerance could not be achieved with the ELRA. Freshwater samples were generally appraisable. Higher salinity levels above 20.5 per thousand would tend towards false negative results. The low inter-test error of 11% makes the ELRA suitable for the detection of estrogenic and anti-estrogenic potentials of single substances, substance mixtures, and of environmental samples. CONCLUSIONS: The ELRA is very fast and reproducible, it can be used for high-throughput screening in a microplate format at low cost, it is robust to microbial contamination, and is less susceptible to cytotoxic interferences than cell culture methods. RECOMMENDATIONS AND PERSPECTIVES: In their established form, the YES and the E-Screen methods are not applicable for liquid phase testing at higher salinity conditions. The salinity-adapted test version of the ELRA described here shows a broader working range for samples. Native water samples of more or less brackish origin or high-salinity effluent samples are testable. Results of tests with sediment associated samples of different salinity will be subject of a forthcoming publication.     

Multi-risk,multi-scale and multi-stakeholder – the contribution of a bow-tie analysis for risk management in the trilateral Wadden Sea Region

Risk management processes increasingly call for enhanced stakeholder participation, and aim to integrate different risk perceptions, concerns and interests. Frequently, this goal is driven by the increased complexity of risk management processes, as risk management processes continuously have to deal with multi-risk situations including impacts resulting from risks of natural hazards and risks caused by misguided social or economic development. Although stakeholder participation is required by different policies, major challenges still arise from the question of how to perform multi-stakeholder participation in practice. In order to find answers, we tested the so-called ‘bow-tie analysis’ as a potential tool to facilitate multi-stakeholder participation with a major effort on integrating stakeholders risk perceptions and interest in the risk management processes. The bow-tie analysis is a commonly used risk assessment technique (IEC 2009) to analyse cause-and-effect pathways of risks, but its application in multi-stakeholder processes in risk management of natural hazards, especially in a European context, is rather new. Using practical experiences from the trilateral Wadden Sea Region we demonstrate the bow-tie analysis’ contribution to coastal risk management processes in this coastal area by facilitating collaborative identification, comprehension and analysis of the management system. The use of a modified bow-tie analysis in collaboration with stakeholders from the Wadden Sea Region proved to be an appropriate framework for enhancing the understanding of risk management processes and fostered disclosure of different perceptions and concerns of multi-risk problematics. The bow-tie can be beneficial as a communication and co-construction tool in risk management processes in a multi-risk context.     

Facilitating biodiversity conservation through partnerships to achieve transformative outcomes

Conservation biology is a mission-driven discipline that must navigate a new relationship between conservation and science. Because conservation is a social and political as well as an ecological project, conservation biologists must practice interdisciplinarity and collaboration. In a comparative study of 7 cases (Jaguars in the Chaco, Grevy's zebra in Kenya, Beekeeping in Tanzania, Andean cats in Argentina, Jaguars in Mexico, Lobster fishing, and Black bears in Mexico), we examined motivations for collaboration in conservation, who can collaborate in conservation, and how conservation professionals can work well together. In 5 case studies, successful conservation outcomes were prioritized over livelihood benefits. In the other 2 cases, livelihoods were prioritized. All case studies employed participatory approaches. There were multiple external actors, including local and Indigenous communities, nongovernmental organizations, agencies, regional and national governments, and international organizations, which enhanced conservation and wider sustainability outcomes. Key collaboration aspects considered across the case studies were time (mismatch between relationship building and project schedules), trust required for meaningful partnerships, tools employed, and transformative potential for people, nature, and the discipline of conservation biology. We developed guidelines for successful collaboration, including long-term commitment, knowledge integration, multiscalar and plural approaches, cultivation of trust, appropriate engagement, evaluation, supporting students, and efforts for transformation.     

A butterfly’s chemical key to various ant forts: intersection-odour or aggregate-odour multi-host mimicry?

Deception is a crucial yet incompletely understood strategy of social parasites. In central Europe, the Mountain Alcon Blue, Maculinea rebeli, a highly endangered butterfly, parasitises several Myrmica ant species. Caterpillars gain access to host nests probably by faking the ants odour. We analysed gas chromatography–mass spectrometry data of body surface hydrocarbons of pre-adoption and hibernated larvae of Maculinea rebeli and of their host species Myrmica sabuleti and M. schencki. Data were ordinated by different methods, based on similarities in the relative quantities of compounds between chromatograms. The two Myrmica species exhibit species-specific profiles. The Maculinea rebeli pre-adoption larva has a complex profile that simultaneously contains species-specific substances of the two investigated host species. This evidence leads to the interpretation that, in central Europe, Maculinea rebeli is predisposed for multi-host use by the chemical signature of its pre-adoption larva. The Maculinea rebeli larva clearly does not rely on an intersection-odour of compounds common to all host ant species, but synthesises an aggregate-odour containing specific compounds of each of the investigated hosts. We term this previously unknown chemical strategy aggregate-odour multi-host mimicry.B.C. Schlick-Steiner and F.M. Steiner contributed equally to this work.     

Extraction and quantification of "condensed tannins" as a measure of plant anti-herbivore defence? Revisiting an old problem

Contents of phenolic compounds in leaf extracts often serve as a measure of plant anti-herbivore defence. This method suffers from the multifunctionality of phenolics and from problems with their colorimetric quantification. Here we present further evidence for the pertinence of these problems. Contents of condensed tannins (CCT) were spectrophotometrically quantified in leaf extracts of 11 closely related mimosoid species, and Spodoptera littoralis caterpillars were reared on artificial diet containing these extracts. The relationship of CCT with caterpillar growth differed considerably among plant species, since both positive and negative correlations were detected. There was, however, a negative correlation of CCT with fungal spore germination, indicating a role of these compounds in resistance to fungi. Detailed knowledge on the structure and biological function of defensive compounds and on the overall composition of leaves is required to estimate a plant's defensive efficacy against a particular group of enemies.     

Charakterisierung von Cyaniden in Böden und industriellen Abfällen mit der Fourier-Transformations-Infrarot-Spektrometrie

Soils on sites of former coking and manufactured gas plants as well as on those of blast furnaces are frequently contaminated with cyanides (CN). For assessing the possible risk arising from these hazardous compounds, it is necessary to analyze a considerable number of samples quickly as well as at low costs. Fourier transform infrared (FTIR) spectroscopy has the advantage of being much easier in handling compared to conventional wet-chemical methods for analyzing cyanides. For testing FTIR spectroscopy in the application mentioned above, pure cyanide compounds as well as contaminated purifier waste, blast furnace sludge, and spiked soils were investigated by the reflexion and the transmission techniques. Applicability of the attenuated total reflexion (ATR) technique was limited, since the absorption bands of the crystal used (diamond) overlapped in the range of the cyanide-specific absorption. Although the environmental samples investigated have unfavorable optical properties, results obtained by the transmission technique (KBr pressed pellets) were much better than those obtained by the reflexion technique. Pure cyanide compounds could be distinguished by their IR spectra because their absorption bands of the cyanide-valence vibration differed in position, shape and intensity. The soil matrix did not interfere with either the intensity or the shape and position of the absorption maxima, as concluded from the IR spectra of spiked soil samples. Applying the transmission technique, cyanides are detected at concentrations larger than 500 mg kg^?1 CN. However, the signal was low when the cyanide concentration was below 1,000 mg kg^?1 CN. The FTIR spectroscopy is capable of detecting cyanide compounds in soils and industrial wastes. Additionally, the method provides information about the cyanide species, which is important in toxicological assessments. FIIR spectroscopy is a suitable method for a first survey and environmental assessment of cyanide contaminated areas.     

Main nutrient compounds in food bodies of Mexican <Emphasis Type="Italic">Acacia</Emphasis> ant-plants

Summary. Myrmecophytic plants use obligate ant mutualists as a constitutive indirect defence mechanism. These plants often produce cellular food bodies (FBs) to nourish their resident ants. Lipids, proteins, and even highly specialised compounds such as glycogen have been reported from FBs, but detailed chemical analyses of FB composition have so far been presented only for Southeast Asian Macaranga and Central American Piper myrmecophytes. Here we report the chemical composition of FBs of five myrmecophytic Acacia (Fabaceae) species from Mexico using HPLC (carbohydrates and proteins) and GC-MS (lipids). Feeding experiments revealed no hints on any use of external food sources by the inhabiting Pseudomyrmex ants. These ants obviously rely completely on FBs and extrafloral nectar provided by their hosts. The total content of nutrients in Acacia FBs was 15-25% of FB dry mass, being much lower than in Macaranga or Piper FBs. Proteins were dominating (8-14 % dm) in Acacia FBs and thus were present in higher amounts than in Macaranga FBs, yet in lower amounts than in Piper. Lipids contributed 1-9 % of dry mass, showing a lower proportion than in FBs of Macaranga or Piper. Carbohydrates made up 3-11 % dm, reaching in most Acacia species the same range as observed in Macaranga and in Piper FBs. Water content was 18-24 % of FB fresh mass, and structural tissue obviously made up a much higher proportion in Acacia FBs than in Macaranga or Piper FBs. Both characters might represent an adaptation to producing FBs unprotected at the leaf tips under dry conditions. Acacia FBs contain all amino acids and all fatty acids that are considered essential for insects, and their contents of lipids and proteins are higher than in the leaves from which they are ontogenetically derived. This indicates a putatively adaptive enrichment of nutritionally valuable compounds in structures functioning as ant-food.     

Zum indirekten Photoabbau chlororganischer Verbindungen durch OH-Radikale in der Atmosphäre

The substituent chlorine is generally considered to be an agent which reduces the reactivity of organic molecules with regard to the indirect photodegradation by OH radicals. A systematic study of selected, representative classes of compounds, however, reveals that the deactivating influence of chlorine on the OH radical reactivity is actually rather moderate. In individual cases, for instance, chlorine substitution can even lead to an increase in this reactivity. This study is based on both experimental data and on the molecular orbital calculations of OH radical reactivity.