Recommendations for the routine sampling of diatoms for water quality assessments in Europe

Many methods for using diatoms for routine monitoring of water quality have been developed in Europe and, in some countries, these are being used to enforce environmental legislation. In order to facilitate their wider use, particularly with respect to European Union legislation, steps are being taken to harmonize methodology. In this paper, the principles and practice of sampling are described in relation to the main habitat types encountered in Europe. Although details of methods and sampling programmes have to be tailored to particular circumstances and the overall objectives of the monitoring, a number of generalizations can be made. Where available, rocks and other hard surfaces are the preferred substrates and methods for sampling these are described. If such substrata are not available, then introduced ('artificial') substrata have many applications. Various types of introduced substrata can be used successfully, so long as some basic precautions are described. Other types of substrata such as macrophytes and macroalgae may also be useful under certain circumstances, although there is less consensus in the literature on the most appropriate methods, and of the validity of comparisons between indices computed from epiphytic and epilithic communities. When designing surveys, it is recommended that as far as possible, extremes of non-water quality factors (e.g. shade, current speed, etc) are avoided, unless these are characteristic of the system under investigation. Detailed guidelines for sampling epilithon are described. Along with the recommendations for sampling other substrata, it is hoped that these provide a framework that can be adapted to most river types in Europe. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic dissection of testicular weight in the mouse with the BXD recombinant inbred strains

Testicular weights were studied in the mouse BXD recombinant inbred (RI) strains. These strains were derived from DBA/2J and C57BL/6J progenitors that differ significantly in their testicular weights (0.224 g ± 0.015 vs. 0.161 g ± 0.03, P < 0.0001). The heritability of testicular weights was calculated to be 0.53, and the minimum number of responsible effective factors was estimated to be 5.7. The total genome scanning of the BXD RI strains with over 1000 markers revealed a quantitative trait locus (QTL) on mouse Chromosome (Chr) 13 near the D13Mit3 marker (LOD score 6.9). This QTL region was designated Twq1 and associated with over 75% of genetic variability. Received: 23 January 1998 / Accepted: 16 March 1998     

Spectroscopic investigation of nickel cation binding with adenine mononucleotides: stability and structure of the 1:2 complex with adenosine 5′-monophosphate

 The interaction of Ni(II) ions with adenine mononucleotides (5′-AMP, 3′-AMP, 2′-AMP, 2′,3′-cAMP, 3′,5′-cAMP) was studied in aqueous solution using Raman spectroscopy and ¹³C and ³¹P NMR paramagnetic relaxation measurements. Macrochelate structures were observed to form for all non-cyclic AMPs, with increasing stability in the series: 3′-AMP < 2′-AMP < 5′-AMP. N7 of adenine was found to be the key site of the Ni(II)-adenine interaction for all non-cyclic AMPs. For 2′-AMP, an alternative binding to the pyrimidine ring may also exist. The dependence of Raman spectra on AMP and Ni(II) concentration confirmed the existence of a stable 1 : 2 Ni(II)-(5′-AMP) complex, besides the 1 : 1 complexes. In this complex, the adenine moieties of both 5′-AMP molecules are situated close to Ni(II), and their relative orientations with respect to the cation are very similar. The paramagnetic relaxation enhancements of the carbons indicate that the nickel ion is not located in the plane of the adenine units, but that the line connecting Ni(II) and N7 deviates strongly from the adenine planes. Phosphates are outer-sphere coordinated by the cation. Findings from both methods have led us to propose possible global architectures of the complex. Received: 26 June 1998 / Accepted: 22 July 1998     

Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus

The surface-located fibrinogen-binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall-anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the α- and β-chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the γ-chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (i) clumping of exponential-phase cells in a solution of fibrinogen, (ii) adherence of exponential-phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial-related infections. However, in wild-type exponential-phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary-phase cultures with fibrinogen. ClfB-dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca²⁺ and Mn²⁺, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low-affinity inhibitory cation binding site.     

Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.     

Sensitivity of seedling recruitment to moss, litter and dominant removal in an oligotrophic wet meadow

The effect of a dominant species, the litter layer, and the moss layer on seedlings and established vegetation was evaluated in two manipulative experiments in an oligotrophic wet meadow (Molinion with some features ofViolion caninae according to phytosociological classification). In the first experiment, the dominant species (Nardus stricta), litter layer, and litter layer together with the moss layer were removed and the response of the established vegetation and seedlings were compared with a control. Results revealed that after one season there was no significant effect of the treatments on established vegetation, but seedling recruitment was significantly affected. The greatest increase of seedling recruitment for many species was observed in the plots with the moss layer removed. In the second experiment, seeds ofSelinum carvifolia were sown into plots subjected to the following treatments: complete vegetation removal, mowing, mowing combined with removal of the moss layer, and an untreated control. The highest number of seedlings was found in plots with the vegetation removed, but the seedling recruitment was nearly as high in plots with the moss layer removed. Low numbers were found in mown plots and the lowest in the untreated control. The results show that seedling recruitment is more sensitive to competition than the established vegetation, at least over one season. This supports the idea of the importance of the regeneration niche for the maintenance of diversity in grassland communities.     

Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing

Genomic sequencing was used to localise 5-methylcytosine residues in individual DNA strands of 5S rRNA genes in tobacco. The density of methylation along the sequence was high in both strands, exceeding the average methylation density of the tobacco genome. Besides methylation of CG and CNG sequences, considerable amounts of mC were found in non-symmetrical sites. Among 69 sequenced clones obtained from leaf DNA we did not detect any non-methylated clone, and Southern blot hybridisation analysis also failed to suggest the presence of methylation-free 5S rDNA units in the tobacco genome. Differences were observed among methylation patterns of individual sequenced clones. This heterogeneity reflects either heterogeneity among individual members of 5S rRNA gene cluster or differences among individual cells. Methylation of CNG and non-symmetrical sites can be efficiently reduced by treatment with dihydroxypropyladenine, an inhibitor of S-adenosylhomocysteine hydrolase.     

History vs. habitat type: explaining the genetic structure of European nine‐spined stickleback (Pungitius pungitius) populations

The genetic structure of contemporary populations can be shaped by both their history and current ecological conditions. We assessed the relative importance of postglacial colonization history and habitat type in the patterns and degree of genetic diversity and differentiation in northern European nine‐spined sticklebacks (Pungitius pungitius), using mitochondrial DNA (mtDNA) sequences and 12 nuclear microsatellite and insertion/deletion loci. The mtDNA analyses identified – and microsatellite analyses supported – the existence of two historically distinct lineages (eastern and western). The analyses of nuclear loci among 51 European sites revealed clear historically influenced and to minor degree habitat dependent, patterns of genetic diversity and differentiation. While the effect of habitat type on the levels of genetic variation (coastal > freshwater) and differentiation (freshwater > coastal) was clear, the levels of genetic variability and differentiation in the freshwater sites were independent of habitat type (viz. river, lake and pond). However, levels of genetic variability, together with estimates of historical effective population sizes, decreased dramatically and linearly with increasing latitude. These geographical patterns of genetic variability and differentiation suggest that the contemporary genetic structure of freshwater nine‐spined sticklebacks has been strongly impacted by the founder events associated with postglacial colonization and less by current ecological conditions (cf. habitat type). In general, the results highlight the strong and persistent effects of postglacial colonization history on genetic structuring of northern European fauna and provide an unparalleled example of latitudinal trends in levels of genetic diversity.