Long-term biomonitoring of lichen and bryophyte biodiversity at Burnham Beeches SAC and global environmental change

Long-term monitoring began 20 years ago at Burnham Beeches Site of Special Interest (SSSI), National Nature Reserve (NNR) and European Special Area of Conservation (SAC) lying 40 km west of London as a consequence of the authorization of an application to extract gravel from an adjacent site lying north of Slough Trading Estate. Dust monitoring (sticky pads) and photographic monitoring, recording and image analysis was instigated in 1992 on Parmelion communities to assess changes in lichen growth, health and community composition. Long-term monitoring identifies that the lichen flora on free-standing trees has undergone rapid expansion from a near dominance by the SO₂-tolerant ‘acidophyte’ species Lecanora conizaeoides and Hypogymnia physodes following reductions in SO₂ concentrations. Long-term influences of low levels of eutrophication, gaseous pollutants (particularly globally rising background ozone concentrations) on lichen and bryophyte communities and succession under changing climatic conditions are unknown. Soil–plant relationships, lichen–invertebrate interactions and a pollution legacy must also be considered.     

Polymeric Microspheres as Protein Transduction Reagents

Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere–protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.Many proteomic techniques can be used to build a picture of a protein with unknown function, but eventually the individual protein''s activity must be studied. Traditional transfection of encoding DNA permits intracellular expression, but often at uncontrolled, nonphysiological levels. Moreover, DNA transfection can neither deliver protein–inhibitor complexes nor readily deliver multiple proteins in a single experiment and thus exploit knowledge from proteomic protein–protein interaction analyses. In contrast, a truly generic protein transduction reagent could theoretically address all possibilities. We believe that polymeric microspheres could fulfill this role, and we have recently synthesized and characterized dual-functionalized, bio-compatible microspheres that permit intracellular tracking (1). Herein, we now report the development of those microspheres into a protein transduction reagent that can carry protein stably, deliver it efficiently to cells, release the protein in the cytoplasm, and concurrently permit fluorescent imaging of transduced cells.Phagocytosis of microspheres was first observed over 30 years ago (2). Perhaps more unexpectedly, uptake of polystyrene microspheres has recently been reported in many other, nonphagocytic cell types, some of which are traditionally considered to be resistant to DNA transfection and/or protein transduction. For example, microspheres are taken up readily by primary immune cells (3), embryonic stem cells (4), human neural stem cells (5), differentiating mouse neural stem cells (5), and several nonphagocytic cell lines (3, 6, 7). In all instances, the reported efficiency of cellular uptake is high, with “beadfection” of up to 90% of cells being typical (4, 5, 8). No additional reagents aside from the microspheres themselves are required in order to promote cellular uptake, and critically, no toxicity has been observed in any of the cell types beadfected, including HEK293T and L929 cells 2 days after beadfection (8), E14g2a embryonic stem cells 3 days after beadfection (4), and mouse and human neural stem cells 30 days after beadfection (5). In the latter case, the microspheres did not have any deleterious effect on the differentiation of human neural stem cells 30 days after beadfection (5).The mechanism of microsphere entry is also nontoxic, and compelling evidence has been published recently that polystyrene-based microspheres (from 0.2 μm to as large as 2 μm) enter cells via a non-endocytosis, energy-independent mechanism (8). Although unusual, such a mechanism is consistent with claims for the commercial reagent Chariot™ (9). Interestingly, a non-endocytic, energy-independent mechanism has also been reported for the entry of rhenium cluster/polymer hybrid particles into HeLa cells (10). Failure of the microspheres to be endocytosed, at least via a clathrin-dependent mechanism, is perhaps to be predicted, as their diameter considerably exceeds that of clathrin-coated vesicles (typically 100 nm). Bradley and co-workers (8) propose that the entry mechanism for polystyrene-based microspheres is one of passive diffusion in which the microsphere interacts with the membrane, anchors, and, after membrane reorganization, enters the cell, resulting in direct cytoplasmic localization.For functional analysis following transduction, the avoidance of endocytosis or phagocytosis is particularly relevant, as endocytosed particles are destined for endosomes and then, normally, for the lysosomes. The lowered pH of the endosome and, more seriously, the acidic and hydrolytic environment of the lysosome risk disruption of the protein structure and/or function. In contrast, for vaccine delivery (where liposomes can be employed), such exposure is advantageous because protein breakdown forms an essential part of antigen presentation. The potential for protein breakdown in endosomes is also irrelevant for the delivery of protein/peptide drugs such as insulin (for which microencapsulation has proven effective for long-term controlled drug release (11, 12)), as these drugs typically function in the extracellular environment, often exerting their effects by binding to membrane-bound receptors. Thus, although vehicles such as liposomes and nanoparticles are employed both extensively and successfully as drug and vaccine delivery vectors in vivo (13–16), they are far from ideal for studying the biological effect of a delivered protein in vitro. Colloidal particles are also endocytosed (17), and therefore these delivery vehicles may present similar disadvantages.Traditionally, protein transduction domains such as HIV TAT (18–20) or other cell-penetrating peptides (21–23) are used to deliver proteins to cells. Whereas positively charged peptides such as TAT are thought to enter the cells via macropinocytosis (reviewed in Ref. 24), a recent publication suggests that at least some cell-penetrating peptide/bio-cargo complexes (siRNA) are endocytosed (25). Here, although the cargoes avoid the lysosomes, acidification of the endosome is required for endosomal escape of the delivered cargo, and indeed, acidification appears to be a recurring requirement for endosomal escape of biomolecular cargoes using cell-penetrating peptides (reviewed in Ref. 24). Consequently, cell-penetrating peptides are unlikely to become generic tools for functional protein delivery.In contrast, the recent demonstrations that polystyrene microspheres can carry a variety of molecular cargoes with them into the cytoplasm (4, 5, 7, 26, 27) make them particularly exciting as potential vectors for delivering functional proteins and/or protein complexes. β-Galactosidase retains its activity when delivered via this route (7), confirming the potential of microspheres to act as generic protein-delivery vehicles. However, delivered proteins have to date remained tethered to the microspheres, and thus existing studies are limited to proteins that are active in the cytoplasm and, critically, retain their activity when immobilized on polystyrene. For the broad-based study of protein function, the subsequent release of the delivered protein within the cell is desirable.An ideal technology would deliver any protein to any cell type and release that protein in the cell, where it could undertake its normal activity. Here we report the first example of such a microsphere-based approach. Protein is delivered on microspheres and then released in the cell by the reducing cytoplasmic environment. This release is mediated by a linker that attaches the protein stably and covalently to the microspheres in vitro but intracellularly is cleaved over a period of hours. It has already been shown that microspheres are taken up with high efficiency by a range of cell types and can carry a variety of cargoes. Because the chemistry of the linker described herein is amenable to linkage with any molecule containing a free amine moiety, the technology provides a new generic platform for in vitro, cell-based delivery of individual proteins, protein complexes, protein mixtures, or other amino-functionalized molecules.     

Developing a diatom monitoring network in an urban river-basin: initial assessment and site selection

There is increasing interest in the restoration of urban river systems because they are affected by multiple point- and diffuse-source impairments. In South Wales, these issues are exemplified by the rivers Taff and Ely, which have a well-documented history of pollution. We developed a network of river-monitoring sites to capture as many sources of impairment as possible and to provide an integrated assessment of basin-wide conditions using established, recently revised and new methods to assess ecological status as required by the Water Framework Directive (WFD). Most of the 34 river sites, except those in upstream areas, had moderate or poor ecological status and low species richness. There were significant correlations between the UK Trophic Diatom Index (TDI), phosphate and nitrate concentrations, but the strongest correlation was with sodium concentrations. Sodium also differentiated best between ecological status classes. The UK TDI, Ecological Quality Ratios and the Indice de Polluosensibilité Spécifique (IPS) correlated strongly, but the IPS reflected chemical conditions less well. There were also possible metal impacts including some upstream sites despite apparently good or high ecological status. These data reveal strong capacity in diatoms for reflecting stressors that affect urban river networks. We recommend (i) further assessments including geographically comprehensive and repeated surveys to underpin local decision-making and (ii) further refinement of the tools currently in use for WFD purposes through the development of specific diagnostic indices or multi-metric methods for a comprehensive assessment of complex catchments affected by multiple impairments.     

Lichen biomonitoring near Karabash smelter town, ural mountains, Russia, one of the most polluted areas in the world

Biogeochemical signatures were investigated in transplanted and native lichens near a major pollution source using sensitive multi-element chemical analysis. Transplants were established across a 60 km transect centred on the smelter town of Karabash, Ural Mountains, Russia. Statistically significant trends in element concentrations were recorded, some below one part per million. Fine metal particles are accumulated from pollution aerosols. Prolonged exposure may lead to cellular damage and enhanced accumulation or element loss. 206Pb: 207Pb isotope ratios are similar to those associated with airborne particles in Europe and Russia; an outlier near Kyshtym with a lower ratio indicates a source with a higher 235U : 238U ratio. The method is discrete, sensitive, able to detect short-term pollution episodes and useful for understanding element cycling, which is of critical importance for human and environmental health.     

Semper's (zoanthid) larvae: pelagic life,parentage and other problems

Semper's larvae were obtained from <300 out of 1800 plankton tows taken in the world's oceans (1964–1993). Zoanthellae (larvae of Sphenopidae) occurred at 217 stations and zoanthinae (larvae of Zoanthidae) at 86, the two larval types showing distributions clearly delimited by a minimum sea temperature (22 °C for zoanthellae, 18 °C for zoanthinae; a statistically significant difference, P<0.001). Length of formalin-fixed zoanthellae was 2–8.6 mm and of zoanthinae 1.5–5.9 mm. Endodermal zooxanthellae were present in 9/24 zoanthinae but in no zoanthellae (of 19). Three larvae contained an endo-commensal/parasitic amphipod. Septa were externally visible in larger zoanthinae and were counted in transverse sections of other larvae, a majority of which (both kinds) had 12 septa, the normal maximum. The pattern was brachycnemic in 40/43 larvae and anomalous (but non-macrocnemic) in three. If macrocnemic genera reproduce by Semper's larvae, they should have been represented in such a large sample. The distribution of adult Epizoanthus was examined: many species are deep sea (recorded down to 5000 m) but shallow-water species are relatively plentiful in, for example, the Adriatic and North Seas. No Semper's larva has ever been recorded from either. Some Parazoanthus species also occur in shallow water, especially associated with western Atlantic reef sponges. If they produce Semper's larvae, these have never been found. It is probable that macrocnemic zoanthids settle from planulae that do not develop into recognizable zoanthellae or zoanthinae.     

Diatoms in Lowland Ponds of Koshi Tappu,Eastern Nepal – Relationships with Chemical and Habitat Characteristics

Epiphytic and epipelic diatom assemblages were studied in relation to water chemistry and habitat character in lowland ponds of Koshi Tappu, Nepal. Epiphytic assemblages from different microhabitats, such as morphologically different plants, roots, stems, filamentous algae and decomposing leaves, within the same ponds were similar. Assemblage composition of epiphytic diatoms reflected gradients in water chemistry and habitat character of the pond with respect to pond vegetation and substratum type, bank profile and land use in the catchment. Epiphytic and epipelic assemblages responded to chemical gradients in the surface water, particularly concentrations of Ca, Mg and Na, but epipelic diatoms also indicated differences in SO₄. Epipelic diatoms were also sensitive to interstitial water chemistry variations in PO₄, Si, Ca and Mg. There were no relationships between species richness, diversity or evenness and gradients in water chemistry and habitat character. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Fragmentation and pre-existing species turnover determine land-snail assemblages of tropical rain forest

Aim The main aims of the study were: (1) to investigate the effect of fragment age in relation to other patch‐ and landscape‐scale measures of forest fragmentation, and (2) to assess the relative importance of fragmentation, habitat degradation (i.e. degradation caused by selective logging and past shifting cultivation) and putative pre‐existing species turnover in structuring current land‐snail assemblages. Location South‐western Sri Lanka. Methods The land‐snail fauna was sampled using standardized belt transects. Fifty‐seven transects were sampled in 21 lowland rain forest fragments (c. 1–33,000 ha). The spatial arrangement of fragments in the study area was explicitly considered in an effort to take into account the non‐random nature of fragmentation and degradation and the possibility that current species composition may reflect patterns of species turnover that existed prior to fragmentation. The data set of 57 land‐snail species and 28 environmental and spatial variables was analysed using canonical correspondence analysis and partial canonical correspondence analysis. Results Fragment age, mean shape complexity (i.e. a landscape‐scale measure of shape complexity), altitude, and the spatial variables x (longitude), y (latitude) and y² explained significant variation in land‐snail species composition. None of the three nominal variables quantifying habitat degradation was significantly correlated with variation in species composition. The independent effects of fragment age and mean shape complexity were similar. The combined effect of the spatial variables alone was larger than the independent effects of fragment age, mean shape complexity or altitude, but was of the same order of magnitude. The total variation explained by the spatial variables was comparable to the total non‐spatial variation accounted for by fragment age, mean shape complexity and altitude. Main conclusions Fragment age was found to be one of only two key determinants (the other was shape complexity at the landscape scale) driving fragmentation‐related changes in community composition. The influence of pre‐fragmentation patterns of species turnover on assemblage structure can be stronger than the effects of fragmentation measures, such as age, and may override the effects of forest degradation. Thus, strong patterns of pre‐existing turnover may potentially confound interpretation of the effects of forest fragmentation and degradation.     

Diatoms as indicators of stream quality in the Kathmandu Valley and Middle Hills of Nepal and India

1. Diatoms are recognised as indicators in temperate streams, but only recently have assessments begun of their value in indicating stream quality in the tropics and sub‐tropics. Here, we extend previous studies by assessing stream diatom assemblages in relation to water quality and habitat character in the Kathmandu Valley, and in the Middle Hills of Nepal and northern India. We also assessed whether the U.K. Trophic Diatom Index (TDI) was sufficiently portable to reveal pollution in Himalayan rivers. In the more urbanised and highly agricultural Kathmandu Valley, we compared diatom response to water quality classes indicated by a local invertebrate index, the Nepalese Biotic Score (NEPBIOS). 2. Thirty and 53 streams in the Kathmandu Valley (2000) and Middle Hills (1994–96), respectively, were sampled in October and November during stable flows following the monsoon. Diatoms were collected in riffles, water samples taken for chemical analysis, and habitat character of the stream channel, bank and catchment assessed using river habitat surveys. In the Kathmandu Valley, macroinvertebrates were collected by kick‐sampling. 3. In total, 113 diatom taxa were found in the Kathmandu Valley streams and 106 in the Middle Hills. Of 168 taxa recorded, 62 occurred only in the Kathmandu Valley, 56 only in the Middle Hills and 50 were common to both areas. Most taxa found only in the Kathmandu Valley belonged to the genus Navicula while most taxa confined to the Middle Hills were Achnanthes, Fragilaria and Gomphonema. 4. In the Kathmandu Valley, richness and diversity increased significantly with K, Cl, SO₄ and NO₃, but declined significantly with Al, Fe, surfactants and phenols. Richness here also varied with habitat structure, being lowest in fast flowing, shaded streams with coarse substrata in forested catchments. In all streams combined, richness increased significantly with Si, Na and PO₄, but declined significantly with increasing pH, Ca and Mg. 5. Diatom assemblage composition in the Kathmandu Valley strongly reflected water chemistry as revealed by cations (K, Na, Mg, Ca), anions (Cl, SO₄), nutrients (NO₃, PO₄, Si), and also substratum composition, flow character and catchment land use. The commonest taxa in base‐poor forested catchments were Achnanthes siamlinearis, A. subhudsonis, A. undata and an unidentified Gomphonema species; Cocconeis placentula and Navicula minima in agricultural catchments; and Mayamaea atomus var. alcimonica, M. atomus var. permitis, and Nitzschia palea at polluted sites near settlements. Diatom assemblages in none‐agricultural catchments of the Kathmandu Valley and Middle Hills were similar, but they contrasted strongly between urban or agricultural catchments of the Kathmandu Valley and the less intensively farmed catchments of the Middle Hills. 6. In keeping with variations in assemblage composition, most streams in the Kathmandu Valley had higher TDI values (33–87, median = 64) and more pollution tolerant taxa (0–78%, median = 16) than streams in the Middle Hills (25–82, median 45, 0–26%, median = 2). TDI values correlated significantly with measured PO₄, Si, and Na concentrations in the Kathmandu Valley, and with Si and Na concentrations in the Middle Hills. There was some consistency between water quality classes revealed by NEPBIOS and diatoms, but also some contrast. Water quality class I–II sites had lower TDI values and were less species rich than water quality II sites, however, there were no significant differences in detrended correspondence analysis (DCA) assemblage scores and relative abundances of pollution tolerant taxa between NEPBIOS classes. 7. While diatoms in the Middle Hills indicate unpolluted or only mildly enriched conditions, they reveal pronounced eutrophication and organic pollution in the densely populated Kathmandu Valley. In addition, diatoms appear to respond to altered habitats in rural agricultural and urban areas. As demands on water resources in this region are likely to increase, we advocate the continued development of diatoms as indicators using methods based on what appear to be consistent responses in the TDI between Europe and the Himalaya.