ALBERT

Notes: A large-scale biomanipulation trial was carried out on Lake Vesijärvi in Finland during 1989–1993. Following the mass removal of coarse fish the biomass of cyanobacteria collapsed from 1.4 g/m−3 to below 0.4 g/m−3, while total phosphorus concentration declined from 45 μg/L to 30 μg/L. No relevant changes in zooplankton communities were observed. The results suggest that the success of food web manipulation as a tool for lake restoration is not necessarily dependent on the grazing rate of zooplankton. The effects of reduced fish-mediated internal loading and recycling of nutrients are in many cases stronger than those of reduced planktivory. Alternative stable states of water quality may also exist in lakes not covered by macrophytes, owing to the changes in the behavior of fish stocks. Year-to-year variation in the littoral zone may cause large oscillations in lake ecosystems—for example, through the recruitment of fish. In addition, the nutrients translocated by fish from the littoral zone may affect the nutrient dynamics of the pelagial plankton community. In terms of phytoplankton species composition and the ratio of phosphorus to chlorophyll a, the water quality in Lake Vesijärvi has improved in a stepwise fashion within the last 10 years. This is probably due to the fact that the five-year mass removal of fish in Enonselkä fulfilled the requirement of sustained management of fish stocks in order to maintain nonequilibrial conditions between alternate stable states. The prediction of the water quality development is obscured, however, by spatial and temporal within-lake variation, which sets high requirements for sampling programs.

Notes: Vesicular-arbuscular mycorrhizal fungi were investigated in the roots of flowering plants and ferns obtained from a variety of Connecticut freshwater wetland habitats. We sampled 290 plants from 89 species of 75 genera disposed among 42 families of flowering plants. All species of mature plants as well as selected young plants on developing shorelines were colonized by mycorrhizal fungi, suggesting that this phenomenon is common in the development of vegetation associated with fluctuating water, nutrient, and oxygen conditions. The purpose of this report is to make soil conservationists, wetland agents, and others concerned with wetlands aware of this relationship as they select plants for use in restoration, and to point out the widespread nature of the endomycorrhizal phenomenon.

Notes: A primary objective of riparian restoration in California is the creation of habitat for endangered species. Four restoration sites in San Diego County were monitored between 1989 and 1993 and evaluated for their suitability as nesting habitat for Vireo bellii pusillus (Least Bell's Vireo), a state and federally endangered obligate riparian breeder. Vegetation structure at each site was quantified annually and compared to a model of canopy architecture derived from Least Bell's Vireo territories in natural habitat. Vireo use of restored habitat was documented through systematic surveys and nest monitoring. By 1993, only one site in its entirety met the habitat suitability criteria of the model, but portions of each site during all years did so. Differences between sites in the time required to develop suitable habitat—well-developed layered vegetation from the ground to under 8m in height)—were attributable largely to variation in annual rainfall. Vireos visited restoration sites to forage as early as the first growing season, but they did not establish territories or nest there until at least part of the site supported suitable habitat as determined from the model. Placement of territories and nests coincided with patches of dense vegetation characteristic of natural nesting areas. Occupation of restored sites was accelerated by the presence of adjacent mature riparian habitat, which afforded birds nest sites and/or foraging habitat lacking in the planted vegetation. Vireos nesting in restored habitat achieved success comparable to that of vireos nesting in surrounding natural habitat, and there was no evidence that productivity was reduced in created areas. These findings indicate that creating nesting habitat for this target species is feasible and suggest that the critical components of vireo nesting habitat have been captured in both the design and quantitative assessment of restoration sites.

Notes: Minnesota, the land of 10,000 lakes, also has more than 4000 abandoned quarry pits and over 200 deep, exhausted iron ore pits. In the past 25 years the iron ore pits have gradually filled with groundwater and surface water, forming lakes on the Cuyuna, Mesabi, and Vermillion Iron Ranges in northeastern Minnesota. Most remain abandoned, but besides creating a small number of recreational parks and fisheries, the regional economic development agency promoted approximately 20 of the pit lakes for economic reclamation by using them for salmonid aquaculture. Intensive net-pen aquaculture was carried out from 1988 to 1995 in the Twin City–South and Sherman pit lakes on the Mesabi Range. A water quality controversy resulted over the potential for long-term degradation of the lakes and regional aquifer. The Minnesota Pollution Control Agency then mandated that aquaculture be terminated in Twin City–South in May 1993 and the lake restored to preaquaculture conditions by 1996. With no management other than artificial aeration for one summer, the lake rapidly recovered to near baseline water quality and returned to an oligomesotrophic (unproductive) status. Within 18 months the phosphorus budget was typical of reference pit lakes in the area and dissolved oxygen in bottom water remained above ∼4 mg O2/L without artificial aeration. Algal growth was low in 1993, due to light limitation from artificial mixing, but it remained low in 1994 without any management due to renewed phosphorus limitation. Inorganic nitrogen initially decreased faster than expected, at a rate similar to its increase during intensive aquaculture. More rapid reductions in water column nutrients might have occurred in 1993 by reducing aeration to allow anoxia in the lower hypolimnion, promoting denitrification and minimizing sediment resuspension, but this was precluded by water quality standards. The “natural” burial of solid wastes under inorganic sediment eroded from the basin walls effectively minimized transport of sediment nutrients to the overlying water. Fallowing for several years provided a simple, effective method for restoration of these pit lakes from aquacultural impacts. No change attributable to aquaculture was observed in the water quality of three nearby pit lakes, including a drinking water source. This fact suggests that there were few or no impacts from off-site migration of aquaculturally enriched water into the regional aquifer.

Notes: Invasion of coastal dunes by Lupinus arboreus (yellow bush lupine) results in soil enrichment and displacement of native plants. Restoration by means of heavy equipment was tested as an alternative to costly manual techniques in a heavily invaded area of relatively flat terrain. Two experiments were conducted in consecutive years at the Eureka Dunes Protected Area in Humboldt County, California. Each experiment consisted of three plots subjected to one of three primary treatments: removal of vegetation with a brush rake, removal of vegetation with a brush rake followed by removal of litter and duff with a plough blade or bucket, and removal of vegetation with tractor-pulled chokers. Plots were then subdivided into smaller secondary treatment plots subjected to one of two treatments or a control. Secondary treatments consisted of weedmat placed for a 1- or 2-year duration. The goal of the treatments was to remove and prevent reestablishment of nonnative vegetation, including but not limited to bush lupine; success was measured by percent cover of recolonizing vegetation 1.5 years after treatment ended. In the first experiment, primary treatment (vegetation removal) but not secondary (prevention of reestablishment) resulted in significant differences in cover by the end of the experiment. The brush rake and plough blade treatment was most successful at preventing reestablishment of nonnative vegetation. In the second experiment, secondary treatment (prevention of reestablishment) but not primary (removal of vegetation) resulted in significant differences at the end of the experiment. Re-invasion increased with the amount of time subplots were left uncovered. The difference in the results of the first and second experiments was attributed to variation in rainfall and, to a lesser extent, to localized variation in species composition. Results suggest that mechanical restoration by means of combination of the brush rake and plough blade primary treatment with the 2-year weedmat secondary treatment would be most successful in meeting the dual goals of removal of nonnative vegetation and prevention of its reestablishment.

Notes: We studied two tallgrass prairies and adjacent restoration areas in northeast Kansas to analyze (1) the invasion of native tallgrass prairie species from native prairie source populations into replanted areas; (2) the establishment of planted prairie species five and 35 years after being sown; and (3) the effects of native prairie species on soil organic matter. For the majority of dominant species, composition differed statistically between sampled areas even though seed rain was available from the native tallgrass prairie remnants. Plant community differences were statistically different between each native prairie area and all respective restoration sites according to the Multiple Response Permutation Procedure. In addition, species richness was greatly reduced in replanted areas compared to adjacent native prairie remnants. Soil carbon isotope ratios indicated that the planting of warm-season grasses resulted in substantial replacement of old soil organic matter by the newly replanted grasses but that it did not create substantial increases of soil organic matter beyond replacement. The lack of accumulation reflects a nutrient-poor system (nitrogen-poor in particular), and the relative absence of native or introduced nitrogen-fixing plant species on the replanted areas may be a significant factor. It appears that restoration of the original highly diverse vegetation component of the tallgrass prairie ecosystem, even when aided by seeding and an adjacent prairie seed source, will occur on carbon- and nitrogen-depleted soils only over very long periods of time (perhaps centuries), if at all.

Notes: We examined the effects of deer browsing, fabric mats, and tree shelters on the growth and survival of Quercus rubra (northern red oak) seedlings planted as part of a reforestation project in southcentral Minnesota. Browsing by white-tailed deer occurred on 68.6% of the seedlings growing without tree shelters, whereas only 3.6% of the trees were browsed by mice or voles and 2.5% were browsed by rabbits. Fabric mats, used to control competition from herbaceous plants for roughly half of the seedlings included in the study, had a detrimental effect overall. Seedlings grown with mats had a greater frequency of deer browsing and a greater chance of dying than seedlings grown without mats. Stem height for seedlings browsed by deer was less overall than for nonbrowsed seedlings, although this pattern varied with use of fabric mats and plot location. The use of plastic tree shelters effectively prevented deer browsing and reduced the mortality rate from 34.6% to 3.2%. Our results indicate that fabric mats should not be used in restoration projects with large deer populations. They also suggest that planting seedlings away from existing forest edges and using seedling protection measures such as tree shelters will increase seedling survival and growth in future restoration projects.

Notes: Wetland ecosystems have ecological significance in areas such as natural waste purification and nutrient cycling. Much has been accomplished in determining the floristic and edaphic characteristics of wetlands, but little is known about key structure-function relationships such as mycorrhizae. We collected descriptive data on the plant and mycorrhizal fungal community associated with a moisture gradient along a rehabilitated prairie fen to assist in ongoing restoration efforts (Zimmerman Prairie, Greene County, Ohio). Analysis of soil samples from the prairie fen indicated that significant levels of arbuscular mycorrhizal fungal infection occurred within both the prairie and fen plant species (infection level means ranging from 20% to 47%). Mycorrhizal root infection was significantly correlated with all edaphic factors tested—soil moisture, organic matter, potassium, phosphorus, calcium, magnesium and pH—from May to October 1994. The plant-mycorrhizal data clearly indicate the presence of mycorrhizae in soils that are saturated or even inundated. The functional role of mycorrhizae in wetlands is still unclear, however. For example, is the species interaction we are observing truly mutualistic or of some other form due to environmental conditions? Arbuscular mycorrhizal fungal relationships in all classes of wetland ecosystems need to be studied further if we are to understand their potential role in the ecological restoration of wetland ecosystems.

Notes: Long-term data are often lacking to effectively assess patterns of lake acidification and recovery. Fortunately, paleolimnological techniques can be used to infer past changes in lakewater acidity and related variables by means of biological indicators, such as diatom valves and chrysophyte scales, preserved in 210Pb-dated sediment cores. We summarize paleolimnological data that we have gathered from 36 Sudbury (Ontario) and 20 Adirondack Park (New York) lakes to estimate the magnitude of lake acidification and any subsequent recovery in these lake systems. In both regions, many lakes were shown to have acidified considerably, some over two pH units, since the 1850s. Although some recovery was noted in both lake regions, Sudbury lakes generally showed larger increases in inferred lakewater pH with recent declines in sulfur emissions. Possible explanations of these differences include the greater decrease in sulfate deposition in the Sudbury area, as well as generally longer residence times of lakes in Sudbury, perhaps allowing for more in-lake alkalinity generation. In addition, Sudbury lakes generally had higher pre-industrial pH levels, suggesting that lakes with higher natural buffering capacities are more likely to recover more quickly with declines in deposition, even if they had been acidified to a great extent.

Notes: The concept of critical load is now widely used in the management of acidified waters. In southern Norway, acidification due to long-range transported air pollutants is one of the most serious environmental problems, affecting an area of 80,000 km2. To preserve and restore biodiversity, Norwegian authorities have chosen liming as a temporary mitigation measure. Critical load estimates were used to estimate the material and financial needs for this large-scale program. In 1995, more than 2000 localities ranging from small lakes to large salmon rivers were limed. Liming costs in 1996 were $18 million (U.S.). Critical load estimates also formed the basis for international negotiations on sulfur emission reductions, resulting in the second sulfur protocol for Europe and North America in 1994. The critical load estimates indicated that acidified areas in Norway would be reduced to 35,000 km2 after the year 2010, after the commitments of the sulfur protocols are met. By that time, estimated liming costs would be reduced by almost 40%. Lime treatment of River Tovdalselva, with a catchment area of 1885 km2, is probably the largest integrated liming project in the world. In 1990 the critical load was exceeded in 98% of the Tovdalselva catchment. After the year 2010 the exceeded area may be reduced to 44% and the liming cost by two-thirds.

Notes: Mesocosm experiments were conducted in the summer of 1996 to quantify the effect of bioturbation by Carcinus maenas (the introduced European green crab) on survival of transplanted Zostera marina (eelgrass). The research grew out of a successful 2.52 ha eelgrass transplant project in the Great Bay Estuary of New Hampshire. At several subtidal sites, green crabs were found to damage transplanted eelgrass by cutting the shoots to the extent that some sites demonstrated poor survival. In three separate experiments, eight replicate mesocosm tanks were transplanted with 36 shoots of eelgrass, and different crab densities were introduced into the tanks. The number of shoots damaged by crabs was significantly higher in tanks with moderate (4.0 crabs/m2), high (7.0 crabs/m2), or very high (15.0 crabs/m2) crab densities than in tanks with low (1.0 crabs/m2) crab densities. Up to 39% of viable shoots were lost within one week of exposure to green crab activities. The mesocosm results demonstrated that green crabs were not directly attracted to eelgrass but that they significantly decreased transplant survival through their activity. Field densities of green crabs were found to exceed the density at which most damage occurred in the experiments, suggesting that this introduced species can be a major determinant of eelgrass transplant survival. The results underscore the major influence that biological components of transplant sites can have on transplant survival, and the need for their consideration in the site selection process.

Notes: Designing strategies to manage rare species’ habitats may involve tradeoffs that include negative short-term impacts to achieve positive long-term success. In managing grasslands, fire is a powerful tool to control invasive weeds and stimulate native plant growth, but it may decimate the invertebrate fauna. To rank potential burn strategies for Icaricia icarioides fenderi (Fender's blue butterfly) habitat, we present an empirically based mathematical model. Parameter estimates are based on experiments conducted by Wilson and Clark from 1994 to 1997. Potential strategies include combinations of times between burn (1, 2, 3, 4, or 5 years) and fractions of a habitat to burn in each fire (1/8, 1/4, 1/3, or 1/2), as well as a strategy of never burning. Burning one-third of the habitat every year maximizes the average annual population growth rate, but, based on maximum likelihood parameter estimates, 8 of 21 strategies led to 95% of simulated butterfly populations persisting for 100 years. In simulations based on the parameters’ lower confidence limits, however, there were some cases in which no strategies led to populations persisting 100 years. In this uncertainty analysis—the effect of changes in parameters based on our confidence in them—we also investigated the rank order of the strategies. This uncertainty analysis indicated that the rank order of burning strategies is most sensitive to our confidence in rates of habitat change after a burn (number of “good” years after a fire and time for habitat to return to pre-burn conditions). Surprisingly, however, the rank order of strategies changes little over a wide range of butterfly demographic rates. Better knowledge of rates of habitat change after a burn would improve our ability to make management decisions substantially more than better knowledge of the butterfly's vital rates.

Notes: Knowledge of the recovery of aquatic communities from lake acidification is limited. Data from studies of crustacean zooplankton communities, however, do reveal some of the major mechanisms important in the biological recovery process. Important influences on recovery include factors related to habitat quality and the ability of organisms to colonize. During recovery, existing species and colonists from internal and external sources interact to form a new community. The relative roles of internal and external influences remain poorly understood. Four general community types can be identified as possible outcomes of the recovery process: the original community, a normal alternate community, a community limited by dispersal, and a community limited by biological resistance. Empirical data indicate that, given suitable water quality, about a decade is sufficient to permit substantial recovery of zooplankton communities if there are no severe physical or biological barriers to restructuring.

Notes: Benthic aquatic insects were collected from rocky nearshore areas (〈 1 m deep) of 17 lakes near Sudbury, Ontario, with a pH range of 4.7–7.3 and a size range of less than 10 ha to over 10,000 ha. These insect communities were composed of taxa common to lake soft-sediments and streams. Direct and indirect effects of lake acidity appeared to be major controls on the structure of these communities, implying that several factors may be involved in restructuring during acidification or recovery. Declines in abundances of several taxa of Ephemeroptera at pH below 5.5 were attributable to acid toxicity, while increases in the abundances of Odonata and Diptera at pH below 5.5 were associated with the absence of fish predators and other indirect effects of acidity. The communities of two experimentally neutralized lakes restructured rapidly within 5 years, approaching but not achieving community structures typical of our near-neutral survey lakes. Neutralization led to recolonization or increased abundance of the acid-sensitive mayfly, Stenacron interpunctatum, and the dragonfly, Boyeria grafiana; however, recolonization by other taxa expected to be present in near-neutral lakes (Stenonema femoratum, Eurylophella, and Basiaeshna janata) was not observed. Consistent with results for the survey lakes, declines in the abundances of the dragonflies Aeshna interrupta, Aeshna eremita, and Leucorrhinia glacialis in the neutralized lakes were associated with reintroductions of Salvelinus fontinalis (aurora trout) and increased fish predation pressure, while reduced abundances of the dipterans Ceratopogonidae, Psectrocladius, and Stackelbergina may be related to indirect effects of acidity other than fish predation. Although community composition varied greatly across the acidity gradient, total species richness and abundance were not correlated with lake chemistry or number of fish species.

Notes: Growth and maturation of transplanted salt marshes is often limited by the availability of nitrogen (N). We examined the role of N2-fixing benthic microbial assemblages (microalgae and associated bacteria) in two restored marshes (1-year-old and 6-year-old marsh) and a natural salt marsh in the Newport River Estuary, North Carolina. Benthic N2 fixation (nitrogenase activity, NA), chlorophyll a (Chl a) concentration, Spartina alterniflora (smooth cordgrass) stem counts, and sediment organic matter content were determined in the three marshes. Significant differences were observed between sites for both Chl a and NA. The 1-year-old marsh always exhibited the highest levels of NA and Chl a. Sediment organic matter content was lowest in the 1-year-old marsh (∼2%), intermediate in the 6-year-old marsh (∼5%), and highest in the natural marsh (∼10%). Carbon and nitrogen analyses were also performed on the 1-year-old marsh sediments, which were depleted in N. A positive correlation was observed between surface sediment N and Chl a. Remineralized, microbially derived N may provide growth-limiting inorganic N to Spartina transplants. N2-fixing microbial assemblages in the 1-year-old marsh may also be an important food source for marsh infauna. Benthic N2-fixing microbial assemblages play a key role in the N economy of restored salt marshes.

Notes: Reintroduction programs are a high-risk conservation strategy for restoring populations of endangered species. The success of these programs often depends on the ability to identify suitable habitat within the species’ former range. Bioclimatic analysis offers an empirical, explicit, robust, and repeatable method to analyze large areas rapidly using a small number of locality records, and in turn predicting (and/or reconstructing) its potential distribution limits. This approach therefore can estimate the broad limits of the distribution of a taxon, using data that may be inadequate for standard forms of statistical analysis. We illustrate the potential value of bioclimatic modeling for reintroduction biology using a case study of the highly endangered Helmeted Honeyeater (Lichenostomus melanops cassidix) from Victoria, southeastern Australia. The results of our analyses assisted us to both predict the former range limits of the Helmeted Honeyeater and determine the broad limits of those areas that may contain potentially suitable sites for future reintroduction programs for the subspecies. The analysis predicted that the range of the Helmeted Honeyeater extends from the Yarra River district east of Melbourne, south to the Western Port Bay and east as far as the Morwell area of Victoria. The climatic characteristics of habitat occupied by the extant population of the Helmeted Honeyeater were found to be unique within its predicted range. We recommend that reintroduction efforts therefore be concentrated within this small area, as has occurred to date.

Notes: In lower-montane ecosystems of Ecuador, Setaria sphacelata (foxtail grass), the predominant introduced pasture species, forms a tussock grassland that reduces soil nitrogen and resists recolonization of forest vegetation. We compared the influence of individual trees or small clusters of nitrogen-fixing (Inga sp., Fabaceae) and non-nitrogen-fixing trees (Psidium guajava L., guava) on the soil and abiotic conditions that affect further regeneration of forest vegetation within pastures. Pasture trees ameliorated air temperature and light intensity to levels similar to those in adjacent intact forest. Beneath Inga, soil NO3−-N was four times higher than in open pasture. Nitrification was five times higher under Inga canopies than in open pastures for both field and laboratory incubations. This suggests that the increased soil N transformations under Inga are derived mainly from improved soil rather than microenvironmental conditions. Psidium canopies slightly increased field nitrification but had no effect under laboratory conditions. We also compared the natural abundance 13C signature and the carbon and nitrogen content of subcanopy soil with adjacent open pasture soil. Inga increased the C and N content of the upper 5 cm of soil and increased by 7% the fraction of soil organic matter derived from C3 plants. The improved soil and abiotic conditions beneath the canopies of N-fixing pasture trees favor the establishment and growth of woody montane species, suggesting that these trees could be used to accelerate forest regeneration within abandoned pastures.

Notes: To understand landslide regeneration and provide information necessary for restoration, we sampled seed rain, seed pool, and plant cover on two Ecuadorian landslides. We trapped 1304 seeds and found that, while most seeds were in the family Asteraceae, there was substantial variation in seed rain among plant families. Four hundred and seventy-five seedlings emerged from soil samples, including nonvascular and vascular families; again, species in Asteraceae dominated, with species in Piperaceae also very common. Plant cover, consisting of members of four fern families and 20 vascular plant families—with species in Asteraceae, Melastomataceae and Poaceae most common—was scored as a percentage of the total plot area. Principal components analysis (PCA) showed that, for all three of these plant life stages (seed rain, seed-propagule pool, plant cover), spatial variation was dominated by differences between the two landslides rather than within-landslide plot differences. PCA also showed that plots separated best on axes defined by the families Cecropiaceae, Urticaceae, Melastomataceae, Papilionaceae, Asteraceae, and Araceae with clumping of families in PCA space suggesting common successional strategies. Another multivariate technique, canonical correspondence analysis (CCA), showed that the combined seed rain and seed pool data could predict the percent cover of the family Verbenaceae and that the current plant cover families could predict Asteraceae seeds and seedlings. Finally, we use our past and present landslide data, along with multivariate modeling results, to suggest strategies for successful landslide restoration.

Notes: The effectiveness of organic mulch as a simple means of enhancing the restoration of disturbed lands by providing a competitive edge to native perennials, such as Artemisia californica (California sagebrush), over exotic annuals, such as Avena fatua (wild oat), was studied by investigating the effect of organic amendments on microbial activity and nitrogen immobilization through both soil analysis and aboveground plant growth. The addition of organic amendment resulted in an increase in microbial activity, a parallel increase in nitrogen immobilization, and no significant differences in total soil nitrogen. It is likely that nitrogen was gradually being removed from its more available form of nitrate and being immobilized in the tissues of the increasing microbial biomass. The survival rate of planted native perennial seedlings of A. californica in organic amended plots was almost double that of control-plot seedlings, and plant volume was significantly higher. When the availability of nitrogen was reduced through increased immobilization, amended plots established an environment more conducive to native perennial shrubs, allowing them to outcompete exotic annuals for water and nutrients. This simple procedure could have major implications for enhancing the restoration of disturbed lands.

Notes: Nearly all mountain lakes in the western United States were historically fishless, but most now contain introduced trout populations. As a result of the impacts of these introductions on ecosystem structure and function, there is increasing interest in restoring some lakes to a fishless condition. To date, however, the only effective method of fish eradication is the application of rotenone, a pesticide that is also toxic to nontarget native species. The objective of this study was to assess the effectiveness of intensive gill netting in eradicating the trout population from a small subalpine lake in the Sierra Nevada, California. We removed the resident trout population and a second trout population accidentally stocked into the study lake within 18 and 15 gill net sets, respectively. Adult trout were highly vulnerable to gill nets, but younger fish were not readily captured until they reached approximately 110 mm. To determine the utility of gill netting as a fish eradication technique in other Sierra Nevada lakes, we used morphometry data from 330 Sierra Nevada lakes to determine what proportion had characteristics similar to the study lake (i.e., small, isolated lakes with little spawning habitat). We estimated that gill netting would be a viable eradication method in 15–20% of the high mountain lakes in the Sierra Nevada. We conclude that although gill netting is likely to be more expensive and time consuming than rotenone application, it is a viable alternative under some conditions and should be the method of choice when sensitive native species are present.

Notes: Mineral extraction activities in the Arctic regions of the world produce long-lasting ecological disturbances. Assisted recovery from such disturbances may require restoration of the tundra thermal regime. We transplanted plugs of entire root zone and live tundra plants to a disturbed site in Alaska oil fields. The dominant species were Carex aquatilis, Eriophorum angustifolium, Dupontia fisheri, Poa glauca, Festuca rubra, Salix ovalifolia, S. reticulata, and Sphagnum spp. We studied plant responses in the plugs to thermal regime manipulations by means of greenhouse and of single- or double-plug treatments. All plugs continued to produce new plants with time and expanded in area and canopy volume. Plants responded differently to treatments and generally reversed those responses when we reversed the greenhouse treatment the third year after transplant. Our small-scale experiment showed that the native thermal regime of a plant community is vital in revegetating a disturbed tundra. But large-scale restoration using transplants requires resources of modern extraction technology, engineering, and planning to salvage the extensive live tundra mats now routinely destroyed under gravel fills of roads, structures, and mine-site stockpiles.

Notes: The ataxonomic phytoplankton composition and abundance (biomass classes) in lakes of differing acidity were examined by flow cytometry. The ataxonomic parameters applied here were photosynthesis pigments and cellular protein content. Up to 1000 cells per second can be assessed by this method. Consequently, enough cells to create biomass spectra can be counted within only a few minutes. Photosynthesis pigment autofluorescence was used to separate algal cells from detritus and to classify the phytoplankton organisms into different pigment groups. Chlorophyll fluorescence ratio (CFR) at different excitation wave lengths proved to be a sensitive tool. As expected, the diversity of CFR-determined pigment groups decreased with increasing acidification. Some groups were acid-insensitive and occurred even at pH below 3.0. However, picoplanktic cyanobacteria (Synechoccocus/Synechocystis-like particles [SLPs]) were absent at pHs below 4.5–4.0, with their accompanying high metal concentrations. Thus, the reappearance of cyanobacterial picoplankton may serve as a first major restoration goal in strongly acidified lakes. Protein staining using fluorescein isothiocyanate enables fast estimates of phytoplankton biomass and establishment of biomass spectra as an estimate of the integrity of plankton communities. Although the phytoplankton investigations presented are only snapshots of the situation on the sampling days, the feasibility of flow cytometrical methods for preparing phytoplankton biomass spectra has been demonstrated. The completeness of such biomass spectra, such as the presence or absence of SLP-picoplankters, as well as the variances around regression lines (linear or parabolic), may serve as goals in restoring lakes acidified to different degrees.

Notes: A large-scale liming program was initiated in Sweden in the mid-1970s to ameliorate the effects of acidification and to restore acidified lakes and rivers to conditions present before acidification. I compared the fish fauna composition and biomass among limed, acidified, and consistently circumneutral lakes. The study focused on (1) recolonization and fish assemblage development and (2) fish biomass and assemblage composition. Time-series data from 14 limed, 5 acid, and 10 circumneutral lakes, and a data set of 323 limed and 346 nonlimed circumneutral lakes, are analyzed. Long-term monitoring revealed that fish species richness increased after liming and was of the same magnitude in limed lakes as in nonlimed circumneutral lakes after 10–20 years. Species proportions differed between limed and nonlimed lakes, and the relative biomass of Perca fluviatilis (Eurasian perch) was in general higher, whereas cyprinid and pelagic species were less important in the limed lakes than in the nonlimed circumneutral lakes. Recolonization of fish species was found to be a crucial factor for the development of the fish assemblages after liming, and in several limed lakes the reintroduction of fish species has accelerated the recovery of pre-acidification fish assemblages.

Notes: A single fall-season prescribed burn and two consecutive fall-season prescribed burns were conducted in 1988 and 1989 to quantify the effects of fire intensity and frequency on woody species in two Willamette Valley wet prairies. Fuel biomass, fuel properties, fire behavior, biomass consumption, and changes in woody species density and height were documented before and after the burns. Before burning, Rosa nutkana (Nootka rose) was the most abundant woody species at both sites. In 1988, burns were significantly more intense, produced more heat per unit area at Fisher Butte than at Rose Prairie, and were fairly complete, with 37% and 35% woody biomass consumption, respectively. In 1989, burns were spotty; only 11% of woody biomass was consumed at Fisher Butte. The one-burn treatment did not affect R. nutkana density in 1 × 15 m transects at either site; the two-burn treatment significantly increased its density in three of the five communities sampled. Burning significantly reduced height growth by eliminating taller individuals while stimulating sprouts from belowground tissues. Redistribution of R. nutkana and trees into shorter height classes indicates the possibility of reducing the visual dominance of woody species if periodic burns were conducted in native Willamette Valley prairie. The low-intensity burns in this study were sufficient to remove the woody shoots under 3 m tall but did not reduce their capacity to regenerate and did not reduce the density of woody species. After decades of fire suppression, two burns were not sufficient to reestablish the desired balance between native herbaceous and woody species or to reduce the presence of introduced woody species. A long-term commitment to the reintroduction of fire as a management tool will be required to maintain native wetland prairies in the Willamette Valley.

Notes: Seed rain was studied during one growing season in an intact high-alpine grassland and in a downhill ski run that had been machine-graded about 26 years earlier. The study plots were located at about 2500 meters above sea level. The number of trapped seeds per square meter in the grassland was significantly larger than in the ski run (930 versus 96, respectively). Alpha diversity in seed rain was about two times higher in the grassland than in the ski run. Seed rain was primarily influenced by site conditions but also varied in space and time, and both the actual number of diaspores and the species composition changed throughout the growing season. Most of the species found in the seed rain occurred in the standing vegetation, but some clearly arrived from outside. Seed rain in both sites was dominated by a few species, and no clear relationships were found between the species abundance in standing vegetation and in the seed rain. The results of this preliminary study contribute to a better understanding of plant behavior in high-alpine sites and will be helpful in planning and implementing restoration work above the timberline.

Notes: Decomposed granite (DG) is often difficult to revegetate because of its low nitrogen (N) content and poor physical properties. Use of soluble fertilizers on DG sites increases plant-available N but may encourage invasive annuals to grow rapidly and exclude perennial species. This study evaluates the effect of N availability on two potential DG revegetation species: an invasive, exotic annual grass (Bromus mollis) and a native perennial grass (Elymus glaucus). Plants in 10-L pots filled with DG were irrigated with all essential elements except N, which was provided in treatments ranging from zero to 1000 μM NO3-N. Shoot biomass and root distribution were measured in monocultures and in mixtures of annual and perennial plants, both when the two species were seeded simultaneously and when the perennials were seeded 50 days prior to the annuals. At the higher N treatments, growth of annuals exceeded that of perennials. At solution N concentrations lower then the 50–100 μM treatments, however, growth of the perennial grasses equaled or exceeded that of the annuals. When seeded simultaneously, both species showed reduced biomass in mixtures to an extent similar to that when each species grew alone. When the perennials were already established, the biomass of annuals was reduced proportionately more than that of perennials, even at the highest N treatment. At low and medium N treatments, root placement of the perennial was deeper than that of the annual. At high N treatments, however, root distribution of both species was similar, and the deep rooting characteristic of the perennial was no longer observed.

Notes: From 1992 to 1995 we experimentally evaluated the effectiveness of several revegetation treatments along a segment of Going-to-the-Sun Highway in Glacier National Park, U.S.A. This segment, reconstructed during the spring and summer of 1992, is bordered by fescue prairie vegetation and is known to be susceptible to invasion by several alien species, including Centaurea maculosa (spotted knapweed) and Phleum pratense (common timothy). We used a split plot study design to evaluate the effectiveness of herbicide and seeding treatments on assisting recovery of native flora and limiting the establishment of alien species. The herbicide treatment consisted of a yearly herbicide spray application of clopyralid (3,6-dichloropicolinic acid). Five seeding treatments were evaluated, three of which included an indigenous graminoid-forb seed mix. Percent canopy coverages of four species groups—alien graminoids, native graminoids, alien forbs, and native forbs—were determined in July 1995. In addition, community-level patterns in sprayed plots and unsprayed plots were compared with a reference site of native fescue prairie. Herbicide treatments decreased mean canopy coverage of alien forbs (treated = 4.2%, untreated = 23.4%) and increased mean canopy coverage of native graminoids slightly (treated = 6.3%, untreated = 4.0%). But herbicide treatments reduced mean coverage of native forbs (treated = 3.9%, untreated = 8.9%) and likely increased coverage of alien graminoids. Treatments that included a fall 1992 seed mix increased native graminoid coverages 2.8–4.6 times, although coverages were still lower than those attained by alien graminoids. Native and alien forb coverage appeared unaffected by seeding treatments. Species composition was less diverse in sprayed plots and more dominated by alien grasses than in unsprayed plots and the reference site. Areas for additional study are suggested, including seed bank assays to determine treatment effects on recruitment of alien versus native species and the use of native graminoids to create low-diversity communities with high canopy coverages to resist establishment of alien species.

Notes: We assessed the vertical growth and mycorrhizal infection of woody plant roots on a closed landfill, using tree and shrub clusters that had been previously installed in patches of increasing size to establish protocols for woodland restoration. The density of the fine roots of shrubs, which had poor-to-moderate mycorrhizal infection, decreased strongly with increasing depth. Oak (Quercus) seedlings planted within and outside patches were assessed for ectomycorrhizal infection. Oak root systems were mycorrhizal, but root-tip proliferation was improved and ectomycorrhizal composition was influenced by woody debris in the mineral soil. Most surviving oaks were found within patches, but all seedlings showed poor growth: most taproots were deflected horizontally above the boundary between surface soil and subsoil layers (〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:10612971:REC6310:ges" location="ges.gif"/〉−15 cm). Abrupt decreases in pH between surface and subsurface horizons (6.9 versus 5.3), together with poor drainage and aeration of the latter soil, were probably responsible for poor root growth. Root growth of greenhouse-grown pine and maple seedlings was similarly restricted in pots packed with topsoil over subsoil material. Our results suggest that many current specifications for the cover of closed landfills will not permit restoration of native woody plant communities because of physical limitations to root growth and infectivity. The structure of the engineered soil must address basic plant growth requirements as well as traditional concerns of drainage and barrier protection.

Notes: I discuss ecological and cultural restoration within the broader context of the critical transition period from the fossil fuel age to the post-industrial global information age. In this cultural evolutionary process, the restoration of natural and cultural landscapes should play a vital role. For this purpose, it has to be guided by a holistic and transdisciplinary systems approach, aiming not only at the organismic but also at the functional and structural restoration of ecological and cultural diversity as total landscape ecodiversity. For the development of suitable restoration strategies, a clear distinction has to be made between different functional classes of natural and cultural solar-powered biosphere and fossil-powered technosphere landscapes, according to their inputs and throughputs of energy and materials, their organisms, their control by natural or human information, their internal self-organization and their regenerative capacities. Not only technosphere landscapes but also intensive agro-industrial landscapes have lost these capacities and are heavily subsidized by fossil energy and chemicals, to the detriment of the environment and human health. They therefore have to be rehabilitated by more sustainable but not less productive agricultural systems based on organic farming. But their natural regenerative capacities can be restored only by regenerative systems, with the help of cultural “neotechnic” information. The promise for an urgently required evolutionary symbiosis between human society and nature in a sustainable post-industrial total human ecosystem lies in the functional integration of such innovative regenerative systems and all natural and cultural biosphere landscapes with healthier and more livable technosphere landscapes. To this goal, ecological and cultural landscape restoration can make an important contribution.

Notes: Large areas of the world's tropical forests are being degraded, with a consequent loss of species diversity. Only some of these are able to recover unaided. Where attempts are being made to restore such forests, the scale of the attempts is usually small. Timber plantations are one of the few means by which large areas of cleared or degraded landscape can be reforested. These usually restore the productive capacity of the landscape but do little to recover biological diversity. But a number of approaches might be used to redesign such plantations so that they would both yield the timber needed to justify the investment and also contain some proportion of their former biodiversity. These approaches include using indigenous species rather than exotic species, creating species mosaics by matching species to particular sites, embedding the plantation monocultures in a matrix of intact or restored vegetation, using species mixtures rather than monocultures, or encouraging the diverse plant understories that can often develop beneath plantations. The degree of ecological restoration possible using these alternatives ranges from modest to significant, although none is likely to achieve complete restoration. Any improvements in regional biodiversity brought about by these changes might be seen as being at the expense of plantation productivity. But many of these approaches have advantages other than just restoring former biodiversity. I discuss some of the trade-offs and implications for plantation managers and restorationists.

Notes: Species richness and sporocarp density of ectomycorrhizal fungi in stands of Pinus sylvestris (Scots pine) in The Netherlands have decreased during the last decades. The lowest species diversity was found in P. sylvestris stands situated in areas with high atmospheric deposition of nitrogen originating from intensive livestock industry. In these stands, litter and humus have accumulated into thick layers, and the herbaceous understory vegetation is dominated by the grass Deschampsia flexuosa (wavy hair grass). Earlier investigations showed negative correlations between the number of species of ectomycorrhizal fungi above ground and the depth of humus layers. Our aim was to investigate whether removal of litter, humus layers, and herbaceous vegetation (sods)—so-called “sod cutting”—increased species diversity above ground and sporocarp density of ectomycorrhizal fungi in P. sylvestris stands of different age. Therefore, three P. sylvestris stands of different ages (planted in 1987, 1963, and 1924) on Haplic Arenosol were selected. In 1990, litter, humus layers, and herbaceous vegetation were removed to create nutrient-poor sandy soils without overlying litter and humus layers. Untreated plots served as controls. Surveys conducted in 1991, 1992, and 1993 indicated that sod cutting enhanced the species diversity and sporocarp density of ectomycorrhizal fungi. These results suggest that sod cutting is a way to restore ectomycorrhizal flora in medium-aged and old stands of P. sylvestris where litter and humus have accumulated.

Notes: Lack of seed dispersal has been shown to be a major factor limiting tropical forest recovery in abandoned pasture land. The goal of this work was to determine whether bird perching structures serve to enhance seed dispersal and seedling establishment in an abandoned pasture in Costa Rica. Two types of perching structures (crossbar and branch) were tested. Bird visitation rates were significantly higher on branch than on crossbar perches. The number of animal-dispersed seeds was significantly higher below branch perches than below crossbar perches or in open pasture. Despite differences in seed rain, percent cover of animal-dispersed plants and the number of seedlings of animal-dispersed plant species were similar below both perch types and in open pasture. Baiting perches with bananas did not increase either bird visitation rates or seed rain. These results suggest that, although bird perching structures increase seed dispersal, they do not overcome other barriers to tropical forest recovery such as seed predation and low seed germination.

Notes: Experimental restoration of Halodule wrightii (shoalgrass) to its former range on Galveston Island, Texas, began in April 1994. We tested the effects of site, planting density, water depth, and fertilizer addition on survival and growth through June 1996. Temperature, salinity, light transmittance, turbidity, and sediment properties at two restoration sites, Redfish Cove and Snake Island Cove, were similar to those in naturally occurring grassbeds in nearby Christmas Bay. Halodule survival, coverage, and new shoot densities were affected by site (significantly higher at Redfish Cove than at Snake Island Cove, which eventually failed), by planting density (significantly higher when planted on 0.25-m or 0.5-m centers rather than on 1.0-m centers), and by water depth (significantly higher when planted in relatively shallow water). Propagation (spreading from transplant units) was significantly greater from 0.25-m or 0.5-m center plantings but was not consistently affected by site or water depth. Fertilizer enhanced propagation but not survival. After two years, Redfish Cove produced belowground biomass similar to that observed in Christmas Bay, but aboveground biomass remained significantly less. Snake Island Cove plant mortality in September 1995 may have been presaged by low root-rhizome carbohydrate levels observed in October 1994, but causes remain unknown. Further restoration of Halodule to Galveston Bay is possible at selected sites, but structural equivalency will take longer than two growing seasons to achieve.

Notes: Aristida beyrichiana (wiregrass) is increasingly being planted in restoration projects across the southeastern coastal plain, with little focus on genetic differences among populations across the region. Local and regional population differentiation for establishment and growth traits were examined in common garden and reciprocal transplant experiments. Seeds from up to 20 plants from each of seven populations were collected in northern and central Florida sites that encompassed gradients of soils, hydrology, and temperature. Reciprocal seed transplants using three of the common garden populations were conducted in two consecutive years. In the common garden, significant population differences were seen in seed weight, seedling emergence and survival, tiller height, number of tillers, the relationship between tiller number and tiller height, and flowering. Variation among maternal families was seen in tiller number and in the relationship between tiller number and tiller height. The reciprocal transplant study did not detect either local adaptation to sites of origin or consistent superiority of one source population or planting site in seedling establishment. These results suggest that the probability of seedling establishment is primarily dependent on environmental conditions rather than genetic differences. Genetic variation for traits related to fitness (e.g., tiller number) may be retained within populations because phenotypically plastic growth responses of seedlings to environmental variation buffer genetic variation against the action of selection. But despite the lack of evidence for genetic influences on initial establishment in wiregrass, our common garden study suggests genetic differences among populations. This result, when combined with previous results indicating local adaptation in later life stages of wiregrass, suggests that restoration efforts involving this species should use local seed sources from sites with similar soil and hydrological conditions.

Notes: In France the alteration of species-rich grasslands is due mainly to agriculture and civil engineering projects (roadways, dams, ski resorts, etc.). The restoration of such ecosystems has been tested in many regions and after many sorts of degradation. According to the level of initial degradation, different responses can be envisioned. Some variations are presented of a general model of restoration and rehabilitation: (1) rejuvenation of fallow land by grazing with rustic animal breeds in wetlands or chalk grasslands in south and northwestern France, (2) restoration by recovery of extensive agricultural management in intensive agricultural areas such as the Rhône or Meuse Valleys, and (3) rehabilitation by appropriate ecological engineering carried out in grasslands degraded by intensification, ski-track, or civil engineering installations. Despite some positive results, these undertakings have not always had the expected effects and therefore should not provide alibis for the destruction of natural grasslands. In general, the success of such undertakings depends on the maintenance in the surrounding areas of protected seed source reservoirs and on the persistence of a diversified landscape pattern permitting connectivity between these seed sources and the restoration or rehabilitation sites.

Notes: The postmining rehabilitation of coastal sand dunes north of Richards Bay (28°43′S, 32°12′E), South Africa, is resulting in the development of a series of known-aged stands of vegetation dominated by Acacia karroo (sweet thorn). Other broad-leaved species are establishing themselves in rehabilitating areas more than 12 years of age. Soils from rehabilitating stands 3–5, 9–12, 13–15, and 16–18 years of age, from two disturbed, unmined stands 30 and 58 years of age, and from a mature unmined stand were examined to assess age-related trends in selected soil properties. Individually, these stands represent a series of different developmental stages of a coastal dune successional sere. Soil organic material, percentage organic carbon and concentrations of sodium, potassium, magnesium, calcium, and nitrogen increased with an increase in habitat regeneration age. Concentrations of most of these elements were lower than those recorded on the 58-year-old unmined and mature unmined stands. Multivariate analyses suggest, however, that the similarity of these values for rehabilitating stands to those for the unmined stands increased with an increase in regeneration age. The growth response of Raphanus sativus (radish) plants, based on mass attained under experimental growing conditions in soil collected from these stands, suggests an increase in soil fertility with an increase in regeneration age.

Notes: We studied the ecological effects of the invasion of coastal dunes by Lupinus arboreus (yellow bush lupine), an introduced species, and used the results to develop manual restoration techniques on the North Spit of Humboldt Bay. Vegetation and soil data were collected in five vegetation types representing points along a continuum of bush lupine's invasive influence. We collected data on the number and size of shrubs, vegetation cover, and soil nutrients. One set of plots was subjected to two restoration treatments: removal of lupine shrubs only, or removal of all nonnative vegetation and removal of litter and duff. Treatments were repeated annually for four years, and emerging lupine seedlings were monitored for three years. Prior to treatment, ammonium and nitrate were found to increase along the lupine continuum, but organic matter decreased at the extreme lupine end. Yellow bush lupine was not the most significant variable affecting variation in soil nutrients. After four years, nonnative grasses, including Vulpia bromoides, Holcus lanatus (velvet grass), Bromus spp. (brome), and Aira spp. (European hairgrass), were significantly reduced in those restoration plots from which litter and duff was removed. Native species increased significantly in vegetation types that were less influenced by lupine. By the third year, soil variables differed among vegetation types but not by treatment. Bush lupine seedling emergence was higher, however, in plots receiving the litter and duff removal treatment. Based on these results, we conclude that bush lupine invasion results in both direct soil enrichment and indirect enrichment as a result of the associated encroachment of other nonnative species, particularly grasses. Although treatment did not affect soil nutrients during the period of this study, it did reduce establishment of nonnative grasses and recruitment of new bush lupine seedlings. Restoration should therefore include litter and duff removal. In areas that are heavily influenced by lupine and contain few native propagules, revegetation is also required.

Notes: A monitoring program was established on San Antonio Terrace at Vandenberg Air Force Base to compare vegetation development at two created wetland sites and six nearby natural wetlands. The reference wetlands were chosen to represent a range of habitats in dune swale wetlands on the Terrace. Vegetation in the reference wetland plant communities varies from low-growing herbaceous marsh species with open canopies to closed canopies dominated by shrub or tree species. Transects and plots for long-term vegetation monitoring were established in all the wetlands, stratified by plant communities in the reference wetlands and by geomorphic location in the newly created wetlands. Quantitative vegetation and environmental data were collected at all the sites; measures included species distributions, species cover, and topographical elevations. Over the first three years of monitoring, variations in groundwater depth at different geomorphic locations in the created wetlands resulted in a variety of physical conditions for plant growth. In the first year, more than 100 plant species were observed, the majority being natives. During the next two years, species richness at the created wetland sites remained relatively stable and was higher than at the reference sites. Statistical comparisons of vegetation parameters by analysis of variance and hierarchical clustering exhibited patterns of increasing similarity between the created and reference wetlands. Long-term monitoring will be continued to track the progress of vegetation at the created sites, and to assess their development relative to the reference wetlands.

Notes: Follow-up studies after whole-ecosystem-stress experiments can provide important insights into the recovery process itself and into basic ecosystem properties. We report here on zooplankton community recovery during the first 5 years following the experimental acidification of Little Rock Lake, Wisconsin, U.S.A. Acidity in the lake’s treatment basin returned quickly to near pre-manipulation levels. Zooplankton population shifts, however, did not support our hypothesis that species that had increased in abundance with acidification would persist and resist the return of the pre-manipulation community. The three species that had proliferated most dramatically under low pH conditions—Daphnia catawba, Tropocyclops extensus, and Keratella taurocephala, returned close to their originally low, pre-acidification population levels during the early stages of acid recovery. Some species that had been reduced during low pH conditions, such as Diaptomus minutus and Daphnia dubia, did not recover to pre-manipulation levels. Overall, the zooplankton community in the treatment basin exhibits little similarity to that in the reference basin, a condition quite different from that which had occurred prior to the imposition of acid stress.

Notes: This review of long-term monitoring and experimental manipulation studies from Ontario, Canada, offers encouraging evidence that lake trout (Salvelinus namaycush) populations can be restored to acidified lakes once the problem of acid deposition is solved in North America. Predicting the rate of chemical and biological recovery remains difficult, because there are several limnological attributes (dissolved organic carbon, flushing rate) and biological attributes (acid sensitivity, species longevity, fish community composition) of lake trout ecosystems that can either enhance or delay the recovery process. Some options for managing recovering lakes are also reviewed, including increased pollution controls, liming, hatchery stocking, and harvest controls.

Notes: We investigated the effects of prescribed fire, herbicide treatment, and sod removal on the eradication of exotic grasses and the establishment of native plant species in 24 experimental restoration plots in three razed residential sites within the boundary of Indiana Dunes National Lakeshore. During 1992–1995, herbicide treatment and sod removal decreased the combined cover of Poa pratensis (Kentucky blue grass) and Agropyron repens (quackgrass) significantly (from 82% to 13%, and 85% to 8%, respectively), whereas fire did not suppress such exotic lawn grasses. In 1993, several opportunistic species, represented by Cyperus spp. (umbrella sedges), Digitaria sanguinalis (crab grass), and Ambrosia artemisiifolia (common ragweed), filled the gaps left by the removal of lawn grasses. For the same period, Detrended Correspondence Analysis revealed a clear vegetation divergence between the control-fire plots and the herbicide-sod removal plots. While Poa pratensis and Agropyron repens continued to dominate the control and fire plots, the planted native species, represented by Schizachyrium scoparium (little blue-stem), Sorghastrum nutans (Indian grass), Rudbeckia hirta (black-eyed Susan), and Monarda punctata (horsemint), began to dominate in the herbicide and sod removal plots from 1994. In both herbicide and sod removal plots, the ground cover of grasses (68%) was much higher than the forbs (10%). The herbicide plots, where exotic species were removed but nitrogen-rich top soils were not removed, showed a higher diversity of planted native species than the sod removal plots (where both exotic species and top soils were removed) and the control-fire plots (where neither was removed). This finding suggests that an optimum but not excessive concentration of soil nitrogen is needed to support a maximum species diversity in such infertile substrate as sandy soil. In addition, the decrease in potassium in all plots, regardless of treatment, suggests that potassium may become a limiting factor for our restored native vegetation.

Notes: “The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music.” Lewis Thomas3Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.

Notes: Pneumocystis carinii, a major opportunistic lung pathogen of AIDS patients, is found in a number of mammals and is proposed to be a member of the fungi. In this work, several members of the highly conserved HSP70 multigene family were characterized from rat-derived P. carinii. Previously, we reported characterization of the ER resident HSP70 homolog known as BiP from prototype (P. c. carinii) and variant (P. c. rattus) strains of the organism. We report here, from P. c. carinii, characterization of Pcsal, an HSP70 homolog that encodes a cognate/stress-induced HSP70 homolog of the SSA subfamily in Saccharomyces cerevisiae. We also identify from both rat strains and from a human isolate of P. carinii (P.c. hominis), a third set of HSP70 homologs that apparently encode a ribosome-associated cytoplasmic HSP70 homologous to the S. cerevisiae SSB subfamily. Our data indicate that Pcsal mRNA, like Pcbip mRNA, bears an intron in the 5′ untranslated region, is induced by heat shock, and suggest that this gene undergoes alternative transcription and splicing. The SSB homologs display significant sequence heterogeneity between P. carinii source strains, supporting the genetic divergence and likely speciation of P. carinii isolates within and between host species. Phylogenetic analysis with the PcSAl protein supports inclusion of P. carinii among the higher fungi.

Notes: Diffusion theory can completely describe the movement of a ciliate along a track of a certain length (L), travelled in a time (t), and with the extremes lying at a distance D. Three important descriptors of this behavior are: (1) the kinetic index (Ik= L/t), namely the average velocity in μm/s, which expresses the state of the “accelerator” of the ciliate; (2) the geometric index (Ig= D/L) measuring the straightness of the track by a dimensionless number. 0 ≤ Ig≤ 1, which expresses the state of the “steering wheel” and represents a sort of “directional efficiency”; and (3) the displacement rate (Rd= D/t), integrating the first two indices and expressing the combined effect of the “accelerator” and the “steering wheel” of the organism with a unique measure (in μm/s), which defines the average displacement rate or the effectiveness of the track in displacing the organism in space. A weighted estimate of general mobility is given by the mobility rate [Rmo= (R̄d.f)creeping- (R̄d.f)swimming], obtained by multiplying the average Rd of the creeping organisms and the average Rd of the swimming organisms by their relative frequencies of occurrence (f), and adding the two products. Values for experimental populations of Oxytricha bifaria (Ciliata, Hypotrichida) maintained at 24, 19, 14, and 9° C demonstrated both the appropriateness and the usefulness of these indices and rates to describe the tracks a posteriori, and to provide measures to reason about their possible adaptive significance.

Notes: The diversity of symbiotic dinoflagellates (SD) from seven coral species (Fungia scutaria, Fungia paumotensis, Lep-tastrea transversa, Pavona cactus, Pocillopora verrucosa, Montastrea curia, and Acropora fonnosa) was studied in a restricted geographical area, the Lagoon of Arue on the island of Tahiti. Their diversity was explored by small subunit ribosomal RNA gene (SSU rDNA) restriction fragment length polymorphism (RFLP). After a nested amplification with SD specific primers, RFLP analyses were performed directly and after a cloning step. The diversity of these different SSU rDNA was estimated in respect to possible technical artifacts. In an axenic culture of SD from the coral Galaxea fascicularis, both heterogeneous SSU rDNAs and artifact molecules were observed as in our SD samples. According to the number of patterns observed, corals Fungia paumotensis, Leptastrea transversa. Pavona cactus, Montastrea curia, and Acropora fonnosa contained one class of SD SSU rDNAs. whereas Fungia scutaria and Pocillopora verrucosa contained three and two classes of SD SSU rDNAs respectively. In the limited geographic area studied. SD from different coral species shared the same pattern, except SD from Montastrea curta, which showed a unique pattern. In addition to the possibility of SD flux among different coral species, specific mechanisms could also be involved in the establishment of a symbiosis.

Notes: We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris, opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70-90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30-45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.

Notes: Morphological features of the rumen ciliate Ostracodinium gracile (Dogiel, 1925) are described from pyridinated silver carbonate-impregnated specimens. Ostracodinium gracile has a characteristic arrangement of infraciliary bands not present in other ophryoscolecid ciliates. Buccal infraciliature is composed of three polybrachykineties. The adoral polybrachykinety does not completely encircle the circumference of the vestibular opening, but arches ventrally from its right to left side. The dorsoadoral polybrachykinety extends laterally along the dorsal side of the vestibular opening. The vestibular polybrachykinety extends along the dorsal wall of the long tubular vestibulum. Dorsal infraciliature consists of the dorsal polybrachykinety that extends laterally along the dorsal side of the body. During binary fission, four primordia, that is ventral, right, left, and dorsal primordia, form in the stomatogenic field and develop into the adoral, dorsoadoral, vestibular, and dorsal polybrachykineties of the opisthe. respectively.

Notes: The phylogenetic diversity of parabasalian flagellates from termite hindguts has been examined by small subunit ribosomal RNA (rRNA) amplification and sequencing. Two species of particular interest, the giant trichomonad Pseudotrypanosoma giganteum and the hypermastigote Trichonympha magna, were isolated from the gut of Porotermes adamsoni by micropipetting. and the rRNA genes from these small populations amplified and sequenced. rRNA genes representing Hypermastigida and the Trichomonadida families Devescovinidae and Trichomonadidae. were also recovered by amplification from whole hindguts of three termites, P. adamsoni, Cryptotermes brevis, and Cryptotermes dudleyi. The parabasalian rRNA genes from C. brevis were found to comprise a unique and extremely heterogeneous lineage with no clear affinities to any known parabasalian rRNAs. In addition, one of the sequences isolated from P. Adamsoni was found to be similar to another uncharacterised rRNA gene from Reticulitermes flavipes. The phylogeny of all known parabasalian small subunit rRNAs was examined with these new sequences. We find many taxonomic groups to be supported by rRNA, but not all. We have found the root of parabasalia to be very difficult to discern accurately, but have nevertheless identified several possible positions.

Notes: The full length coding sequence of the Euglena gracilis actin gene was determined by RT-PCR of Euglena gracilis mRNA. Conserved regions in the actin amino acid sequence were used as guides for the synthesis of degenerate primers. Sequence was obtained for 1.238 nucleotides, of which 1.131 were coding for 377 amino acids. Sequence comparisons showed a similarity with other actins of 56% to 80%. Even though most of the actin amino acid sequence was conserved, some regions showed high divergence, i.e. the DNase I-binding loop at the N-terminal region. The construction of a phylogenetic tree based on actin sequences from different organisms placed Euglena gracilis in a cluster with Trypanosoma brucei and Leishmania major.

Notes: Trachipleistophora anthropophthera n. sp., was found at autopsy in the brain of one and in the brain, kidneys, pancreas, thyroid, parathyroid, heart, liver, spleen, lymph nodes, and bone marrow of a second patient with AIDS. The parasite is similar to the recently described T. hominis Hollister, Canning, Weidner, Field. Kench and Marriott, 1996, in having isolated nuclei, meronts with a thick layer of electron dense material on the outer face of their plasmalemma and sporogony during which spores are formed inside a thick-walled sporophorous vesicle. In contrast to T. hominis, this species is dimorphic as it forms two kinds of sporophorous vesicles and spores: Type I-round to oval polysporous sporophorous vesicle. 7-10 μm in size, usually with eight spores (3.7 × 2.0 μm), thick endospores, subterminal anchoring disc and anisofilar polar filaments forming seven thicker and two thinner terminal coils. This type of sporophorous vesicle is associated with 25-30 nm filaments extending into the host cell cytoplasm. Type II—smaller, bisporous sporophorous vesicle (4-5 times 2.2-2.5 μm) with two, nearly round, thin-walled spores, 2.2-2.5 × 1.8-2.0 μm in size, having 4-5 isofilar coils. No outside filamentous elements are associated with the bisporous sporophorous vesicle. Both types of sporophorous vesicles were common in the infected brain tissue and could be found within the same cell. The newly described species, together with T. hominis and previously reported Pleistophora-like parasites from human muscle, likely represent a group of closely related human microsporidia.

Notes: To obtain additional information on the phylogenetic relationships within the family Trypanosomatidae (order Kinetoplastida), we have sequenced the small subunit ribosomal RNA genes from the endosymbiont containing species Herpetomonas roitmani TCC080, Herpetomonas sp. TCC263, Crithidia oncopelti ATCC 12982 and a partial large subunit rRNA gene from H. roitmani. The small subunit sequences in the two isolates of Herpetomonas are very similar but not identical, and so are their restriction digest profiles of kinetoplast DNA. The size of minicircles in both isolates is 4.2 kilobases. The inferred ribosomal RNA phylogenetic trees shows the genera Herpetomonas and Crithidia as polyphyletic. Endosymbiont-bearing herpetomonads cluster with the endosymbiont-bearing crithidias and a blastocrithidia to form a monophyletic clade, whereas the endosymbiont-free members of these genera are found elsewhere in the tree. These data support the hypothesis of a monophyletic origin of endosymbiosis in trypanosomatid evolution and also suggest that a taxonomic revision is needed in order to better describe the natural affinities in this family.

Notes: We compared 16S-like ribosomal RNA (rRNA) coding regions of samples of the solitary spumellarian radiolarian Thalassicolla nucleata collected from the Sargasso Sea and the Pacific Ocean. Sequences derived from these locations showed variability in both length and base-pair composition. This level of sequence variability is similar to the degree of variability reported in the literature for species- or even genus-level distinctions. Explanations for our results include multiple alleles for the rRNA gene, or the existence of multiple species of Thalassicolla that are morphologically indistinguishable. The seven existing descriptions of Thalassicolla species, including T. nucleata, are discussed in view of these molecular findings and with reference to our current understanding of the physiology and life cycle of the spumellarian radiolaria.

Notes: We describe the transmission and pathogenic effects of a novel, feminising microsporidium, probably a Nasema species, on its crustacean host Gammarus duebeni. The parasite prevalence in the field was high (46% of females were infected) and the parasite was transovarially transmitted to 91% of embryos of infected females. The impact of the parasite on the host was assessed by means of a host breeding experiment. The parasite feminised 66% of infected host young and was transovarially transmitted by these individuals to the next host generation. The parasite differed from other feminising microsporidia in G. duebeni in that early embryos had a high parasite burden (288 parasites per embryo) and the infection was pathogenic, causing a reduction in both the growth rate of young hosts and in adult size. This study suggests that feminising microsporidia are a diverse group in which a variety of host/pathogen relationships have evolved.

Notes: The lipophosphoglycan-like (LPG-like) molecules of E. histolytica virulent strains are clearly distinct from those of the avirulent E. histolytica and E. dispar strains. Abundant ‘LPG’ levels are apparently limited to virulent strains, while lipophosphopeptidoglycans (‘LPPG's) are common to both virulent and avirulent strains of E. histolytica and E. dispar. It is therefore conceivable that ‘LPPG’ performs a function that is essential to survival within the host, while the ‘LPG’ performs a more specific function related to virulence.

Notes: Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactosc/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E. coli, retained at least some of its native conformation.

Notes: Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.

Notes: The presence of (Ca2/calmodulin (Ca2/CaM)-dependent protein kinase (TcCaM K) and some stage-specific substrates that appeared during morphogenesis of the parasite Trypanosoma cruzi were identified. Western blot analysis using a polyclonal antibody against rat brain CaM K type II recognized the same subunit composition (52, 59/62 kDa) observed for the mammalian enzyme, as well as the previously characterized TcCaM K found in epimastigote forms. Differential protein phosphorylation profiles were observed after enzyme activation in the stages of T. cruzi. Co-immunoprecipitation of stage-specific substrates with the TcCaM K suggested that the enzyme might be involved in the phosphorylation of a different set of proteins through the life cycle. Three phosphoproteins, pp105 and pp87 from epimastigotes and pp23 from trypomastigotes were identified as potential substrates for TcCaM K. The characterization of these endogenous stage markers might be a useful tool to understand the developmental cycles of these pathogenic protozoa.

Notes: The new analytic approach to the behavior of ciliates represented by the ethogram was used to study the locomotion of Oxytricha bifaria at different temperatures, isotropically applied according to a new protocol. It was shown that under these experimental conditions as the temperature dropped in stages from 24°C to 19°C to 14°C to 9°C, the general mobility of experimental populations decreased, as indicated by 1) the decreasing percentage of mobile organisms, 2) their decreasing velocity, 3) their prolonged backward creeping, and 4) the increasing length of their immobilization periods. The ethogram more particularly revealed that decreasing temperatures induced 1) the appearance of rightward arcs (several of them being travelled by specimens sliding on the substrate). 2) the reduction of both the radius and the velocity along the normal leftward arcs (A-) and the segments (S), and 3) the symmetric increase of both the central angle and the duration of the A- and S. These changes were reversibly induced: they disappeared when the temperature returned to 24°C. Moreover. a new behavioral pattern, the prolonged Side Stepping Reaction, was found. Helicoidal swimming, occurring only at 14°C, was analyzed. Among all of the behavioral parameters, nine were shown to change dramatically between 14°C and 9°C, demonstrating that the linear cooling of the populations induced clear non-linear effects.

Notes: The cystic and merogonic stages of the haemogregarine Hepatozoon sipedon, infecting Northern water snakes (Nerodia sipedon sipedon) and Northern leopard frogs (Rana pipiens), respectively, in Ontario, Canada, were investigated by transmission electron microscopy. Cysts, which were observed in the liver of Northern leopard frogs (Rana pipiens) after these anurans ingested mosquitoes (Culex pipiens) containing oocysts of the parasite, harboured two cystozoites, each of which contained a large crystalloid inclusion anterior to the nucleus. Two types of meronts were observed in snakes that were fed the liver of infected frogs. Macromeronts, which matured in endothelial cells of the liver approximately 16 d after snakes ingested infected frogs, contained about 50 large macromerozoites. Macromerozoites emerged from macromeronts, entered the bloodstream of the snake, and reinfected endothelial cells. Micromeronts, which matured about 34 d post-inoculation, contained about 150 micromerozoites that infected erythrocytes and transformed into gamonts. The ultrastructural features of micromeronts and macromeronts differed only slightly: immature macromeronts and macromerozoites contained numerous amylopectin and lipid inclusions, whereas immature micromeronts and micromerozoites did not contain amylopectin inclusions and featured fewer, smaller lipid inclusions. A comparison of cystic stages among Hepatozoon species in different groups of vertebrates is presented with respect to their structure and evolutionary significance.

Notes: A monoclonal antibody was obtained that reacts with many different proteins (14-200 kDa) of Amoeba proteus. By indirect immunofluorescence microscopy we found the antigens to be dispersed throughout the cytoplasm but were more concentrated in the nucleus. The antibody cross-reacted with proteins of Tetrahymena, Xenopus embryo, and mouse macrophages. Using the antibody as a probe we cloned a cDNA of 1.2 kb coding for ubiquitin in five repeats. Amino acid sequences of ameba's polyubiquitin showed the most variations among the nineteen polyubiquitins of other organisms compared. The well-conserved 20Ser and 55Thr residues were replaced with Gly and Ser. respectively. The 28Ala residue found in most organisms was replaced with Gln or Glu in the amoeba. Amoebae contained two ubiquitin-mRNAs that could be detected by Northern blot analysis using the cDNA as a probe. In an analysis for specificity, the antibody reacted with polyubiquitin and ubiquitin-fusion proteins larger than 14 kDa but not with monomeric ubiquitin. The antibody is a useful probe in the detection and characterization of proteins ubiquitinated in response to cellular stresses.

Notes: The phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of the lower termite Reticulitermes speratus was examined by a strategy that does not rely on cultivation. From mixed-population DNA obtained from the termite gut, small subunit (16S-like) ribosomal RNA sequences were directly amplified by the polymerase chain reaction method using primers specific for eukaryotes. Comparative sequence analysis of the clones revealed two kinds of sequences, one from the termite itself and the other from a symbiotic protist. A fluorescent-labeled oligonucleotide probe for the latter sequence was designed and used in whole-cell hybridization experiments to provide direct visual evidence that the sequence originated from a large hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that this hypermastigote and trichomonads shared a recent common ancestry.

Notes: Acanthamoeba spp. are free-living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B6C3F1 mice after intranasal challenge with Acanthamoeba-induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with 3H-uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chro-mium-51 from radiolabeled macrophages. Acanthamoeba culbensoni, which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba-mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba-specific antibodies increased the amebicidal activity of activated macrophages. Macrophage-mediated destruction was by contact-dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor α. interleukin 1α, and interleukin 1β, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.

Notes: Degenerate primers for two regions of sequence homology in the myosin head domain were used in a polymerase chain reaction screen of Tetrahymena thermophila genomic DNA to amplify a 765 bp fragment that was cloned and sequenced. Based on the presence of conserved, myosin-specific sequences, the 765 bp PCR product was identified as a fragment of a myosin gene, the first to be discovered in ciliated protozoa and herein referred to as MYO1. An inverse polymerase chain reaction strategy was used to obtain additional sequence data that included the entire head domain of MYO1. Alignment of the predicted amino acid sequence of the MYO1 head domain with known myosin sequences identified the ATP-binding site, a phosphorylation site, and other myosin-specific consensus regions. In a northern blot analysis, a 765 bp MYO1-specific probe detected a 6.6 kb transcript under highly stringent hybridization conditions. Phylogenetic analysis revealed that the predicted protein encoded by MYO1 is not a member of any of the previously defined myosin classes and therefore represents a presumptive new myosin class.

Notes: Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56° C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4° C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.

Notes: Mucins secreted from the gastrointestinal epithelium form the basis of the adherent mucus layer which is the host's first line of defense against invasion by Entamoeba histolytica. Galactose and N-acetyl-D-galactosamine residues of mucins specifically inhibit binding of the amebic 170 kDa heavy subunit Gal-lectin to target cells, an absolute prerequisite for pathogenesis. Herein we characterized the secretory mucins isolated from the human colon and from three human colonic adenocarcinoma cell lines: two with goblet cell-like (LS174T and T84) and one with absorptive cell-like morphology (Caco-2). By Northern blot analysis the intestinal mucin genes MUC2 and MUC3 were constitutively expressed by confluent LS174T and Caco-2 cells, whereas T84 cells only transcribed MUC2 and not MUC3 mRNA. 3H-glucosamine and 3H-threonine metabolically labeled proteins separated as high Mr mucins in the void (Vo 〉 106 Da) of Sepharose-4B column chromatography and remained in the stacking gel of SDS-PAGE as depicted by fluorography. All mucin preparations contained high amounts of N-acetyl-glucosamine, galactose, N-acetyl-galactosamine, fucose and sialic acid, saccharides typical of the O-linked carbohydrate side chains. Mucin samples from the human colon and from LS174T and Caco-2 cells inhibited E. histolytica adherence to Chinese hamster ovary cells, whereas mucins from T84 cells did not. These results suggest that genetic heterogeneity and/or posttranslational modification in glycosylation of colonic mucins can affect specific epithelial barrier function against intestinal pathogens.

Notes: Detergent lysates of Entamoeba histolytica trophozoites contained high levels of β-N acetyl-D-glucosaminidase. β-N acetyl-D-galactosaminidase and α-D-galactosidase activity, and lower but significant levels of five other glycosidases. Although these activities should have been capable of largely degrading the oligosaccharide side-chains of human colonic mucin, in fact only about one third of high MW mucin was degraded in 72 h and trypsin alone produced a similar effect. There was no evidence that these glycosidases were excreted and we conclude that they are unlikely to represent significant virulence factors for E. histolytica.

Notes: The microtubular stabilizing agent docetaxel (Taxotere™) is known to inhibit the intraerythrocytic development of Plasmodium falciparum. To investigate the mechanism(s) of inhibition, we analyzed the structural organization of the mitotic spindle by immunofluorescence and electron microscopy. When 30 μM docetaxel was applied for five hours on ring forms, alterations in the mitotic spindles leading to abnormal nuclear divisions were observed. At the trophozoite- and schizont-stage, docetaxel pulses prevent mitosis by stabilizing microtubular structures associated with the mitotic apparatus, giving abnormal spindles. However, this inhibition did not interfere with parasite DNA synthesis indicating the absence of a checkpoint that couples exit from mitosis with proper spindle assembly as observed in higher eukaryotic cells. In parallel, intraerythrocytic concentration of docetaxel was measured in parasitized erythrocytes, after incubation of cells with 3H-docetaxel for five hours. It was found to be 14-fold increased at the ring-stage of infected erythrocytes compared to normal ones, 170-fold increased at the trophozoite-stage and 1,500-fold increased at the schizont-stage. Our data show that, even though the overall intracellular concentration of docetaxel is low in docetaxel-pulsed rings, the agent might be sufficient to disturb the spindle organization. However, the existence of targets for docetaxel other than mitotic spindle microtubules. i.e. erythrocyte membrane components, could interfere with mitotic spindle formation

Notes: The phylogenetic relationships and taxonomic affinities of coccidia with isosporan-type oocysts have been unclear as overlapping characters, recently discovered life cycle features, and even recently discovered taxa. continue to be incorporated into biological classifications of the group. We determined the full or partial 18S ribosomal RNA gene sequences of three mammalian Isospora spp., Isospora felis, Isospora ohioensis and Isospora suis, and a Sarcocystis sp. of a rattlesnake, and used these sequences for a phylogenetic analysis of the genus Isospora and the cyst-forming coccidia. Various alveolate 18S rDNA sequences were aligned and analyzed using maximum parsimony to obtain a phylogenetic hypothesis for the group. The three Isospora spp. were found to be most closely related to Toxoplasma gondii and Neospora caninum. This clade in turn formed the sister group to the Sarcocystis spp. included in the analysis. The results confirm that the genus Isospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae. Furthermore, there appear to be two lineages within the Sarcocystidae. One lineage comprises Isospora and the Toxoplasma/Neospora clade which share the characters of having a proliferative phase of development preceding gamogony in the definitive host and an exogenous phase of sporogony. The other lineage comprises the Sarcocystis spp. which have no proliferative phase in the definitive host and an endogenous phase of sporogony.

Notes: The pellicle of Paramecium has three two-dimensionally arrayed systems that occupy separate but closely paralleling planes. All three systems are now distinguishable by their differing immunological properties. This study focused on the two deeper systems. The infraciliary lattice lies innermost and labels with centrin-specific antibodies. The middle system, the striated bands, is specifically labeled with a monoclonal antibody that we have raised to a 110 kDa conical antigen in P. multimicronucleatum. This antibody labels a similar geometric cortical pattern in at least two species, P. multimicronucleatum and P. tetraurelia. Centrin-specific structures appear to be net-like in the above two species but show a more interrupted pattern in P. caudatum. The cytostomal cord is an essentially unbranched extension of the net-like infraciliary lattice and, like it, is centrin-specific. The cord has a unique association with the alveolar sacs which suggest these calcium-storing compartments contribute to the calcium fluxes required for contraction of the cord. A structural rather than a contractile function is favored for the striated bands, based solely on their morphology.

Notes: Protargol-stained cultured samples of Strombidinopsis spiniferum and S. acuminatum revealed a kind of enantiotropic mode of cell division. Stomatogenesis may be divided into eight stages. Stage 1 was the appearance of an anterior anarchic field of a few kinetosomes, probably originating apokinetally near a somatic kinety on the dorsal side of the cell. In Stage 2, the anarchic field becomes denser and larger by kinetosomal replication. The kinetosomes become aligned as oral polykinetids (OPk's) in Stage 3. In Stage 4, the paroral (PO) emerges when most of the anarchic field has transformed into OPk's. In Stage 5. all OPk's are apparently formed, and the anlage resembles a barrel, lacking a few staves. In Stage 6, the barrel rotates approximately 90° transversely to the cell's axis, concomitantly the outer end of the barrel splays open. In Stage 7, the somatic kineties start to divide, the anlage has opened to the cell surface, and the part with the internal OPk's spirals into the oral cavity while the OPk's form a closed circle. At this stage, one or two large macronuclei were seen preparing to divide. In Stage 8, the adult or interphase stage, the cytopharynx is formed. Based on our results, the original redescriptions of S. spinifierum and S. acuminatum must be modified in three respects: the cells have a PO; the OPk's are composed of three rows of kinetosomes (uncertain for S. acuminatum), not two as originally reported; and the number of internal OPk's is not always 3, but ranges from 3-4 in S. spiniferum and 3-5 in S. acuminatum.

Notes: Abstract This review surveys the kinds of protein complex that participate in cell communication and identifies, where possible, general principles by which they form and act. It also advances the notion that biophysical constraints imposed by macromolecular crowding and diffusion have had a controlling influence on the evolution of cell signaling pathways. Complexes associated with the bacterial aspartate receptor, with eucaryotic tyrosine kinase receptors, with T-cell receptors, and with focal contacts are examined together with proteins that serve as adaptors, anchors, and scaffolds for signaling complexes. The importance of diffusion in controlling the numbers and locations of signaling complexes is discussed, as is the special role played by membranes in signaling pathways.

Notes: Abstract The substrates for the essential biological processes of transcription, replication, recombination, DNA repair, and cell division are not naked DNA; rather, they are protein-DNA complexes known as chromatin, in one or another stage of a hierarchical series of compactions. These are exciting times for students of chromatin. New studies provide incontrovertible evidence linking chromatin structure to function. Exceptional progress has been made in studies of the structure of chromatin subunits. Surprising new dynamic properties have been discovered. And, much progress has been made in dissecting the functional roles of specific chromatin proteins and domains. This review focuses on in vitro studies of chromatin structure, dynamics, and function.

Notes: Abstract Retroviral protease (PR) from the human immunodeficiency virus type 1 (HIV-1) was identified over a decade ago as a potential target for structure-based drug design. This effort was very successful. Four drugs are already approved, and others are undergoing clinical trials. The techniques utilized in this remarkable example of structure-assisted drug design included crystallography, NMR, computational studies, and advanced chemical synthesis. The development of these drugs is discussed in detail. Other approaches to designing HIV-1 PR inhibitors, based on the concepts of symmetry and on the replacement of a water molecule that had been found tetrahedrally coordinated between the enzyme and the inhibitors, are also discussed. The emergence of drug-induced mutations of HIV-1 PR leads to rapid loss of potency of the existing drugs and to the need to continue the development process. The structural basis of drug resistance and the ways of overcoming this phenomenon are mentioned.

Notes: Abstract Biochemical and genetic approaches have identified the molecular mechanisms of many genetic reactions, particularly in bacteria. Now a comparably detailed understanding is needed of how groupings of genes and related protein reactions interact to orchestrate cellular functions over the cell cycle, to implement preprogrammed cellular development, or to dynamically change a cell's processes and structures in response to environmental signals. Simulations using realistic, molecular-level models of genetic mechanisms and of signal transduction networks are needed to analyze dynamic behavior of multigene systems, to predict behavior of mutant circuits, and to identify the design principles applicable to design of genetic regulatory circuits. When the underlying design rules for regulatory circuits are understood, it will be far easier to recognize common circuit motifs, to identify functions of individual proteins in regulation, and to redesign circuits for altered functions.

Notes: Abstract Cytochrome c oxidase, the terminal enzyme of the respiratory chains of mitochondria and aerobic bacteria, catalyzes electron transfer from cytochrome c to molecular oxygen, reducing the latter to water. Electron transfer is coupled to proton translocation across the membrane, resulting in a proton and charge gradient that is then employed by the F0F1-ATPase to synthesize ATP. Over the last years, substantial progress has been made in our understanding of the structure and function of this enzyme. Spectroscopic techniques such as EPR, absorbance and resonance Raman spectroscopy, in combination with site-directed mutagenesis work, have been successfully applied to elucidate the nature of the cofactors and their ligands, to identify key residues involved in proton transfer, and to gain insight into the catalytic cycle and the structures of its intermediates. Recently, the crystal structures of a bacterial and a mitochondrial cytochrome c oxidase have been determined. In this review, we provide an overview of the crystal structures, summarize recent spectroscopic work, and combine structural and spectroscopic data in discussing mechanistic aspects of the enzyme. For the latter, we focus on the structure of the oxygen intermediates, proton-transfer pathways, and the much-debated issue of how electron transfer in the enzyme might be coupled to proton translocation.

Notes: Abstract During the past thirty years, deuterium labeling has been used to improve the resolution and sensitivity of protein NMR spectra used in a wide variety of applications. Most recently, the combination of triple resonance experiments and 2H, 13C, 15N labeled samples has been critical to the solution structure determination of several proteins with molecular weights on the order of 30 kDa. Here we review the developments in isotopic labeling strategies, NMR pulse sequences, and structure-determination protocols that have facilitated this advance and hold promise for future NMR-based structural studies of even larger systems. As well, we detail recent progress in the use of solution 2H NMR methods to probe the dynamics of protein sidechains.

Notes: Abstract The hammerhead ribozyme is a small catalytic RNA that cleaves a target phosphodiester bond in a reaction dependent on divalent metal ions. Crystal structures of the hammerhead reveal the tertiary fold of an enzymatic "ground state" of the molecule; however, they do not clarify the catalytic mechanism of the ribozyme, presumably because a significant conformational rearrangement is required to reach an enzymatic transition state. The structural domains seen in the hammerhead can be related to sequence or structural motifs in transfer and ribosomal RNAs, suggesting that they represent tertiary building blocks that will be found in large, complex RNAs.

Notes: Abstract Pleckstrin homology (PH) motifs are approximately 100 amino-acid residues long and have been identified in nearly 100 different eukaryotic proteins, many of which participate in cell signaling and cytoskeletal regulation. Despite minimal sequence homology, the three-dimensional structures are remarkably conserved. This review gives an overview of the PH domain architecture and examines the best-studied examples in an attempt to understand their function.

Notes: Abstract The actin cytoskeleton is a highly dynamic network composed of actin polymers and a large variety of associated proteins. The main functions of the actin cytoskeleton are to mediate cell motility and cell shape changes during the cell cycle and in response to extracellular stimuli, to organize the cytoplasm, and to generate mechanical forces within the cell. The reshaping and functions of the actin cytoskeleton are regulated by signaling pathways. Here we broadly review the actin cytoskeleton and the signaling pathways that regulate it. We place heavy emphasis on the yeast actin cytoskeleton.

Notes: Abstract Proteins that control mitochondrial dynamics in yeast are being identified at a rapid pace. These proteins include cytoskeletal elements that regulate organelle distribution and inheritance and several outer membrane proteins that are required to maintain the branched, mitochondrial reticulum. Interestingly, three of the high molecular weight GTPases encoded by the yeast genome are required for mitochondrial integrity and are potential regulators of mitochondrial branching, distribution, and membrane fusion. The recent finding that mtDNA mixing is restricted in the mitochondrial matrix has stimulated the hunt for the molecular machinery that anchors mitochondrial nucleoids in the organelle. Considering that many aspects of mitochondrial structure and behavior are strikingly similar in different cell types, the functional analyses of these yeast proteins should provide general insights into the mechanisms governing mitochondrial dynamics in all eukaryotes.

Notes: Abstract Cells respond to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing transcription of genes encoding ER resident proteins. The information is transmitted from the ER lumen to the nucleus by an intracellular signaling pathway called the unfolded protein response (UPR). Recent work has shown that this signaling pathway utilizes several novel mechanisms, including translational attenuation and a regulated mRNA splicing step. In this review we aim to integrate these recent advances with current knowledge about maintenance of ER composition and abundance.

Notes: Abstract Following the genomic localization and subsequent identification of the breast cancer susceptibility genes, BRCA1 and BRCA2, the basic patterns of cancer risk associated with mutations in these genes have been defined. In addition, preliminary insights into the prevalence of mutations and their contributions to cancer incidence have been acquired. Features of breast and other cancers that develop in these genetic syndromes have now been investigated and shown to differ from sporadic versions of the same neoplasms. However, several areas are complex and require further clarification. There remain discrepancies between published cancer risk estimates. Furthermore, there may be variation in cancer risk between different mutations in the same gene and there is preliminary evidence that genetic and nongenetic influences may modify risks. Finally, it is probable that the genes underlying a substantial component of susceptibility to breast cancer remain to be identified.

Notes: Abstract The gaseous hormone ethylene induces diverse effects in plants throughout their life cycle. Ethylene response is regulated at multiple levels, from hormone synthesis and perception to signal transduction and transcriptional regulation. As more genes in the ethylene response pathway are cloned and characterized, they illustrate the precision with which signaling can be controlled. Wounding, pathogenic attack, flooding, fruit ripening, development, senescence, and ethylene treatment itself induce ethylene production. Ethylene binding to receptors with homology to two-component regulators triggers a kinase cascade that is propagated through the CTR1 Raf-like kinase and other components to the nucleus. Activation of the EIN3 family of nuclear proteins leads to induction of the relevant ethyleneresponsive genes via other transcription factors, eliciting a response appropriate to the original stimulus.

Notes: Abstract Epitope tagging is a recombinant DNA method by which a protein encoded by a cloned gene is made immunoreactive to a known antibody. This review discusses the major advantages and limitations of epitope tagging and describes a number of recent applications. Major areas of application include monitoring protein expression, localizing proteins at the cellular and subcellular levels, and protein purification, as well as the analysis of protein topology, dynamics and interactions. Recently the method has also found use in transgenic and gene therapy studies and in the emerging fields of functional genomics and proteomics.

Notes: Abstract The leptotene/zygotene transition of meiosis, as defined by classical cytological studies, is the period when homologous chromosomes, already being discernible individualized entities, begin to be close together or touching over portions of their lengths. This period also includes the bouquet stage: Chromosome ends, which have already become integral components of the inner nuclear membrane, move into a polarized configuration, along with other nuclear envelope components. Chromosome movements, active or passive, also occur. The detailed nature of interhomologue interactions during this period, with special emphasis on the involvement of chromosome ends, and the overall role for meiosis and recombination of chromosome movement and, especially, the bouquet stage are discussed.

Notes: Abstract In 1970, Perutz tried to put the allosteric mechanism of hemoglobin, proposed by Monod, Wyman and Changeux in 1965, on a stereochemical basis. He interpreted their two-state model in terms of an equilibrium between two alternative structures, a tense one (T) with low oxygen affinity, constrained by salt-bridges between the C-termini of the four subunits, and a relaxed one (R) lacking these bridges. The equilibrium was thought to be governed primarily by the positions of the iron atoms relative to the porphyrin: out-of-plane in five-coordinated, high-spin deoxyhemoglobin, and in-plane in six-coordinated, low-spin oxyhemoglobin. The tension exercised by the salt-bridges in the T-structure was to be transmitted to the heme-linked histidines and to restrain the movement of the iron atoms into the porphyrin plane that is necessary for oxygen binding. At the beta-hemes, the distal valine and histidine block the oxygen-combining site in the T-structure; its tension was thought to strengthen that blockage. Finally, Perutz attributed the linearity of proton release with early oxygen uptake to the sequential rupture of salt-bridges in the T-structure and to the accompanying drop in pKa of the weak bases that form part of them. Almost every feature of this mechanism has been disputed, but evidence that has come to light more than 25 years later now shows it to have been substantially correct. That new evidence is reviewed below.

Notes: Abstract To date, high-resolution structures have been solved for five different architectural proteins complexed to their DNA target sites. These include TATA-box-binding protein, integration host factor (IHF), high mobility group I(Y)[HMG I(Y)], and the HMG-box-containing proteins SRY and LEF-1. Each of these proteins interacts with DNA exclusively through minor groove contacts and alters DNA conformation. This paper reviews the structural features of these complexes and the roles they play in facilitating assembly of higher-order protein-DNA complexes and discusses elements that contribute to sequence-specific recognition and conformational changes.

Notes: Abstract Eukaryotic protein phosphatases are structurally and functionally diverse enzymes that are represented by three distinct gene families. Two of these, the PPP and PPM families, dephosphorylate phosphoserine and phosphothreonine residues, whereas the protein tyrosine phosphatases (PTPs) dephosphorylate phosphotyrosine amino acids. A subfamily of the PTPs, the dual-specificity phosphatases, dephosphorylate all three phosphoamino acids. Within each family, the catalytic domains are highly conserved, with functional diversity endowed by regulatory domains and subunits. The protein Ser/Thr phosphatases are metalloenzymes and dephosphorylate their substrates in a single reaction step using a metal-activated nucleophilic water molecule. In contrast, the PTPs catalyze dephosphorylation by use of a cysteinyl-phosphate enzyme intermediate. The crystal structures of a number of protein phosphatases have been determined, enabling us to understand their catalytic mechanisms and the basis for substrate recognition and to begin to provide insights into molecular mechanisms of protein phosphatase regulation.

Notes: Abstract Exciting progress has been made in the last decade by those who use physical methods to study the structure of the ribosome and its components. The structures of 10 ribosomal proteins and three isolated ribosomal protein domains are known, and the conformations of a significant number of rRNA sequences have been determined. Electron microscopists have made major advances in the analysis of images of ribosomes, and microscopically derived ribosome models at resolutions approaching 10A are likely quite soon. Furthermore, ribosome crystallographers are on the verge of phasing the diffraction patterns they have had for several years, and near-atomic resolution models for entire ribosomal subunits could emerge from this source at any time. The literature relevant to these developments is reviewed below.

Notes: Abstract Identification of biomolecules in complex biological mixtures represents a major challenge in biomedical, environmental, and chemical research today. Chemical separations with traditional detection schemes such as absorption, fluorescence, refractive index, conductivity, and electrochemistry have been the standards for definitive identifications of many compounds. In many instances, however, the complexity of the biomixture exceeds the resolution capability of chemical separations. Biosensors based on molecular recognition can dramatically improve the selectivity of and provide biologically relevant information about the components. This review describes how coupling chemical separations with online biosensors solves challenging problems in sample analysis by identifying components that would not normally be detectable by either technique alone. This review also presents examples and principles of combining chemical separations with biosensor detection that uses living systems, whole cells, membrane receptors, enzymes, and immunosensors.