ALBERT

Notes: Successful long-term wetland restoration efforts require consideration of hydrology and currounding land use during the site selection process. This article describes an approach to initial site selection in the San Luis Rey River watershed in southern California that uses watershed-level information on basin topography and land cover to rank the potential suitability of all sites within a watershed for either preservation of restoration. This approach requires the use of a geographic information system (GIS)to map relative wetness and land cover within a watershed. Relative potential wetness values were derived from U.S. Geological Survey (USGS) 30-m digital elevation models by calculating the flow that would potentially accumulate at all 30-m × 30-m pixels within the watershed. Land cover was derived from a Landsat scene covering the 1500 Km2 study area. We ranked sites (contiguous groups of pixels 〉 1 ha with similar land cover) in terms of their potential for restoration or preservation based on their wetness values (Iow, medium, and high), size, and proximity to existing riparian vegetation. Sites with medium or high wetness values and extant vegetation were identified as potential preservation sites. Agiricultural or barren sites with medium to high wetness were identified as potential restoration sites. Approximately 5500 ha (3.67% of the total watershed) were prioritized for preservation or resloration.

Notes: This historical and conceptual overview of riparian ecosystem restoration discusses how riparian ecosystems have been defined, describes the hydrologic, geomorphic, and biotic processes that create and maintain riparian ecosystems of the western USA, identifies the main types of anthropogenic desturbances occurring in these ecosystems, and provides an overview of restoration methods for each disturbance type. We suggest that riparian ecosystems consist of two zones: Zone I occupies the active floodplain and is frequently inundated and Zone II extends from the active floodplain to the valley wall. Successful restoration depends n understanding the physical and biological processes that influence natural riparian ecosystems and the types of disturbance that have degraded riparian areas. Thus we recommend adopting a process-based approach for riparian restoration. Disturbances to riparian ecosystems in the western USA result from streamflow modifications by dams, reservoirs, and diversions; stream channelization; direct modification of the riparian ecosystem; and watershed disturbances. Four topics should be addressed to advance the state of science for restoration of riparian ecosys-tems: (1) interdisciplinary approaches, (2) a unified framework, (3) a better understanding of fundamental riparian ecosystem processes, and (4) restoration po-tential more closely related to disturbance type. Three issues should be considered regarding the cause of the degraded environment: (1) the location of the causative disturbance with respect to the degraded riparian area, (2) whether the disturbance is ongoing or can be elim-inated, and (3) whether or not recovery will occur nat-urally if the disturbance is removed.

Notes: A two-stage system for selecting stream reaches and riparian communities for restoration was applied to the 80-km San Luis Rey River below the Lake Henshaw dam in southern California. In the first satge, data from topographic quadrangles and aerial photographs were analyzed to define and classiy reaches. These analyses concluded that (1) 28 km of the river and adjacent floodplain were suitable for second-stage evaluation of restoration needs and (2) 32 km met criteria for reference conditions at the stream reach scale and should be protected from further impacts. The remaining 20 km of the river and floodplain were considered unsuitable for restoration to reach-scale reference conditions; individual sites may be restored under existing regulatory review. Second-stage field sampling provided data on vegetation and floodplain landforms and substrate from more thatn 3000 plots within the 28 km of river and 1120 ha of floodplain selected for further Study. Classification of floristic samples stratified by landform/substrate class indicated six primary riparian communities on the floodplain, some associated with particular floodplain landform/substrate classes and others ubiquitous. Reference conditions for these communities were interpreted from the data. There were two major departures from reference conditions: tree-dominated communities were less extensive than historic levels and exotic plants had significantly invaded some landforms and communities, displacing natural com-munities. General goals would include restoration of tree communities and removal of exotics, with further consideration of site-specific objectives. The results included estimates of the areas by community type re-quiring restoration. The approach was developed for streams in the semi-arid western United States, but it may be adapted for use elsewhere.

Notes: This study compares the results of Olson and Harris (1997) and Russell et al. (1997) in their work to prioritize sites for riparian restoration in the San Luis Rey River watershed. Olson and Harris defined reaches of the mainstem and evaluated the relative potential for restoration and protection based on cover of natural vegetation, land use, and connectivity. Then they used data on geomorphic conditions, plant species composition, and community structure to prescribe strategies for restoration. Russell et al. used a modeling approach within a geographic information system to combine data on wetness and land use/land cover to identify areas with potential for protection and restoration. They prioritized the areas based on patch size and proximity to extant riparian habitat. The main-stem and associated floodplain defined by Olson and Harris was more than twice the size of the area defined by Russell et al., because Olson and Harris considered the entire valley floor, whereas Russell et al. used a wetness index to identify saturated zones within the floodplain. For seven of the twelve management units delineated along the mainstem, the two studies agreed on a strategy of restoration or protection. They differed on two. No comparison could be made of the three units for which Olson and Harris used project review, a unique category. Agreement of the results is due to the similarity of criteria used to identify and rank sites for protection and restoration; disagreement is due primarily to the level of resolution of the data. Both approaches have potential for use in watershed-level planning. The predictive power of the two approaches may be maximized when they are used in a complementary fashion.

Notes: We present a conceptual model for identifying restoration sites for riparian wetlands and discuss its application to reaches within the Upper Arkansas River basin in Colorado. The model utilizes a Geographic Information System (GIS) to analyze a variety of spatial data useful in characterizing geomorphology, hydrology, and vegetation of riparian wetland sites. The model focuses on three basic properties of riparian wetland sites: relative soil moisture, disturbance regime, and vegetative characteristics. A relative wetness index is used to define nominal soil moisture classes within the watershed. These classes generally coincide with uplands (low), channel margins (moderate), and channels or open water (high). Vegetative conditions are characterized using color infrared aerial photographs. Land cover types are grouped into five major land cover classes: riparian, moist herbaceous, bare ground, upland, and open water. Disturbance regime is characterized by a reach-based index of specific power (ω). Preliminary results indicate that reaches within the Upper Arkansas River basin can be classified as high energy (ω≥ 8 W/m2) or low energy (ω≤ 3W/m2), using discharge estimates that reflect the 10-year flood event. Field surveys of channel and floodplain conditions show that high-energy reaches (ω≥ 8 W/m2) are characterized by sites where the channel occupies a large proportion of the valley bottom. By contrast, low-energy reaches (ω≤ 3 W/m2) are characterized by meandering channels with wide alluvial valleys. Restoration potential is evaluated as a combination of nominal scores from wetness, land cover, and disturbance indices. Application of these methods to field sites within the Upper Arkansas River basin identifies a wide range of riparian wetland sites for preservation or restoration. Potential sites within identified reaches are prioritized using size and proximity criteria.

Notes: A major goal of population biologists involved in restoration work is to restore populations to a level that will allow them to persist over the long term within a dynamic landscape and include the ability to undergo adaptive evolutionary change. We discuss five research areas of particular importance to restoration biology that offer potentially unique opportunities to couple basic research with the practical needs of restorationists. The five research areas are: (1) the influence of numbers of individuals and genetic variation in the initial population on population colonization, establishment, growth, and evolutionary potential; (2) the role of local adaptation and life history traits in the success of restored populations; (3) the influence of the spatial arrangement of landscape elements on metapopulation dynamics and population processes such as migration; (4) the effects of genetic drift, gene flow, and selection on population persistence within an often accelerated, successional time frame; and (5) the influence of interspecific interactions on population dynamics and community development. We also provide a sample of practical problems faced by practitioners, each of which encompasses one or more of the research areas discussed, and that may be solved by addressing fundamental research questions.

Notes: Cool smoke treatments were applied to unmined Eucalyptus marginata (jarrah) forest soils, rehabilitated bauxite mine soils, and broadcast seed to determine if enhancement in germination could be effected with a view toward maximizng the establishment of species in bauxite mines in Western Australia.Forest sites showed a 48-fold increase in total germinants from the soil seed bank when treated with aerosol smoke. Newly returned bauxite mine soils showed a greater than threefold increase in total germinants after the same treatment. There were also significant increases in the number of species germinating in response to the aerosol smoke treatment in both the forest and the mined soils. Similarly, application of smoked water to the soil seed bank in previously mined sites elicited a significant positive germination response, increasing total germinants and species numbers by 56 and 33%, respectively.Treatment of mixed seed lots with aerosol smoke before broadcast resulted in highly significant improvement in germination when compared to untreated seed. Both total number of germinants, and number of species emerging from mined sites were positively influenced (85% and 34% increases, respectively).Ten target species were used to determine the relative effectiveness of different methods of smoke treatment on the germination of broadcast seed. Nine of the species involved displayed a promotive effect with at least two of the treatments. Generally, however, aerosol smoking of seed before broadcast proved to be the more effective approach. As a result of these findings, all broadcast seed for use in Alcoa's bauxite mined areas in the southwest of Western Australia is now routinely smoke treated before application.

Notes: Understanding Cladium jamaicense (sawgrass) seedling establishment is an important component of an Everglades restoration program because the degree of sawgrass recovery and concurrent Typha domingensis (cattail) decline will be used to evaluate restoration success. To understand sawgrass recovery at locations with increased soil nutrients, we tested the effects of transplanting sawgrass seedlings to pots at different densities and investigated how nutrient additions affect seedling growth. Survivorship of seedlings transplanted into moist commercial potting soil at three densities ranged from 61% to 95%. After 6 months, maximum survivorship (90%) occurred at medium densities (2–4 seedlings per pot 16 cm in diameter). Nutrient additions, totaling 6.5 N g/m2, 9.8 P g/m2, 6.5 g/m2, were applied approximately 4 months after seedlings were transplanted. The biomass of the plants receiving nutrient additions (pulsed) was significantly higher (by over 30%) than plants with no nutrient addition (control). Photosynthetic rates for nutrient-enriched plants (measured 6-weeks after the nutrient additions) were significantly greater (by 32–45%) than for control plants. Instantaneous leaf water use efficiency increased significantly (by more than 20%) in pulsed plants. The results suggest that preventing root damage is crucial for the success of trans planted sawgrass seedlings and that nutrient additions enhanced seedling growth.

Notes: I evaluated responses by 16 native woody species to differential soil compaction and density of ground cover. The trees and shrubs studied represent sites in southern Illinois that commonly have restrictions to root growth from soil or drainage conditions. The study site was a restored surface coal mine in southern Illinois with a rooting medium compacted by grading and a dense ground cover of pasture species. Soil compaction was alleviated in half the study area before tree planting by mechanically ripping the soil to a depth of 1.2 m. Roots of half the trees and shrubs were dipped in a Terra® slurry before planting, and the ground cover around all planting spots was afterwards sprayed with herbicide. In year 2 after planting the ground cover in half of the unripped and half of the ripped area was further controlled by repeated application of herbicides. Ripping significantly increased height growth of all trees combined and all species individually in each year of the study. Second-year control of ground cover increased height growth of all trees combined and of seven species individually. Some species were damaged by herbicides. Terra® had little evident effect on species performance. Animal damage reduced early survival and growth, especially of Acer (maple) and Cornus (dogwood) species, and later growth of Quercus rubra (red oak). Removal of ground cover with herbicides tended to increase deer browse. Soil ripping, herbicide application, and choosing tree species unattractive to deer can be recommended to increase success in planting trees for forest restoration.

Notes: Woodlands dominated by Eucalyptus spp. in temperate southeastern and southwestern Australia have been extensively cleared for agriculture and are often badly degraded by livestock grazing. This has resulted in the loss of biodiversity and widespread land degradation. The continuing decline of these woodlands has become a concern for the conservation of biodiversity, and there is a growing interest among farmers, land managers, and researchers in developing techniques for restoring them. Currently few scientific guidelines exist for undertaking woodland restoration programs. We use a state and transition model to develop hypotheses on restoration strategies for salmon gum (Eucalyptus salmonophloia) woodlands. We consider that this approach provides a suitable framework for organizing knowledge and identifying areas where further information is needed, and hence provides a useful starting point for a restoration program. The model has the potential to provide a tool for land managers with which they can assess the action and effort needed to undertake woodland restoration in agricultural landscapes.

Notes: Most of the world's forest has been cleared, cultivated, and then often abandoned. In many instances these areas have changed to successionally arrested grasslands, shrublands, or fernlands maintained by frequent fires and high herbivore populations. Many studies have shown that various herbaceous, nitrogen-fixing legumes can protect soil surfaces, retain soil moisture, improve soil fertility, and retard ground fires. Our objective was to ascertain if some of these species can potentially inhibit herbivory and satisfactorily establish in these arrested grassland areas to serve as sites for reforestation. We evaluated the potential for four species of nitrogen-fixing legumes (Calapogonium mucunoides, Centrosema pubescens, Desmodium ovalifolium, and Pueraria phaseoloides) to establish on exposed soil within successionally arrested grasslands of Panicum maximum and Cymbopogon nardus in the central hills of Sri Lanka. Four different sites within rectangular grassland areas were cleared of graminoids and sown with seed of each legume. Half of each clearing was protected from browsing rabbits and porcupines, and half was not protected. After 6 months, certain plots were destructively sampled to determine dry biomass gain for each species and treatment. Analyses of variance were performed to test for differences among sites, treatments, and species. All three factors revealed differences, indicating that species must be matched to site. On sites with high amounts of herbivory, D. ovalifolium had the greatest dry biomass gain after 6 months of growth, possibly because of its relatively low nitrogen and moisture content. Where herbivory was absent, P. phaseoloides and C. muconoides had the greatest dry biomass gain. Dry biomass gain of all four legume ground covers was low on sites with lowest pH and nutrient concentrations. Under conditions of low relative fertility and low pH, establishment of the tested legumes failed. Though soil moisture availability was not measured, we speculate that these low fertility sites were also prone to drought. Findings support the site-specific establishment of legume species for purposes of reforestation and watershed protection in central Sri Lanka. This work is applicable to other regions particularly dominated by successionally arrested grasslands with similar circumstances in other parts of south and southeast Asia.

Notes: Oyster cultch was added to the lower intertidal fringe of three created Spartina alterniflora marshes to examine its value in protecting the marsh from erosion. Twelve 5-m-wide plots were established at each site, with six randomly selected plots unaltered (non-cultched) and cultch added to the remaining (cultched) plots. Within each cultched plot, cultch was placed along the low tide fringe of the marsh during July 1992, in a band 1.5 m wide by 0.25 m deep. Marsh-edge vegetation stability and sediment erosion were measured for each plot from September 1992 to April 1994. Significant differences (p 〈 0.05) in marsh-edge vegetation change were detected at the only south-facing site after a major southwester storm. Significantly different rates of sediment erosion and accretion also were observed at this same site. Areas upland of the marsh edge in the cultched areas showed an average accretion of 6.3 cm, while noncultched treatment areas showed an average loss of 3.2 cm. A second site, with a northern orientation, also experienced differential sediment accretion and erosion between treatment type, caused instead by boat wakes that were magnified by the abutment of a dredge effluent pipe across the entire front fringe of the site. During this period we observed significant differences in sediment accumulation, with the areas upland of the marsh edge in the cultched treatment having an average accretion of 2.9 cm and the noncultched an average loss of 1.3 cm.

Notes: Books reviewed in this article: The Significance and Regulation of Soil Biodiversity. Harold P. Collins, G. Philip Robertson, and Michael J. Klug, editors

Notes: Declines in native plant and animal communities have prompted new interest in the restoration of aquatic and riparian ecosystems. Past restoration activities typically have been site specific, with little thought to processes operating at larger scales. A watershed analysis process developed in the Pacific northwest identifies four operating scales useful in developing restoration priorities: region, basin, water-shed, and specific site. Wtershed analysis provides a template for restoration practitioners to use in prioritizing restoration activities. The template identifies seven key steps necessary to understand and develop restoration priorities: (1) characterization, (2) identification of key issues and questions, (3) documentation of current conditions, (4) description of reference conditions, (5) identification of objectives, (6) summary of conditions and determination of causes, and (7) recommendations. When a similar process was used in the Uinta Mountains, Utah, and in the Sluslaw National Forest, Orgon, specialists were able to identify key habitat conditions and habitat forming processes and then to establish restoration priorities and implement the appropriate activities. Watershed analysis provides a valuable set of tools for identifying restoration activities and is currently being used throughout the Pacific Northwest to develop management strategies and restoration priorities. Although the analysis requires significant time, money, and personnel, experience suggests that watershed analysis provides valuable direction for managing aquatic and riparian resources.

Notes: Landscape ecology focuses on questions typically addressed over broad spatial scales. A landscape approach embraces spatial heterogeneity, consisting of a number of ecosystems and/or landscape structures of different types, as a central theme. Such studies may aid restoration efforts in a variety of ways, including (1) provision of better guidance for selecting reference sites and establishing project goals and (2) suggestions for appropriate spatial configurations of restored elements to facilitate recruitment of flora/fauna. Likewise, restoration efforts may assist landscape–level studies, given that restored habitats, possessing various patch arrangements or being established among landscapes of varying diversity and conditions of human alteration, can provide extraordinary opportunities for experimentation over a large spatial scale. Restoration studies can facilitate the rate of information gathering for expected changes in natural landscapes for which introduction of landscape elements may be relatively slow. Moreover, data collected from restoration studies can assist in validation of dynamic models of current interest in landscape ecology. We suggest that restoration and landscape ecology have an unexplored mutualistic relationship that could enhance research and application of both disciplines.

Notes: Restoration ecologists use reference information to define restoration goals, determine the restoration potential of sites, and evaluate the success of restoration efforts. Basic to the selection and use of reference information is the need to understand temporal and spatial variation in nature. This is a challenging task: variation is likely to be scale dependent; ecosystems vary in complex ways at several spatial and temporal scales; and there is an important interaction between spatial and temporal variation. The two most common forms of reference information are historical data from the site to be restored and contemporary data from reference sites (sites chosen as good analogs of the site to be restored). Among the problems of historical data are unmeasured factors that confound the interpretation of historical changes observed. Among the problems of individual reference sites is the difficulty of finding or proving a close match in all relevant ecological dimensions. Approximating and understanding ecological variation will require multiple sources of information. Restoration, by its inherently experimental nature, can further the understanding of the distribution, causes, and functions of nature's variation.

Notes: Books reviewed in this article: Water Quality: Management of a Natural Resource James Perry and Elizabeth Vanderklein

Notes: The U.S. Bureau of Reclamation created a shallow, 110-m channel to provide habitat for two endangered fishes, Cyprinodon elegans (Comanche Springs pupfish) and Gambusia nobilis (Pecos gambusia), at the site of the fishes' former natural habitat. The ciénega (marsh) associated with Phantom Lake Spring in Jeff Davis County, Texas, was destroyed by the creation of an irrigation canal system. In 1993, the endangered fishes were stocked into the refuge with individuals from the irrigation canals, and in the case of C. elegans, hatchery stocks. The condition of habitat, status of fish populations, and fish ecology within the refuge were then monitored for two years. The abundance and density of both species increased in accordance with aquatic plant development. Cyprinodon elegans abundance peaked after one year and stabilized at an average density of 14.7/m2 by the end of our study. Juvenile C. elegans were always rare, which may indicate that the population reached the refuge's carrying capacity and that recruitment is low. Gambusia nobilis was the most abundant fish in the refuge (average density 96/m2), used the entire refuge, and outcom-peted nonindigenous G. geiseri. The two Gambusia species used similar habitats but showed almost no dietary overlap. High densities of aquatic plants reduced the amount of open water areas necessary for C. elegans. The refuge will sustain the two endangered fishes at this historic site of endemism while maintaining flow to the irrigation system; however, the refuge is not equivalent to a restored ciénega.

Notes: No guidelines are currently available that advise which soil properties of arable land can be used to suggest suitable locations for heathland reconstruction. This paper reviews studies comparing soil properties of heathland or semi-natural grassland with those of adjacent arable fields, investigations in the autecology of the dominant heathland plant, Calluna vulgaris (common heather), and long-term experiments of fertilizer inputs on arable soils. Three properties must be assessed before the suitability of a field can be determined: extractable phosphorus, exchangeable calcium, and pH. A number of other nutrients may also be important, but evidence is currently insufficient to substantiate this. Natural changes in levels of extractable phosphorus, exchangeable calcium, and pH appear to be very slow, so nutrient stripping and acidification will be necessary where recommended levels are exceeded to successfully restore heathland vegetation.

Notes: Ants are widely used as bioindicators in Australian land assessment and monitoring programs, particularly in relation to ecosystem restoration following mining. Little is known, however, about the relationship between ant community development and key ecological processes such as nutrient cycling. We have examined the relationship between ant species richness and soil microbial biomass at 17 sites subject to disturbance by mining in the Kakadu region of Australia's Northern Territory. The number of ant species recorded ranged from 7 at an unvegetated site undergoing restoration to 43 at a site that was undisturbed except for edge effects. Soil microbial biomass ranged from 19.3 to 134.3 μgC/g. Ant species richness was positively correlated with soil microbial biomass (r= 0.638), more so than was plant species richness (r= 0.342 for total plant species, r= 0.499 for woody species only). Our findings demonstrate a correlation between aboveground ant activity and belowground decomposition processes at disturbed sites, thereby providing support for the use of ants as indicators of restoration success following disturbance. Interestingly, when a range of undisturbed sites in the region was considered, a negative rather than positive relationship between ant richness and soil microbial biomass was found. This illustrates the importance of distinguishing between variation within a habitat due to disturbance and variation across different habitats when searching for indicators of ecological change.

Notes: Bottomland hardwood forests in the southcentral United States have been cleared extensively for agriculture, and many of the remaining forests are fragmented and degraded. During the last decade, however, approximately 75,000 ha of land—mainly agricultural fields—have been replanted or contracted for replanting, with many more acres likely to be reforested in the near future. The approach used in most reforestation projects to date has been to plant one to three overstory tree species, usually Quercus spp. (oaks), and to rely on natural dispersal for the establishment of other woody species. I critique this practice by two means. First, a brief literature review demonstrates that moderately high woody species diversity occurs in natural bottomland hardwood forests in the region. This review, which relates diversity to site characteristics, serves as a basis for comparison with stands established by means of current reforestation practices. Second, I reevaluate data on the invasion of woody species from an earlier study of 10 reforestation projects in Mississippi, with the goal of assessing the likelihood that stands with high woody species diversity will develop. I show that natural invasion cannot always be counted on to produce a diverse stand, particularly on sites more than about 60 m from an existing forest edge. I then make several recommendations for altering current reforestation practices in order to establish stands with greater woody species diversity, a more natural appearance, and a more positive environmental impact at scales larger than individual sites.

Notes: We evaluated the ecological and reproductive characteristics of plant species occurring in severely disturbed areas that were revegetated with exotic grasses. We identified those species with the best combination of attributes that increase their probability of success in degraded lands. Fifteen degraded areas were studied in two different bioclimatic regions, a high premontane humid bioclimate and a low premontane humid bioclimate. The frequency of native colonizing species and the presence of arbuscular mycorrhizal colonization in their roots were evaluated. The sexual and breeding system, pollination mode, fruit set, and dispersal syndrome of ten of the most frequent colonizing species were also studied. The floristic survey of the colonizing species revealed a similarity to the reported flora of the treeless savannas that are dominant in the region. Bioclimatic conditions prevailing in the degraded lands seem to be an important factor for the presence of colonizing species and for species richness. All colonizing species studied were mycorrhizal, and for this reason the restoration program in these degraded areas should take mycorrhizae into account, reintroducing them or manipulating the soils to increase the mycorrhizal inoculum. We suggest Scleria cyperina and Trachypogon plumosus to start or promote the natural succession in the degraded areas from La Gran Sabana. Because their frequency is high and their reproductive system is less dependent on biotic factors, these species stand out in the studied areas.

Notes: Disturbance of coastal sage scrub in southern California has led to extensive displacement of native shrubs by exotic annual grasses. The initial conversion from shrubland to exotic grassland is typically associated with disturbance caused by intense grazing, high fire frequency, or mechanical vegetation removal. While native shrubs have been shown to recolonize annual grasslands under some conditions, other annual grasslands are persistent and show no evidence of shrub recolonization. This study examined the mechanisms by which annual grasses may exclude native shrubs and persist after release from disturbance. Grass density was manipulated in experimental plots to achieve a series of prescribed densities. Artemisia californica, a dominant native shrub, was seeded or planted into the plots and responses to the grass density treatments were measured over two growing seasons. A. californica germination, first season growth, and survival were all negatively related to the density of neighboring annual grasses. The most probable mechanism underlying the reduction of first season growth and survival was depletion of soil water by the grasses. The effects of the grasses on A. californica were no longer significant in the second season. The results of this study indicate that Mediterranean annual grasses reduce recruitment and can persist by inhibiting post-disturbance establishment of A. californica from seed. Although succession alone may not return disturbed annual grasslands to their former shrubland composition, the results suggest that restoration can be achieved by using container plantings or grass removal followed by seeding.

Notes: This study evaluated the genetic consequences of a reintroduction of the endangered annual plant Cordylanthus maritimus ssp. maritimus to Sweetwater Marsh (San Diego County, California). A survey of 21 enzyme loci in natural populations revealed that genetic diversity is very low and is primarily found as rare alleles at a few loci, making this species especially susceptible to the loss of alleles and heterozygosity through genetic drift. The reintroduction was performed in 1991 and 1992 by sowing seeds (collected from Tijuana Estuary) in numerous small patches of suitable habitat. For this study, leaf tissue was collected from all plants in all patches during flowering in 1995 and surveyed for genotype at the three enzyme loci that are polymorphic at Tijuana Estuary. Rare alleles were absent in 27 out of 30 patches for Pgm-1, in 17 out of 30 patches for Pgm-2, and in 10 out of 11 patches for Mdh-1. In all, half of the patches lacked any rare allele. Rare alleles tended to occur in patches with few individuals. Overall rare allele frequency was lower than in the colonies from which seeds were collected at two of the three loci, and heterozygosity was reduced. The Sweetwater Marsh population is at risk of losing most of its genetic variation at enzyme loci through the extinction of patches with few individuals. Future reintroduction attempts should attempt to create contiguous sets of patches or to periodically reseed existing patches to reduce the loss of genetic variation.

Notes: Books reviewed in this article: Riparian Ecosystem Recovery in Arid Lands: Strategies and References. Mark K. Briggs Land Restoration and Reclamation: Principles and Practice James A. Harris, Paul Birch, and John Palmer

Notes: Effects of waterlogging were studied in the field and under glasshouse conditions on two clonal lines of Eucalyptus camaldulensis Dehnh (river red gum), which are used in the rehabilitation of damaged agricultural catchments in Western Australia. The plantation of 9-year-old trees was in a position that covered a range of waterlogging and salinity conditions. Up-slope the water table was deeper (0.65–1.5 m), whereas the water table was closer to the ground surface down-slope (0.45 m in winter; 1.25 m in summer). Salinity was greater downslope and increased at the end of the dry summer, remaining high until diluted by the winter rains. Trees of both clonal lines were smaller downslope and used less water over the year. Clone M80 used more water in winter; clone M66 more in summer. In the field, the roots of clone M80 were evenly distributed through the soil profile, while roots of clone M66 decreased with increasing depth. Production of new root terminals varied with season. Greatest production was in spring and early summer, with much lower production over late autumn and winter. Only clone M66 produced new root terminals at depth (60–75 cm) during the drier months of late summer and early autumn. At this time, saline ground-water was the main source for water uptake. To explore clonal differences more closely, the effects of prolonged waterlogging were studied under glasshouse conditions. Clone M80 grew similarly under freely drained and continuously waterlogged conditions for the experimental period (21 weeks). The response under continuously waterlogged conditions was achieved through adventitious root production. By contrast, growth of clone M66 was suppressed under continuous waterlogging, a response associated with the lack of adventitious root production. The results from field and glasshouse studies suggest that clone M80 is more adapted to waterlogging by relatively fresh water than clone M66, but that clone M66 may use water of higher salinity than clone M80. Clone M80 would be better suited to higher positions in partially cleared catchments, where rainfall provides relatively fresh soil water. Clone M66 is better suited to lower catchment positions due to its ability to utilize more saline groundwater. Restoration of the water balance of damaged agricultural catchments can be best managed by matching specialized genotypes with particular catchment positions.

Notes: I conducted small-mammal trapping surveys on a desert scrub restoration site in Palm Springs, California, to document concomitant recovery of the rodent community. These surveys were conducted following quantitative vegetation sampling efforts that indicated that a predefined successful restoration criterion of 15% total shrub cover had been met throughout most of the area. But shrub cover, native shrub cover, herb cover, native herb cover, total cover, and total native cover remained significantly lower in the restoration area than in undeveloped desert scrub immediately surrounding the site. Native herb species richness was also generally lower in the restoration area. Despite these vegetation differences, rodent diversity, evenness, and abundance were very similar between the restoration and natural areas (they were consistently slightly higher in the restoration area). More diverse microhabitats, proximity to water, and reduced competition with harvester ants may have contributed to this outcome. If ecosystem restoration is the goal, reestablishment of a faunal community in restored habitat, rather than surpassing a predefined percent cover of vegetation, may be a better indicator of success, because plant cover proved to be a poor predictor of mammal success.

Notes: Books reviewed in this article: California Rivers and Streams: The Conflict Between Fluvial Processes and Land Use. Jeffery F. Mount Evolution and the Aquatic Ecosystem: Defining Unique Units in Population Conservation. J. L. Nielson, editor

Notes: Closed or abandoned landfills represent significant land areas, often in or near urban centers, that are potential sites for ecological restoration of native woodlands. But current guidelines in many jurisdictions do not allow for the installation of trees or shrubs above landfill clay caps, although these plants have many environmental, functional, and aesthetic advantages, including a rapid start to community succession. Typical closure procedures for capped landfills include only a grass cover to control moisture infiltration and impede soil erosion. The main concern that limits the application of a woody cover to a closed landfill is that roots may penetrate and weaken the clay cap. As part of a comprehensive experimental program on woodland restoration, we installed 22 tree and shrub species on Staten Island, New York (the Fresh Kills Sanitary Landfill). We found no evidence that roots of the transplanted woody plants penetrate caps used on these landfills. Root growth requirements and dynamics stop penetration of these materials. Anoxic and acidic conditions were found in the sandy subsoil above the cap, as indicated by corrosion patterns on steel test rods. Also, the intensity of mycorrhizal infection on the experimental plants was high in the surface soil and decreased progressively with increasing soil depth. The potential vertical rooting depth during this time period was greater than that occurring over the clay cap. This was shown from data collected on a nearby control site, where seven of the species were installed on an engineered soil lacking a clay barrier layer, and roots of all seven species penetrated deeper than on the landfill. The engineered landfill soils are poor growth media for roots, and below ground constraints that limit restoration on these sites must be addressed.

Notes: The Northern Rocky Mountain Wolf Recovery plan proposed reintroduction of Canis lupus (gray wolf) to Yellowstone National Park and central Idaho as part of a wolf restoration plan for the northern Rocky Mountains of the United States. Strong opposition from some factions within the region forestalled the action for two decades. An environmental impact statement, conducted in 1992–1994 with extensive public input, culminated in a proposal to reintroduce wolves designated as “non-essential—experimental” under Section 10 (j) of the federal Endangered Species Act. This approach, approved by the Secretary of the Interior in 1994, provided for wolf restoration while allowing management flexibility to deal with concerns of the local public. A reintroduction plan was developed in the summer and fall of 1994. Acquiring, holding, transporting, and releasing suitable wolves for reintroduction presented a myriad of technical and logistical challenges that required effective planning and coordination by all participants. In January 1995, 29 wolves were captured in Alberta and transported to Yellowstone National Park (14) and central Idaho (15). Idaho wolves were freed immediately upon arrival; Yellowstone wolves (three family groups) were held in acclimation pens in the park until late March. Most Idaho wolves traveled extensively within the area intended for them, averaging 82 km net distance away from release sites after 5 months (range = 30–220 km), and three male-female pairs formed by July. After 5 months in the wild, at least 13 of 15 Idaho-released wolves were alive within the intended area, as were 13 of 14 Yellowstone wolves; one wolf was known to have been illegally killed in each area. No livestock were killed. Wolves released into Yellowstone Park continued to live as packs, stayed closer to their release sites (x = 22 km at end of June), and settled into home ranges; two packs produced a total of nine pups. The progress of the reintroduction program in its first year far exceeded expectations. Reintroductions of about 15 wolves to each area for 2–4 more years are scheduled, but the project may be shortened because of early successes. Future reintroduction planners can expect sociocultural issues to pervade the effort, but they can be optimistic that, from a biological standpoint, reintroduction of wolves has strong potential as a restoration technique.

Notes: This paper summarizes the accumulation of nutrient capital during the natural afforestation of iron-smelting slag. The study site was the Beckley Furnace (North Caanan, Connecticut, U.S.A.) slag dump, which was abandoned around 1918. Currently, a Pinus strobus (white pine)-dominated forest occupies the site. Our primary objective was to determine the relationships among stand development and belowground nutrient pools to evaluate controls on the rates at which nitrogen, calcium, magnesium, and potassium accumulate in the developing forest. Along a developmental gradient of increasing stand basal area, we measured (1) aboveground biomass, (2) root biomass, (3) above-ground nutrient capital, (4) belowground nutrient capital, (5) soil weight, (6) soil organic carbon weight, (7) soil moisture, and (8) understory richness and density. Regression analysis suggests, as expected, that the addition of organic matter controls the accumulation of above- and belowground nutrients, soil water, and understory plant composition and distribution. The rather rapid rate of nitrogen accumulation suggests that allochthonous as well as autochthonous organic matter is an important source of nutrients for the developing forest soil and vegetation. Compared to those of other northeastern forests, soils in the most developed areas at the Beckley site have accumulated more than enough labile nutrient capital in 75 years to support a forest typical of the region. In the most developed stands, understory composition indicates mesic soil conditions, and that within the next 75 years or so the Beckley slag dump will be floristically very similar to the surrounding forest.

Notes: Extensive land loss, which is mostly wetland loss, has taken place during this century in the Mississippi River delta and other river deltas. Our purpose was to evaluate the effectiveness of constructing “artificial” crevasses, or cuts in the natural levee, made by the U.S. Fish and Wildlife Service in the Delta National Wildlife Refuge (DNWR) to slow or reverse this type of land loss. Land growth of the crevasses was determined from aerial photographs and was related to crevasse-site characteristics. The newly constructed crevasses create emergent wetlands after 2 years of subaqueous growth at about 4.7 ha/year and an average cost of $21,377 per crevasse. The present total cost per hectare declines with age as new land builds, and it will equal $48 per hectare if all the open water in the receiving ponds fills in. At these rates, the net land loss rates in the DNWR measured from 1958 to 1978 would be compensated for by the building of 63 crevasses, 24 of which are already in place.

Notes: This paper describes an ecosystem approach (the Bradbury framework) to prioritizing watersheds for watershed restoration and salmon recovery, and gives an example of its application. The framework was applied at three spatial scales (in descending urder) to prioritize (1) river basins within the north coast geographic area of Oregon (USA), (2) watersheds within the Tillamook Bay basin, and (3) restoration activities at the watershed level, Implementing the framework identified the Nehalem and Tillamook Bay basins as high priority for the north coast of Oregon. Within the Tillamook Bay basin, the Wilson, Kilchis, and Trask river watersheds emerged as high priority. Preliminary analysis indicated that controlling sediment sources by addressing upland road conditions and allowing floodplain and riparian ecosystems to recover are highest priority protection and restoration activities within the Tillamook Bay basin. The sample application demonstrates that an ecosystem approach (the Bradbury framework) is particularly advantageous where data are limited, although previous identification of relatively intact areas is required. Implementing the framework is intended to lead to restoration of native species, but it may not provide immediate assistance for some species or populations of concern.

Notes: This paper describes a two-stage system for prioritizing stream reaches and riparian communities along a given river for protection or restoration. The system uses associations between geomorphology and riparian vegetation at stream reach and community scales as a basis for defining reference conditions. First-stage reach classification involves collecting and analyzing data from topographic maps and aerial photographs. These data, along with judgment-based criteria for ranking reaches relative to reference conditions, are used to classify stream reaches as suitable for protection, recommended for mitigation or restoration within existing site-specific regulatory procedures, or requiring further analysis to evaluate community-scale restoration needs. Second-stage field sampling is conducted on the reaches needing further analysis to determine the riparian communities present, the associations between communities and floodplain landforms, and reference community conditions. This stage requires collection of fields data on geomorphic conditions, plant species composition, and plant community structure. Cluster analysis or a comparable technique is used to classify plant communities associated with floodplain landforms and identify reference conditions for each landform. Community structure and species compositiotion are compared to reference conditions to define restoration possibilities at the community scale. The combined results from stream reach and community scale analysis provide a strategy for protecting and restoring riparian resources for a whole river. Implementation requires further site-specific information on hydrology, geomorphology, and other factors.

Notes: In this study, we examined the site-specific effects of a large episodic flood on the riparian vegetation within the floodplain of the San Luis Rey River in southern California. Using multispectral airborne videography, we quantified percent cover of riparian vegetation, cultivated agricultural land, urban surfaces, upland vegetation, bare soil, and water within 22 sections of flood-plain, both before and after a large flood (January 13, 1993). We also quantified the amount of these cover types within bands of the watershed 1 km wide × 5 km long directly upstream of each floodplain site. The amount of riparian vegetation destroyed by the flood within each section varied from nearly zero to almost 40% of pre-flood coverage. The magnitude of loss in riparian vegetation was most strongly related to the amount of riparian vegetation initially present in the floodplain and the amount of urban surfaces in the nearby watershed. These results suggest that riparian vegetation within the San Luis Rey River floodplain is generally at high risk of destruction from large floods, and that this risk is exaggerated in areas with high urban development. We infer from these results that sites near existing large areas of intact riparian vegetation and away from urban development will have the highest potential for successful long-term restoration.

Notes: Airborne multispectral videography has become a useful tool for classifying and mapping riparian vegetation because its spectral and digital format has high spatial resolution. This paper explains the processing steps and procedures required to use this type of spectral imagery for riparian vegetation classification and mapping. A description of the Utah State University airborne system is given, along with the image processing steps used for developing finalized products, because these are used in other articles in this volume. Examples of riparian applications using high-resolution imagery are included.

Notes: Nine Articles in the special issue of Restoration Ecology addrssing the subject of site selection for riparian restoration activities were critically examined for this review. The approaches described make significant and original contributions to the field of riparian restoration. All are interdisciplinary to some extent, often combining the fields of hydrology, geomorphology, and biology in the design of restorationss. A common component among the articles is that they take a broad view, if not a watershed view, of restoration site selection. The approaches can be generally descrobed as top-down strategic approaches to siting restorations, as opposed to the more methods- and site-driven bottom-up, or tactical, approach. All the articles recognize the importance of developing endpoints related to the ecological function of riparian ecosystems. they succeed in their quest for these indicators of ecological function to varying degrees. The most common indica-for used in these papers is riparian vegetation. Several additional elements of scientific investigation, if successfully pursued, could provide vital information and advance our understanding of riparian restoration: developing interdisciplinary approaches more fully; defining endpoints and reference conditions; implementing multiple scale approaches; viewing restorations as experimental ecosystem manipulations; developing a philosophy regarding exotic species; incorporating geographic information systems more often; and integrating science, society, and politics. the foundation provided by the contributions in this issue should provide a strong basis for the rapid advancement of future research in the area of riparian restoration.

Notes: Successional models are used to predict how restoration projects will achieve their goals. These models have been developed on different spatial and temporal scales and consequently emphasize different types of dynamics. This paper focuses on the restoration goal of self-sustainability, but only in the context of a long-term goal. Because of the temporal scale of this goal, we must consider the impact of processes arising outside of the restoration site as of greater importance than restoration itself. Because ecological systems are open, restoration sites will be subjected to many external influential processes. Depending on the landscape context, the impact of these processes may not be noticeable, or, at the other extreme, they may prevent the achievement of restoration objectives. A second issue is to emphasize the nature of processes in the long term, that they are a complex of characteristics such as magnitude, frequency, and extent. Ecological systems are only adapted to a range of values in each of these characteristics. Restoration often combines goals that are of different scales. Models appropriate to these goals need consideration.

Notes: The ecosystem perspective provides a framework within which most other aspects of the ecology of restoration can be incorporated. By considering the ecosystem functions of a restoration project, the restorationist is forced to consider the placement of the project in the landscape—its boundaries, its connections or lack thereof to adjoining ecosystems, and its receipts and losses of materials and energy from its physical surroundings. These characteristics may set limits on the kind(s) of biotic communities that can be created on the site. The ecosystem perspective also gives restorationists conceptual tools for structuring and evaluating restorations. These include the mass balance approach to nutrient, pollutant, and energy budgets; subsidy/stress effects of inputs; food web architecture; feedback among ecosystem components; efficiency of nutrient transfers, primary productivity and decomposition as system-determining rates; and disturbance regimes. However, there are many uncertainties concerning these concepts, their relation to each other, and their relationships to population- and community-level phenomena. The nature of restoration projects provides a unique opportunity for research on these problems; the large spatial scale of restorations and the freedom to manipulate species, soil, water, and even the landscape could allow ecosystem-level experiments to be conducted that could not be performed otherwise.

Notes: Community ecological theory may play an important role in the development of a science of restoration ecology. Not only will the practice of restoration benefit from an increased focus on theory, but basic research in community ecology will also benefit. We pose several major thematic questions that are relevant to restoration from the perspective of community ecological theory and, for each, identify specific areas that are in critical need of further research to advance the science of restoration ecology. We ask, what are appropriate restoration endpoints from a community ecology perspective? The problem of measuring restoration at the community level, particularly given the high amount of variability inherent in most natural communities, is not easy, and may require a focus on restoration of community function (e.g., trophic structure) rather than a focus on the restoration of particular species. We ask, what are the benefits and limitations of using species composition or biodiversity measures as endpoints in restoration ecology? Since reestablishing all native species may rarely be possible, research is needed on the relationship between species richness and community stability of restored sites and on functional redundancy among species in regional colonist “pools.” Efforts targeted at restoring system function must take into account the role of individual species, particularly if some species play a disproportionate role in processing material or are strong interactors. We ask, is restoration of habitat a sufficient approach to reestablish species and function? Many untested assumptions concerning the relationship between physical habitat structure and restoration ecology are being made in practical restoration efforts. We need rigorous testing of these assumptions, particularly to determine how generally they apply to different taxa and habitats. We ask, to what extent can empirical and theoretical work on community succession and dispersal contribute to restoration ecology? We distinguish systems in which succession theory may be broadly applicable from those in which it is probably not. If community development is highly predictable, it may be feasible to manipulate natural succession processes to accelerate restoration. We close by stressing that the science of restoration ecology is so intertwined with basic ecological theory that practical restoration efforts should rely heavily on what is known from theoretical and empirical research on how communities develop and are structured over time.

Notes: Conceptual and logistical challenges associated with the design and analysis of ecological restoration experiments are often viewed as being insurmountable, thereby limiting the potential value of restoration experiments as tests of ecological theory. Such research constraints are, however, not unique within the environmental sciences. Numerous natural and anthropogenic disturbances represent unplanned, uncontrollable events that cannot be replicated or studied using traditional experimental approaches and statistical analyses. A broad mix of appropriate research approaches (e.g., long-term studies, large-scale comparative studies, space-for-time substitution, modeling, and focused experimentation) and analytical tools (e.g., observational, spatial, and temporal statistics) are available and required to advance restoration ecology as a scientific discipline. In this article, research design and analytical options are described and assessed in relation to their applicability to restoration ecology. Significant research benefits may be derived from explicitly defining conceptual models and presuppositions, developing multiple working hypotheses, and developing and archiving high-quality data and metadata. Flexibility in research approaches and statistical analyses, high-quality databases, and new sampling approaches that support research at broader spatial and temporal scales are critical for enhancing ecological understanding and supporting further development of restoration ecology as a scientific discipline.

Notes: Along rural roadsides of the Sacramento Valley of California, we seeded native and non-native perennial grasses to gauge their potential value in roadside vegetation management programs. In trial I (polycultures), three seeded complexes and a control (resident vegetation only) were tested. Each seeded plant complex included a different mix of perennial grasses seeded into each of several roadside topographic zones. The seeded levels of plant complex were: native perennial grasses 1 (8 species); native perennial grasses 2 (13 species); and non-native perennial grasses (3 species). In trial II, plots were seeded to monocultural plots of 15 accessions of native Californian and three cultivars of non-native perennial grasses. Plots in both trials were seeded during January 1992 and evaluated for three successive years.

Notes: We studied the utility of gap formation and soil disturbance as methods to enhance establishment of plant species in the understory of a northern Kentucky forest where Lonicera maackii (Amur honeysuckle) produced dense thickets. In May 1994, gaps (5 m diameter) were cut in the shrub thicket. In adjacent areas, the shrub canopy remained intact. Subplots were established where soil was either turned with a spade to a depth of 15 cm or not disturbed. We monitored plant establishment for three growing seasons (1994, 1995, and 1996). Shrub removal increased light availability to about 10% of full sun. Gap formation had a significant (p 〈 0.05) and positive influence on total plant density (exclusive of L. maackii), and soil disturbance did not (p 〉 0.05). After three growing seasons, the most important species were L. maackii, Alliaria petiolata, Parthenocissus quinquefolia, Vitis vulpina, and Acer negundo. Of these species, only V. vulpina showed significantly (p 〈 0.05) higher densities in gaps. Other less important species such as Phytolacca americana, Campsis radicans, and Eupatorium rugosum occurred almost exclusively in gaps. Of the 44 taxa observed in this study, most were generalist species that also occur in early successional habitats. Long-term dominance of the understory by L. maackii has likely modified system attributes with corresponding effects on community development. Shrub removal provides a window of establishment for various plant species, but successful restoration may require further management species availability and to control new invaders.

Notes: Many forest roads are being closed as a step in watershed restoration. Ripping roads with subsoilers or rock rippers is a common practice to increase the infiltration capacity of roads before closure. When considering the effectiveness of ripping for reducing runoff and erosion and the potential reduction in slope stability by saturating road fills, it is important to know how ripping changes the infiltration capacity of forest roads. Hydrographs from simulated rainfall on 1 × 1 m plots were analyzed to find the saturated hydraulic conductivity, an indicator of infiltration capacity. I examined saturated hydraulic conductivity for three treatments on two different soils. One road was built in a soil derived from the metamorphic belt series geology of northern Idaho, a soil noted for its high rock fragment content. The second road was built in a sandy soil derived from decomposed granitics of the Idaho batholith. On each soil, five plots were installed on a road before ripping, and nine plots were installed on the same road segment following ripping, four covered with a heavy straw mulch and five without. Three half-hour rainfall events with intensities near 90 mm/hr were simulated on each plot. Results show that ripping increases hydraulic conductivities enough to reduce risk of runoff but does not restore the natural hydraulic conductivity of a forested slope. The unripped road surfaces had hydraulic conductivities in the range of 0–4 mm/hr, whereas ripped roads were in the range of 20–40 mm/hr after the second event. Surface sealing and tilled soil subsidence processes are important in reducing the hydraulic conductivity of the soils with repeated wetting. Subsidence appears to be important on the granitic soil, whereas surface sealing was more important on the belt series soil.

Notes: Rainforest restoration is a relatively new endeavor, and few attempts have been made to assess the success of such restoration efforts in terms of the reestablishment of an ecosystem. Small plantings of rainforest tree species have been carried out adjacent to mature rainforest at Lake Barrine National Park in North Queensland, Australia, since 1988. The aim of this project was to assess the leaf litter invertebrate fauna of these plantings as indicators of the success of the restoration process. Plots planted in 1988, 1989, and 1990, as well as adjacent mature rainforest, were sampled in the wet and dry seasons of 1994. Invertebrates were extracted from leaf litter samples with Berlese Funnels and sorted to order. Diversity, the abundance of different size classes and orders of invertebrates, and the abundance of different functional groups were examined. In most respects the 1988 plot was found to differ little from the mature rainforest plots, whereas the 1990 plot lacked small and predatory invertebrates, especially in the dry season. The 1989 plot was intermediate in invertebrate abundance and diversity. The use of partially deciduous trees in the 1989 and 1990 plots, resulting in lower canopy cover at the driest time of the year, may have contributed significantly to the differences found between the early and later plantings. It is recommended that trees that provide good canopy cover year-round be used as dominant species in plantings to facilitate the development of a leaf litter ecosystem that can be sustained throughout the year.

Notes: Tidal wetland mesocosms at Tijuana River National Estuarine Research Reserve failed to elucidate effects of hydrologic treatments (excluded, impounded, and fully tidal systems) for most parameters measuring Salicornia virginica (pickleweed). Although soil salinity increased where tidal flushing was excluded for 10 months (salinities rose ∼20 to 50%), pickleweed cover and algal chlorophyll did not differ among treatments. Effects were seen only in pickleweed growth rates (∼30% decrease where tides were excluded) and normalized difference vegetation index (NDVI) measurements. We failed to show any differences between impounded and fully tidal conditions, because the mesocosms had coarse sediments, and impounded water drained easily via subsurface flow. However, the problems that we encountered with the mesocosms led to the following advice for future wetland restoration projects: (1) Mesocosms are useful for testing restoration techniques before an actual restoration project takes place. (2) Mesocosms should be used to test factors that may lead to more successful restoration in the future, including planting techniques, substrate conditions, and hydrology. (3) Mesocosms should be used to develop new assessment methods for monitoring wetland ecosystems. Because of the ability to control some environmental parameters while maintaining seminatural conditions, mesocosms offer great potential for the future evaluation of experimental restoration techniques.

Notes: Habitat protection is a major component of the Exxon Valdez oil spill restoration process. The acquisition of private lands, or partial interests in private lands, is intended to promote natural recovery of spill-injured resources and services by removing the threat of additional development impacts. The Comprehensive Habitat Protection Process is the method that was designed to achieve this objective. Over one million acres within the oil spill affected area were evaluated, scored, and ranked by a multi-criteria evaluation process. Initially, lands were divided into large parcels encompassing entire bays and watersheds. Criteria were then used to assess the habitat and human-use values associated with each parcel and the protection benefit that acquisition would provide for 19 injured resources and services. This process has been the basis for the acquisition of 41,549 acres of land on Afognak Island and 23,800 acres on the Kenai Peninsula and for agreements that, if consummated, will result in the acquisition of fee or lesser rights on over a half million acres of land in the Kodiak Archipelago, on the Kenai Peninsula, and in Prince William Sound. All of these lands or rights, if acquired, will be incorporated into parks or refuges or otherwise managed in a manner that will facilitate the recovery of the resources and services injured by the oil spill.

Notes: Agricultural set-aside and compensation land provide restoration ecologists with opportunities to re-create semi-natural habitats. Restoration sites often have high soil fertility and inadequate seed banks of desirable species. Sowing additional seed is a proven method for establishing chalk grassland vegetation. If seed of local provenance is required, it may be collected by hand or by using specialized machines. Ultimately, the mix collected must provide seed suitable for recreating vegetation similar to that of the donor site. We examine the ability of a vacuum machine to meet this requirement by comparison with hand collection, and we discuss possible effects on invertebrates. Microscope analysis and glasshouse and field trials were used to compare the abundance of seed of different species in harvested mixes with the vegetation composition of the donor site. Seed heads of individual species were examined to determine the number of viable seeds per head and attack rates by phytophagous insects. The mix contained seed of over half the species recorded on the donor site. The seed of taller, more common species was overrepresented in the mix, at the expense of some smaller, mat-forming plants. After one season, however, the vegetation of the field trial plots was of the same type as that of the donor site, although the proportions of the constituent species differed slightly and certain species were absent. Mechanical collection is more efficient than hand collection. Endophagous invertebrates are unlikely to be affected by the machine. Seed collection requires a combination of methods, precise timing, and careful planning to provide a full range of species and to minimize impacts on plant and invertebrate populations.

Notes: Abandoned limestone quarries are hostile environments for plant and invertebrate colonization and establishment. The length of time taken for successful establishment by natural processes may be unacceptable for reclamation purposes; several techniques are used to reduce the time scales involved. A new technique, restoration blasting, aims to replicate natural daleside landforms by selective blasting of modern production quarry faces. We compare the flora and invertebrate fauna of restoration-blasted sites, hydro-seeded with daleside species, with naturally regenerating disused quarries and a natural daleside. Restoration-blasted sites were found to have less plant cover, more bare ground, fewer orders of invertebrates, and generally fewer animals within each order than the other two types of site. The disused quarries tended to have intermediate characteristics between the restoration-blasted sites and the natural daleside. The age of the site may be important in determining the plants and invertebrates occurring there. This may be related to the time available for establishment or a greater degree of settlement or stability within the biotic and abiotic components of the site. Although most of the results indicate that time since establishment may be important, some variations occur. In particular, the development of vegetation cover in areas grazed by rabbits is problematic. These results are important in the assessment of successful reclamation because the invertebrate fauna may contribute greatly to the overall system. Both plant and animal communities appear to be establishing well on the sites reclaimed by restoration blasting. Further monitoring will identify the speed at which such environments achieve the desired aim of replicating daleside communities and the communities best able to be sustained following this technique.

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: . Gram- symbiotic X-bacteria present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein (S29x) into the host's cytoplasm across bacterial and symbiosome membranes. The S29x protein produced by E. coli transformed with the s29x gene is also rapidly secreted into the culture medium. Inside amoebae, S29x enters the host's nucleus as detected by confocal and irnmunoelectron microscopy, although it is not clear if S29x is selectively accumulated inside the nucleus. The deduced amino-acid sequence of S29x has a stretch of basic amino acids that could act as a nuclear localization signal, but there is no signal peptide at the N-terminus and the transport of S29x is energy independent. The functions of S29x are not known, but in view of its prominent presence inside the amoeba's nucleus, S29x is suspected to be involved in affecting the expression of amoeba's nuclear gene(s).

Notes: . We used a photoactivatable fluid-phase marker to isolate a new collection of thermosensitive endocytosis mutants in the cellular slime mold Dicfyostelium discoideum. All the strains were thermosensitive for growth on bacteria or axenic medium at 27° C. Initial rates of endocytosis rapidly decreased upon incubation at the restrictive temperature, but surprisingly most of the strains showed a transient recovery of activity with prolonged exposure to 27° C. Endocytosis and exocytosis activities were uncoupled for some of the cell lines at 27° C whereas the others had to be shifted to 29° C. Further molecular analysis of these mutants could lead to the discovery of new proteins involved in endocytosis and its regulation.

Notes: . Monoclonal antibodies against spores of Glugea atherinae were obtained after lymphocytic hybridization made from immunized mouse splenocytes. Screening using an indirect enzyme linked immunosorbent assay (ELISA), revealed seven monoclonal antibodies with an intense but variable reaction with the spores of fish microsporidia, and a moderate reaction with those of an insect microsporidium (Nosema sp.). The reaction was weaker with spores of Encephalitozoon intestinalis found in HIV' patients. FITC and Dot Blot confirmed the majority of these results. After biotinylation of the seven antibodies, inhibition tests allowed the localization of two different recognition domains on the spores of Glugea atherinae. The multiple antigenic determinants and their probable polysaccharide nature seem to be in accord with the class IgM of the antibodies produced. This work confirms the potential of these antibodies for microsporidian taxonomy and diagnosis, especially the use of Mabs 12F9 and 12H5 for detection of spores in stools of HIV+ patients.

Notes: . Leidyana canadensis n. sp. (Apicomplexa: Eugregarinida) is described from larvae of the eastern hemlock looper, Lambdina fiscellaria (Lepidoptera: Geometridae) collected near St. Stephen, Charlotte County, New Brunswick, Canada. Gamonts solitary, located between host ventricular peritrophic membrane and ventricular epithelium. Protomerite very broadly ellipsoid with transverse posterior margin; length 43.7 μm, width 42.7 μm; protomerite-deutomerite septum strongly constricted. Deutomerite narrowly obovoid with anterior transverse margin; length 186.6 μm, width 58.7 μm; total length 227.1 μm. Nucleus spherical to broadly ellipsoid; length 26.3 μm, width 20.2 μm; placement abaxial in the posterior 2/3 of the deutomerite. Nucleus often obscured in late gamonts. Association late, caudofrontal, ephemeral and leading directly to syzygy. Gametocysts roughly spherical; diameter 216.7 μm; hyaline coat increasing diameter to 359.1 μm. Gametocysts mature and dehisce through six short spore tubes within 48 hours. Oocysts axially symmetric, dolioform in dorsal aspect, compressed in the plane perpendicular to the major axis, very uniform in size and shape; length along major axis 5.2 μm, terminal width 1.8 μm, medial width 3.8 μm; extruded in chains.

Notes: . Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze-fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris, the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin-cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.

Notes: . Cultivation of Pneumocystis carinii from rabbit onto cell monolayers was intended. Three cell types were used: rat lung derived cell line L2 and canine kidney derived cell line MDCK, and primary epithelial rabbit lung cells derived from 20 days old foetuses. These primary cells were also immortalized by transfection of the coding sequences for the large and small T antigens of the SV40 virus. P. carinii were extracted from lungs of corticoid treated young rabbits and were purified on Ficoll. In vitro development of P. carinii was assessed by counting the number of attached parasites on cells after staining. No development was observed on L2 and MDCK cell lines. On the contrary, a development was observed on rabbit derived cells with a threefold increase of attached parasites on the third day of culture. Immortalized cells allowed also multiplication of attached P. carinii. These results are similar to those obtained with culture of rodent P. carinii onto cell monolayers.

Notes: . A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila, Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate rearrangement junctions. We have mapped and sequenced two additional rearrangement variants and find that both the left and right can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product.

Notes: . In recognizing food particles, Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradietns. Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H+-ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis.

Notes: . In situ hybridization using fluorescent oligonucleotide probes complementary to unique regions of 16S rRNA molecules provides a way of identifying the food vacuole contents of bactivorous protists. Laboratory experiments with Tetrahymena showed rRNAs in food vacuoles are degraded slowly enough to permit their use as hybridization targets for such probes. A probe specific for a hypervariable region of the small subunit rRNA of an unnamed proteobacterium abundant in a local lake was then synthesized. It was used to probe the food vacuoles of the ciliates present in fixed water samples collected from the same lake. The vacuoles of several filter-feeding ciliates bound the probe, indicating that such probes can be used to identify the food vacuole contents of ciliates collected from natural samples.

Notes: . Oxymonas has the characteristic structures and organization of other oxymonads including two separated pairs of basal bodies/flagella, a preaxostylar lamina, a paracrystalline axostyle, and an absence of mitochondria and Golgi. Like other Oxymonadinae genera it possesses a long proboscis, the rostellum which is terminated by the holdfast. Like the genera Pyrsonympha and Streblomastix, Oxymonas possesses a holdfast which permits it to attach to the cuticle of the termite hind-gut. This holdfast is subdivided into rhizoids and is filled with microfilaments. The rostellum is variable in length and contains two distinct microtubular bundles. One bundle is composed of convoluted microtubular ribbons which originate at the base of the holdfast and extend posteriorly along the rostellum and before penetrating into the cell body. The second bundle is composed of flexuous free microtubules which originate at different levels of the rostellum, increasing in number from top to base. They occupy the axial part of the rostellum and incorporate into the axostylar rows at the basal body/flagellar level. Microtubules of the paracrystalline axostyle are cross-linked by bridges forming parallel rows like in the contractile axostyles of other oxymonads such as Pyrsonympha and Saccinobaculus. Most of the microtubules of the axostyle originate at the flagellar/preaxostylar level but some originate from the axial flexous free microtubules of the rostellum, as indicated above. The possibility of an extension/retraction of the rostellum, suggested by other authors, is discussed.