ALBERT

Notes: Livestock have been excluded from riparian zones along many streams in western North America in an effort to restore aquatic and riparian habitat degraded by livestock grazing. Within these exclosures, channel adjustment to elimination of grazing pressure may lag behind plant recovery because of the time required to deposit sediment along the vegetated banks of the stream channel. Moreover, unless grazing is eliminated from the watershed, the channel within the exclosure must still accommodate increased runoff and sediment loads from upstream. This hydrologic regime may prevent a return to predisturbance channel morphology. Cross sections of the North Fork Cottonwood Creek in the White Mountains of California showed no significant difference in channel width within and downstream of a 24-year-old exclosure, despite a lush growth of stream bank vegetation that gives the impression of a narrower channel within the exclosure.

Notes: Many felids are threatened by loss of habitat, lack of genetic diversity, and over-exploitation. The reintroduction of bobcats (Felis rufus) to Cumberland Island, Georgia provided an opportunity to reintroduce a mid-sized felid without the concern for species survival that is paramount with endangered species. We captured bobcats from the coastal plain region of Georgia, briefly held them in captivity, and released them on Cumberland Island. We describe and evaluate the protocols and techniques used to accomplish the reintroduction. Future reintroductions of felids should consider the problem of post-release dispersal, although our island was relatively isolated and inhibited dispersal. Also, any reintroduction effort should invest effort and resources into post-release monitoring of the population. Empirical knowledge about the effects of spatial distribution, genetics, population dynamics, especially mechanisms of population regulation, behavior, and environmental conditions on the viability of populations is critical to the conservation of endangered species. Future research of the bobcats on Cumberland Island will be able to address aspects of the population and genetic dynamics of a small, insular felid population.

Notes: We studied the use of mineland wetlands by birds and the relationship between avian communities and wetland characteristics. Data were collected from 20 wetlands in Pike County, Indiana, and included wetland size, depth, water conductivity and salinity, aquatic macroinvertebrate abundance, vegetation, and bird use. Principal component analysis showed that physical variables could be explained by two principal component scores and that wetlands could be grouped on the basis of size and conductivity. Principal component analysis could not reduce vegetation variables to fewer principal component scores, meaning that wetland vegetation characteristics were independent of one another and did not show any trend. Most wetlands had low invertebrate density, and wetlands with higher invertebrate density had low invertebrate diversity. Wetlands with similar habitat characteristics (physical, vegetative, and invertebrate) did not necessarily show similarities in bird assemblages. Bird similarity index values ranged from 0 to 59%, implying that each wetland has its own bird community. Stepwise multiple regression analysis (α= 0.05) relating bird use and habitat characteristics showed that bird species richness increased with the species richness of submergent vegetation and was correlated negatively with the species richness of emergent vegetation. There was no significant relationship between bird species richness or bird species diversity and wetland size. The number of species within different avian guilds correlated with different habitat characteristics. The species richness of submergent plants was a factor that correlated positively with the number of species of several guilds (dabblers, wading birds, and plunge divers). Wetland age was not a factor that determined bird use.

Notes: Grass seeding is widely used for erosion control, but its consequences for soil and regeneration following fire have been measured only infrequently. This study investigates the effect of grass seeding on the type and extent of plant cover; soil moisture percentage; and moisture stress, survival, growth, and root-tip and mycorrhiza formation of Pinus lambertiana (sugar pine) seedlings in a clearcut intensely burned by wildfire. One-year-old containerized sugar pine seedlings were planted in seeded and nonseeded areas in Spring 1988 and 1989 in the Longwood Fire area of southwest Oregon. In 1988, tree seedlings in grass-seeded plots experienced intense competition from the grass, reduced root-tip and mycorrhiza formation, low levels of soil moisture to meet evapotranspirational demand, high levels of mortality, and reduced growth. In 1989, however, the opposite was true: tree seedlings in nonseeded plots experienced competition from invading native annuals and perennials, low levels of soil moisture in summer, and higher levels of mortality. The studies we report here further indicate that, in an area characterized by extended summer drought, annual ryegrass impeded regeneration of sugar pine during the first season following the fire. Native species cover and richness have been significantly reduced in the seeded area and may affect long-term soil stability, productivity, and conifer restoration. Seeding of annual ryegrass at high rates under these conditions would seem ill advised.

Notes: Books reviewed in this article: Reintegrating Fragmented Landscapes towards Sustainable Production and Nature Conservation. R. J. Hobbs and D. A. Saunders. editors Restoring Acid Waters: Loch Fleet 1984–1990. G. Howels, and T.R.K. Dalziel, editors

Notes: In prairie restoration, use of seeds from nonlocal sources has been of concern to restorationists. We examined the specificity between vesicular-arbuscular mycorrhizal fungi obtained from a single location and little bluestem obtained from three localities. Seed was obtained from three sources: (1) a commercial seed supplier in Nebraska, (2) Sand Ridge State Forest (SRSF), Mason County, Illinois, the site from which the experimental soil containing the mycorrhizal inoculum was obtained, and (3) Sand Prairie Scrub Oak Nature Preserve (SPSO), 32 km southwest of SRSF. Plants were grown in three substrates: (1) autoclaved soil, (2) autoclaved soil to which a mycorrhizal fungal-free sieving of nonautoclaved soil was added, and (3) nonautoclaved soil. All plants grown in nonautoclaved soil were colonized by mycorrhizal fungi, whereas none of those grown in other substrates were colonized. Plants grown from SRSF seeds produced significantly (p 〈 0.05) more biomass than those grown from Nebraska seeds (X̄± SE, SRSF = 0.54 ± 0.04 g, SPSO = 0.49 ± 0.03 g, Nebraska = 0.37 ± 0.03 g). Plants grown in nonautoclaved soil, regardless of seed source, produced less biomass (0.27 ± 0.02 g) than plants grown in autoclaved soil (0.58 ± 0.03 g) or autoclaved soil plus sievings (0.59 ± 0.03 g).The results provide no clear indication of a host-endophyte specificity. However, the data suggest that the local genotypes of S. scoparium are better adapted to their native soil environment than are genotypes from other localities.

Notes: Glasshouse trials, using trickle irrigation and increasing levels of NaOH-induced alkalinity, identified species that could be expected to tolerate the high-pH conditions of bauxite processing waste residue sites. Of 29 taxa tested, the most tolerant were Casuarina obesa, Melaleuca lanceolata, M. armillaris, M. nesophila, Eucalyptus loxophleba, E. halophila, E. platypus, Tamarix aphylla, and a particular clone of E. camaldulensis; E. spathulata, E. tetragona, E. preissiana, E. gomphocephala, E. diptera, and E. occidentalis proved to be relatively sensitive to severe alkaline conditions. Tolerance appeared to relate to an ability to maintain root membrane function, nutrient uptake balance, and ultimately root tissue structure while under increasing levels of alkalinity stress. Species normally inhabiting alkaline soils tended to have increased growth rates in nutrient irrigation conditions between pH 8 and 10 compared with control plants irrigated with nutrient solutions of pH values near 7.4. However, once the irrigation solutions reached pH 12 and the buffering capacity of the soil appeared to be exceeded, the condition of susceptible plants rapidly declined and death followed. Sensitive plants initially showed symptoms related to nutrient deficiency, followed by wilting and death as the root systems failed. Field trial conditions in the bauxite residue impoundments at Kwinana, Western Australia, include soils with pH values as high as 11.00. In general, the relative survival and growth of seedlings after eight months were predicted by the response under glasshouse trial conditions. Appropriately designed stress trials can be important ecological techniques in choosing species most capable of surviving difficult environmental conditions in the rehabilitation of damaged landscapes.

Notes: In ecological restoration, nonindigenous species can pose a major problem because they are often aggressive and can overwhelm native species, thus altering ecosystem structure. This article identifies the circumstances in which prospects for use of restoration technology in controlling invaders are favorable or unfavorable, the factors that make certain species good colonizers, and the characteristics that make ecosystems susceptible to invasion. It discusses prospects for using restoration technology in controlling nonindigenous species by influencing hydroperiod, photo-period, thermoperiod, edaphic conditions, and availability of biological control agents so as to produce ecological conditions that are inhospitable to invaders. The limitations of restoration are discussed, as well as specific ecological situations in which it is likely to be the method of choice for control of nonindigenous species. Use of fire, flooding, manual removal, shading, substrate removal, and herbicide application as control techniques in conjunction with restoration efforts are considered. Specific examples, including the techniques employed, indicate the potential for controlling nonindigenous species in the process of ecosystem restoration.

Notes: Adequately evaluating the success of coastal tidal marsh restoration has lagged behind the actual practice of restoring tidally restricted salt marshes. A Spartina-dominated valley marsh at Barn Island Wildlife Management Area, Stonington, Connecticut, was tidally restricted in 1946 and consequently converted mostly to Typha angustifolia. With the re-introduction of tidal flooding in 1978, much of the marsh has reverted to Spartina alterniflora. Using a geographical information system (GIS), this study measures restoration success by the extent of geographical similarity between the vegetation of the restored marsh and the pre-impounded marsh. Based on geographical comparisons among different hydrologic states, pre-impounded (1946), impounded (1976), and restored (1988) tidal marsh restoration is a convergent process. Although salt marsh species currently dominate the restored system, the magnitude of actual agreement between the pre-impounded vegetation and that of the restored marsh is only moderate. Further restoration of the salt marsh vegetation may be limited by continued tidal restriction, marsh surface subsidence, and reduced accretion rates. General trends of recovery are identified using a gradient approach and the geographic pattern’ of vegetation change. In the strictest sense, if restoration refers only to vegetation types that geographically replicate preexisting types, then only 28% of the marsh has been restored. Restoration in a broader sense, however, representing the original salt marsh vegetation regardless of spatial position, amounts to 63% restored. Unrestored marsh, dominated by Typha angustifolia and Phragmites australis, remains at 37%. By emphasizing trends during vegetation recovery, this evaluation technique aims to understand the restoration process, direct future research goals, and ultimately aid in future restoration projects.

Notes: Books reviewed in this article: The Earth in Transition: Patterns and Processes of Biotic Impoverishment. A collection of papers from a symposium held in Woods Hole, Massachusetts, October 1986. George M. Woodwell, editor Restoration of Aquatic Ecosystems: Science, Technology, and Public Policy. National Research Council.

Notes: Results are presented using vegetative shoots and bryophyte sods to restore floristically impoverished high arctic wet sedge-moss meadows that had suffered intense damage from vehicle activity during the period 1960–1967. Clonal transplants of Carex aquatilis var. stans, a native sedge, were planted with and without bryophyte sods in vehicle ruts in 1972. After nearly two decades, there was less Carex cover in the planted ruts with flowing water than in the contiguous controls. This pattern was slightly reversed in planted plots with standing water. Reinvasion of Eriophorum angustifolium occurred in treated ruts, but cover was less in both treatments than in controls in 1990. The unexpected recruitment of Eriophorum scheuchzeri from the seed bank in moss-sodded plots is discussed in terms of its local and regional importance. Total plant cover in restored ruts was nearly equal to that of controls, but biomass was somewhat less than that in control plots. Plots with bryophytes were environmentally distinct, due primarily to increases in organic mat depth relative to controls. After 18 years, restoration efforts resulted in increased plant cover in treated ruts compared to naturally recovering ruts.〈blockFixed type="quotation"〉The composition of no two patches of vegetation is precisely the same [and] neither are the seed banks. Successsion on different patches of disturebed ground in the same locality frequently proceeds quite differently because of such differences.—J. Miles, Vegetation Dynamics, 1979

Notes: Books reviewed in this article: Directing Ecological Succession. James O. Luken Soil Restoration: Advances in Soil Science, vol. 17. Rattan Lai and B. A. Stewart, editors

Notes: The Pinelands National Reserve and UNESCO Biosphere encompass a large portion of southern New Jersey's Pine Barrens. Within the core preservation zone of these Reserves lies the Warren Grove Weapons Range, a military installation where exercises during the past 50 years have devastated portions of the indigenous pygmy pine-oak forest. In 1987, restoration efforts were initiated to identify materials and techniques that promote a diverse and productive native plant community atop drastically disturbed portions of the range. We used trial plantings to examine fertilizer and sewage compost fertility amendments, the effect of different native plant mixtures (including the dwarfed race of pitch pine, Pinus rigida), the influence of the ectomycorrhizal fungus Pisolithus tinctorius on the growth of pines and associated species, and mulch applications to conserve moisture and add organic matter. Following two growing seasons, test plantings exhibited 25% of the vegetation production found in the surrounding pine-oak community, 50% canopy closure, and levels of diversity comparable to the reference site. Maximum biomass and cover were achieved following the application of 16 Mg/ha compost and the establishment of pitch pine seedlings. Pitch pine was the dominant species in all plots where it was planted, with herbaceous species comprising the balance of the developing vegetation. Amendments of seeded grasses, P. tinctorius, and mulch influenced species composition but failed to enhance total plant production. We recommend restoring drastically disturbed sites in the pine plains with cultural input of compost to the spoils and planting of pitch pines and other woody species to accelerate the structural blending of reforested sites with the surrounding native vegetation.

Notes: A general model is presented describing ecosystem degradation to help decide when restoration, rehabilitation, or reallocation should be the preferred response. The latter two pathways are suggested when one or more “thresholds of irreversibility” have been crossed in the course of ecosystem degradation, and when “passive” restoration to a presumed predisturbance condition is deemed impossible. The young but burgeoning field of ecological restoration, and the older field of rehabilitation and sustainable range management of arid and semiarid lands (ASAL), are found to have much in common, especially compared with the reallocation of lands, which is often carried out without reference to pre-existing ecosystems. After clarifying some basic terminology, we present 18 vital ecosystem attributes for evaluating stages of degradation and planning experiments in the restoration or rehabilitation of degraded ecosystems. Finally, we offer 10 hypotheses concerning ecological restoration and rehabilitation as they apply to ASAL and perhaps to all terrestrial ecosystems.

Notes: Experiments were conducted in the field and the greenhouse to determine whether vesicular-arbus-cular mycorrhizae affect growth and competition between the native perennial Stipa pulchra and the introduced annual Avena barbata. Soils in the greenhouse were steam-sterilized, and in the field they were treated with the fungicide benomyl. Stipa pulchra showed decreased shoot dry mass and increased root mass when inoculated, while A. barbata showed the opposite response, increased shoot mass and decreased root mass. Mycorrhizal A. barbata also produced more seeds. Mycorrhizae did not alleviate the negative effects of competition of A. barbata on S. pulchra, as has been demonstrated for other pairs of weedy and nonweedy species. The same three species of mycorrhizal fungi were present in annual and perennial grasslands, but their relative composition was different. When inoculum from the two grassland types were tested in the field, the fungal species began to revert within five months to the species composition found in grasslands of the host plant. This indicates that, once annual grassland has been revegetated with the native S. pulchra, the original fungal species composition may return relatively quickly. Where A. barbata dominates, inoculation with mycorrhizal fungi alone will not suffice for establishing S. pulchra, and the usual practices for control of weed competition need to be employed.

Notes: Herbaria are potentially important repositories of living seeds that could be useful for recovery of rare plant species. To examine this capacity, we tested seed germination of rare milkweed (Asclepias) and milkvetch (Astragalus) species representing different collection dates and different herbaria. These groups have contrasting seed characteristics, with greater potential for longevity in the nonpermeable hard-coated milkvetch seeds. Twelve-year-old Asclepias lanuginosa seeds failed to germinate. However, we achieved 45% germination from three-year-old Asclepias meadii seeds, but germination dropped to 0% after ages of four to five years. Astragalus neglectus seeds germinated from 97-, 48-, and 28-year-old herbarium specimens, and Astragalus tennesseensis seeds germinated from a four-year-old collection. Seedlings produced from these experiments were incorporated into ex situ garden populations for recovery or restoration of rare species populations. Different herbarium pest control techniques may have significant bearing on the viability of seeds stored on herbarium specimens. Microwaving can cause precipitous loss of seed viability, while deep-freezing appears to allow some seeds to remain viable. Potentially live seeds of rare species should be stored under conditions that enhance their long-term viability.

Notes: The microbial community in a soil stripped and stored during opencast coal mining was analyzed. There were significant effects of soil disturbance on the microbial community: in particular, there were large decreases in the total microbial biomass, as determined by ATP analysis, and numbers of fungal propagules as a result of the store construction process, but there was no significant effect on the numbers of bacteria. During the subsequent months of storage there was a flush in the numbers of bacteria, with gram-negative bacteria showing an increase of nearly 700% in comparison to the control. During this time there was a steady accumulation in the amount of ammonium in the deepest part of the soil store, indicating the onset of anaerobiosis. These changes may be interpreted in terms of lifestyle strategy theory (Grime 1979). The bacteria exhibit behavior typical of R-strategists, or ruderal species, taking advantage of the nutrients made available by the death of fungal biomass during store construction. Fungi respond as C-strategists, or competitors, and they are severely affected by store construction-and unable to persist deep in the anaerobic part of the store. In contrast, anaerobes, S-strategists or stresstolerators, are able to survive under the same conditions. These changes have serious implications for the restoration of systems using stored topsoil as a resource. The microbial population has been altered in terms of its size and composition. Many of the fungi required for adequate breakdown and incorporation of organic matter will be absent, and the soils will be generally poor in microbial biomass. This will lead to inadequate nutrient cycling and poor soil structural stability, two factors essential for the restoration of a self-sustaining ecosystem.

Notes: In order to extend quarrying near Thrislington Plantation, County Durham, England, 8.5 ha of magnesian limestone grassland was relocated over a period of eight years from October 1982. The effects of this on the flora and invertebrate fauna were examined within the Festuca-Helianthemum community at five plots relocated at different times. Plants were sampled with a point-quadrat, and invertebrates by pitfall trapping. Comparisons were made between age of the relocation, numbers of species and individuals, and diversity of flora and invertebrates. The plots were examined using the percentage similarity measure. The plots showed an initial change in some aspects of community structure for flora and invertebrate fauna, followed by a “recovery” period. This was particularly evident in the numbers of species and species diversity of plants and in the numbers of individuals and species diversity of invertebrates. Bare ground, left by the relocation process, was still evident between relocated turfs in the early plots, but it was successfully colonized by resident species in later plots. These results have implications for the future management of this and similar sites, particularly with respect to the emphasis placed on subsequent monitoring and the need to consider invertebrate faunas when implementing management strategies.

Notes: 〈blockFixed type="quotation"〉We now recognize that humans have the power to alter the planet irreversibly, on a global scale. Humans must be concerned with the condition of the planet that is passed to future generations./〉

Notes: The recent publication of Restoration of Aquatic Ecosystems: Science, Technology, and Public Policy has generated much scientific, public, and political discussion. Although the book emphasizes the restoration of entire aquatic ecosystems, discussion of senescent dams and human-made reservoirs is absent. The important societal and ecological roles of reservoirs warrant a closer examination of the potential ecological restoration of aging reservoirs. Problems with long-term reservoir management include lack of long-term management strategies, sedimentation, hazardous waste accumulation, impacts of recreational use, and the creation of new aquatic and riparian habitats. Policy conflicts may arise when habitats created in the reservoir are destroyed to restore the downstream habitats or when created habitats upstream undergo successional changes that impact the commercial or recreational value of the reservoir. Rare or endangered species may also create similar conflicts. The establishment of an ecological restoration bonding program that includes environmental education and conservation prior to new dam construction may aid in resolving potential conflicts in the future.

Notes: Books reviewed in this article: The Uses of Ecology: Lake Washington and Beyond W. T. Edmonson

Notes: Sagittaria latifolia Willd. is commonly used for wetland enhancement, restoration, and creation. It is a C3 species that is widely distributed in southeastern Canada and the eastern half of the United States. It provides habitat and food benefits to waterfowl and improves water quality in wetlands. Monoecious and dioecious varieties occur in the U.S. that exhibit different life history characteristics. Clonal spread occurs through growth of rhizomes and tubers. S. latifolia grows in a wide range of fresh water and soil conditions. It persists in stabilized water levels at depths of less than 50 cm and few drawdowns. The species tolerates and assimilates high levels of nutrients and heavy metals. There is a limited data base on the installation and management of the species. Tubers and plants are preferred plant materials for field establishment. Herbivory by insects, waterfowl, and other animals may greatly reduce planting success. Future studies relevant to improvement of propagule storage, planting conditions, and management of mature plants for wetland projects are suggested.

Notes: A model of ecosystem degradation and three possible responses to it—restoration, rehabilitation, and real-location—is applied to ongoing projects in the arid mediterranean region of southern Tunisia, the subhumid mediterranean region of central Chile, and the semiarid tropical savannas of northern Cameroon. We compare both nonhuman and human determinants of ecosystem degradation processes in these contrasted regions, as well as interventions being tested in each. A number of quantifiable “vital ecosystem attributes” are used to evaluate the effects of ecosystem degradation and the experimental responses of rehabilitation on vegetation, soils and plant-soil-water relations. We argue that attempts to rehabilitate former ecosystem structure and functioning, both above- and below ground, are the best way to conserve biodiversity and insure sustainable long-term productivity in ecosystems subjected to continuous use by people in arid and semi-arid lands of “the South.” The success of such efforts, however, depends not only on elucidating the predisturbance (or slightly disturbed) structure and function of the consciously selected “ecosystem of reference,” but also on understanding and working with the socioeconomic, technical, cultural, and historical factors that caused the degradation in the first place.

Notes: Results of a short-term study of the revegetation of illegally rock-plowed wetlands in the East Everglades are reported. Comparisons of the plant communities on a restored site, an unrestored site, and the natural control areas directly adjacent to these sites were made using line intercept transects. On the site where removal of the rock-plowed material and grading of the surface to below original elevation were required for restoration, less than 20% of plant cover was of nonwetland species, and the occurrence of exotic species was low. On the rock-plowed site where no restoration efforts were performed, 61% of plant cover was of nonwetland species, and there was a higher occurrence of exotic species compared to the restored rock-plowed and the control sites.

Notes: The extreme species richness of native shrubland vegetation (kwongan) near Eneabba, Western Australia, presents a major problem in the restoration of sites following mineral sand mining. Seed sources available for post-mining restoration and those present in the native kwongan vegetation were quantified and compared. Canopy-borne seeds held in persistent woody fruits were the largest seed source of perennial species in the undisturbed native vegetation and also provided the most seeds for restoration. In undisturbed vegetation, the germinable soil seed store (140–174 seeds · m−2) was only slightly less than the canopy-borne seed store (234–494 seeds · m−2), but stockpiled topsoil provided only 9% of the germinable seeds applied to the post-mining habitat. The age of stockpiled soil was also important. In the three-year-old stockpiled topsoil, the seed bank was only 10.5 seeds · m−2 in the surface 2.5 cm, compared to 56.1 to 127.6 seeds · m−2 in fresh topsoil from undisturbed vegetation sites. In the stockpiled topsoil, most seeds were of annual species and 15–40% of the seeds were of non-native species. In the topsoil from undisturbed vegetation, over 80% of the seeds were of perennial species, and non-native species comprised only 2.7% of the seed bank. Additional seeds of native species were broadcast on restoration areas, and although this represented only 1% of the seed resources applied, the broadcast seed mix was an important resource for increasing post-mining species richness. Knowledge of the life-history characteristics of plant species may relate to seed germination patterns and assist in more accurate restoration where information on germination percentages of all species is not available.

Notes: The composition and structure of ant communities were used to assess the success of the preliminary restoration program at Ranger uranium mine in the seasonal tropics of northern Australia. Ants were surveyed at eight sites, including two relatively undisturbed control sits, within the Ranger lease. The revegetated sites represented a range of variables likely to influence restoration success: revegetation age (two, four, and eight years), proximity to undisturbed sites (which act as potential sources of recolonization), and burning treatment. Revegetation at most sites was dominated by fast-growing species of Acacia. There was a clear succession of ant species across revegetated sites. Initial colonization was by species of Iridomyrmex, but as plant cover and litter development increased these were replaced by broadly adapted, opportunist species, especially the introduced Paratrechina longicornis. Ant recolonization was very slow at isolated sites, with only 12 species present after eight years (the oldest site available). This compares with 21 species after only four years at a site located close to potential sources of recolonization. The ant community at this site, however, was very similar to that at another site located close to colonization sources, but eight years old. Ant succession therefore appeared to have stalled at this point, with species richness and composition bearing little resemblance to that at control sites. The heavy shade and litter produced by acacias were considered to be the major impediment to further change. Results from a site that had undergone a prescribed burn after two years, thereby breaking dominance by acacias and allowing for the establishment of a wide variety of plant taxa, suggest that such management practices may promote further colonization by ant species.

Notes: Soil microbial activity and soil nutrients were monitored on a revegetated coal surface mine in southwestern Wyoming from the initial planting in 1982 through 1987. Total soil nitrogen (N) and organic matter did not change during this period. However, despite no changes in available phosphorus (P) concentrations, the total P declined over 50% during the five-year period, with no apparent reduction in the loss rates. The greatest loss was in the bound inorganic P pool. Moisture appeared not to limit microbial mass-C. Microbial mass-C was higher under shrubs than in interspaces and increased with time. Total organic matter did not increase. Thus, the ratio of microbial mass-C to organic matter-C increased during the study period. This suggests that the input of readily decomposable substrate may limit microbial activity. During the study period, all above-ground litter was removed by wind. Root production in the surface soils was low and highly variable and, in this habitat, probably did not contribute largely to the organic matter status. These data suggest that despite an apparent recovery of many parameters used to indicate reclamation or restoration success, the soil-bound P pools could be undergoing a loss. Microbial-C and organic matter changes indicate a system that is not approaching equilibrium within the required monitoring period of most restoration efforts. These parameters could eventually reduce the recovery potential of restored sites.

Notes: A series of experiments designed to demonstrate the potential of using managed, attached algal production to permanently remove excess phosphorus from agricultural run-off is described. The experiments were carried out on a secondary canal in the New Hope South region of the Florida Everglades Agricultural Area from October, 1991, to May, 1992. Natural algal populations of periphyton, including species of the genera Cladophora, Spirogyra, Enteromorpha, Stigeoclonium, and a variety of filamentous diatoms such as Eunotia and Melosira, were grown on plastic screens in raceways, under a wave surge regime. Considerable biomass production of algae occurred, and the resulting algal canopy also trapped plankton and organic particulates from the water column. A seven- to eight-day harvest interval was determined to be optimal, and both hand harvesting and vacuum harvesting were employed. The vacuum device is applicable to large scale-up. In source water having total phosphorus concentrations of 0.012–0.148 ppm, mean macro-recovery dry biomass production levels of 15–27 g/m2/day were achieved. The lower rates occurred in the winter, the higher rates in the late spring. Two techniques were employed to reduce losses of fine material at harvest during the March to May period. Gravity sieving increased mean dry production levels to 33–39 g/m2/day. The mean phosphorus content of harvested biomass ranged from 0.34% to 0.43%. Total phosphorus removal rates during the spring period of average solar intensity and low nutrient supply, by methods demonstrated in this study, ranged from 104 to 139 mgTP/m2/day (380–507 kgP/ha/year). Over the incoming nutrient range studied, phosphorus removal was independent of concentration and was 16.3% of total phosphorus for 15 m of raceway. Up-stream-downstream studies of overflowing water chemistry (total P, total dissolved -P, orthophosphate -P) showed highly -significant reductions of all phosphorus species. Total phosphorus reduction closely correlated with phosphorus yield from biomass removal. Yearly, minimum phosphorus removal rates are predicted that are 100–250 times that achieved both experimentally and in long-term, large-area wetland systems. Engineering scale-up to systems of hundreds of acres is being studied.

Notes: The goal of this study was to use terrestrial arthropods to help evaluate the progress of a riparian restoration effort along the San Luis Rey River in California by comparing characteristics of the arthropod community at the reconstructed system to those of a naturally occurring riparian woodland used as a reference site. Insects and other arthropods were sampled throughout 1989–1991 using pan trapping and sweep sampling of dominant plants. Assemblages of taxa were monitored as indicators of functional groups that influence ecosystem processes: pollinators, herbivores, predators, parasites, and detritivores. Relative abundances of indicator assemblages were compared between sites to evaluate the establishment and maintenance of processes critical for the natural function of the reconstructed riparian ecosystem. A major objective of this project was to create habitat for the Least Bell's Vireo, so a group of potential prey items was designated to indicate vireo food resources. Over 230,000 arthropods were identified to order or family and by size. Insect communities developed rapidly at the restored habitat. Although the abundance of all arthropods was lower at the reconstructed site than at the reference site, the same orders were present after three years in similar proportions at both locations, and mean abundances were within an order of magnitude of each other. Abundance of certain groups, such as detritivores, suggested that arthropods had propagated rapidly at the restoration site, a possible indication of resiliency. Relatively low numbers of other arthropods, such as predators and parasites, at the reconstructed site indicated the need for continued monitoring. The decline of pollinators and herbivores by 1991 at the reconstructed site suggested that they may have immigrated or been introduced with transplanted vegetation in 1989, but have had difficulty colonizing the site. Although the Least Bell's Vireos were seen foraging at the restored site after three years, no nests were found. Nesting is anticipated, however, as the site matures.

Notes: The ultrastructure of the life cycle of Vairimorpha necatrix (Kramer) was studied in Heliothis (Helicoverpa) zea larvae raised at temperatures ranging from 15° C to 30° C. In contrast to the description of the type species, both patterns of development (Nosema-like and Thelohania-like) that characterize the genus Vairimorpha were present at all temperatures examined. No temperature-related differences in the ultrastructure of Vairimorpha necatrix were observed; however, the effects of temperature variation were apparent in 1) the time between inoculation with V. necatrix and the initiation of the parasite life cycle and 2) the duration (or length) of the parasite life cycle. An additional form of the parasite, that of a diplokaryon in a sporophorous vesicle, was present during early sporogony. The nuclei of the diplokaryon separated followed by cytokinesis, forming two monokaryotic parasites, and eventually two plasmodia, within the sporophorous vesicle. This pattern of development is characteristic neither of Nosema nor Thelohania, nor has it been described for Vairimorpha.

Notes: Observations by electron microscopy were conducted on Coelosporidium periplanetae (Lutz & Splendore, 1903) Swarczewsky, 1914, an extracellular sporogenic protist in the lumen of the Malpighian tubules of Blatta orientalis Linn., 1758 and other domiciliary cockroaches. Results generally agreed with earlier studies by light microscopy but also allowed a more complete characterization of the species. Some outstanding features of the protist were: amoeboid multinucleate stages (plasmodia) capable of producing cytoplasmic projections for attachment to microvilli of tubules, endogenous formation of spores, differentiation of “generative” and “somatic” nuclei in plasmodia undergoing spore formation, polarized early sporoblasts (a nucleus on one half and a chondriome on the other), and biconcave, ovoid, non-polarized spores that are retained until maturation by the plasmalemma and residual cytoplasm of the original plasmodium. The new combination Nephridiophaga periplanetae is proposed based on this new, updated information. The family name Nephridiophagidae Sprague, 1970 is resurrected to include N. periplanetae and eleven other species of protists in the Malpighian tubules of arthropods, mostly insects in the orders Dictyoptera and Coleoptera. According to its characteristics, the family Nephridiophagidae cannot be included into any of the currently recognized phyla of protists. It is suggested that it should be temporarily treated as an incertae sedis group of Protista.

Notes: Direct observations of schizonts and agamonts releasing megalospheres clarified the asexual phase of the life cycle of Peneroplis planatus and made it most probable that this species has a paratrimorphic life cycle. Specimens with maximum lengths between 837 and 3,503 μm released about 500 to 1,500 megalospheric juveniles, which possessed two chambers (proloculi and flexostyles) prior to emergence from the parental shell. The presence of gamonts was not shown and was only implied by the occurrence of the agamonts. Since agamonts and schizonts have been found from December to May and since asexual reproduction occurs in spring in Elat, sexual reproduction probably occurs at another time of year (June to December). More detailed studies of this species need to be conducted throughout the year to improve our knowledge of the life cycle of this species.

Notes: . Ditrichomonas honigbergii n. g., n. sp. is a small trichomonad flagellate that has three emergent flagella arising from four basal bodies, a parabasal apparatus (single dictyosome with associated striated flagellar rootlets), a microtubular axostyle, a short undulating membrane, and hydrogenosomes. Cultures of D. honigbergii were isolated from the sediments of a freshwater lake and there is no known metazoan host. Cells form walled cysts with internalized flagella and go through all phases of the life cycle (excystment, binary division, encystment) without any perturbations to the culture medium. Ditrichornonas honigbergii is capable of ingesting and digesting bacteria by phagocytosis. These facts suggest that D. honigbergii may be a free-living inhabitant of oxygen-reduced environments. The structure of D. honigbergii is similar to that of retortamonads and the relationship of trichomonads to other amitochondrial protists is discussed.

Notes: . Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature, and axonemes isolated from nonmotile mutants lack approximately 90% of their outer dynein arms. Electrophoretic analyses of axonemes isolated from nonmotile mutants (oad axonemes) indicate they contain significantly fewer of the 22 S dynein heavy chains that axonemes isolated from wild-type cells (wild-type axonemes) contain. The 22 S dynein heavy chains that remain in axonemes isolated from nonmotile, oad mutants are assembled into 22 S dynein particles that exhibit wild-type levels of ATPase activity. Two-dimensional gel electrophoresis of oad axonemes show that they are deficient in no proteins other than those proteins thought to be components of 22 S dynein. This report is the first formal proof that outer dynein arms in Tetrahymena cilia are composed of 22 S dynein.

Notes: . A cell culture of Paramecium with a precise negative gravitaxis was exposed to 4 times 10-6g during a parabolic flight of a sounding rocket for 6 min. Computer image analysis revealed that without gravity stimulus the individual swimming paths remained straight. In addition, three reactions could be distinguished. For about 30 s, paramecia maintained the swimming direction they had before onset of low gravity. During the next 20 s, an approximate reversal of the swimming direction occurred. This period was followed by the expected random swimming pattern. Similar behavior was observed under the condition of simulated weightlessness on a fast-rotating clinostat. Control experiments on the ground under hyper-gravity on a low-speed centrifuge microscope and on a vibration test facility proved that the observed effects were caused exclusively by the reduction of gravity.

Notes: . The nuclear apparatus of H. vermiculare consists of a single moniliform macronucleus and about 25 micronuclei. the micronuclei are about 3 μm in diameter and characterized by a meshwork of thick condensed chromatin. Mitosis is intranuclear and acentric as in all other ciliates. In metaphase, interpolar and chromosomal microtubules are abundant and the length of the micronuclei increases to about 5 μm. In late anaphase, interzonal microtubules become prominent and the spindle elongates to about 50 μ. In meta- and anaphase, the microtubules of the spindle are attached to the polar vesicles, and in anaphase, chromosomes become attached to it. In contrast to most other eukaryotes, micronuclear mitosis is not strictly bound to cell division in H. vermiculare. While most of the micronuclei divide prior to cytokinesis, others retain their interphasic shape or degenerate. In addition, some micronuclei divide in the interdivision period, i.e. between two successive divisions of the cell and macronucleus. Mating cells of H. vermiculare become joined to each other in the cilia-free region covering the cytostome. In the course of conjugation, the cell membranes and the underlying oral filamentous sheaths of both cells fuse, thus uniting the endoplasm of both cells in the mouth region. Synaptonemal complexes in the meiotic chromosomes are more distinct in H. vermiculare than in most other dilates. the micrographs presented here depict dearly the central filament, transverse elements, and other substructures.

Notes: . Miamiensis avidus, a marine scuticociliate, undergoes microstome to macrostome transformation. This process is induced by prey ciliates. the morphogenesis of this process was examined using scanning electron microscopy and light microscopy of quantitative protargol-stained specimens. Several stages are identified and described.

Notes: . Scanning electron microscopic observations of feeding plasmodia show three characteristic features: 1) extension of multilobed pseudopodia protruding from the leading edge of the plasmodium as it advances onto the surface of a food particle, 2) confluence of the lobes to form a sheath-like pseudopodium attached to the surface of the food particle, and 3) protrusion of small nodules with thin lamellar projections from the leading edge of the plasmodium. Sections through freeze-dried preparations of the feeding plasmodium exhibit a highly convoluted under surface in contact with loosened starch grains that appear to be released by extracellular digestion. the cytoplasm, viewed by transmission electron microscopy, contains branched, internally penetrating canals (ca. 2 μm wide) enclosing engulfed starch grains. Starch grains in the deeper part of the canals are more electron dense and appear to be digested. Micropseudopodia (70-80 nm dia.), projecting from the surface of the canals, protrude toward and into the ingested starch grains. Digestive marker enzyme (acid phosphatase) activity was detected cytochemically in food particles penetrated by micropseu-dopodia indicating a digestive role for these structures not reported previously.

Notes: . Intestinal microsporidiosis in patients diagnosed with acquired immunodeficiency syndrome (AIDS) and having chronic diarrhea was first reported in 1985 and the associated microsporidian was named Enterocytozoon bieneusi. the intracellular developmental cycle of E. bieneusi in enterocytes has been demonstrated and many cases have been reported worldwide. This report presents the life cycle of a second intestinal microsporidian, associated with the same symptoms, in five AIDS patients. This new microsporidian also infects enterocytes but its pathology and morphology differ from that of E. bieneusi. It involves lamina propria macrophages, fibroblasts, and endothelial cells and can disseminate to infect other parts of the body, e.g. the kidney and gall bladder. the parasite cycle includes development of rounded uninucleate and elongated bi- or tetranucleate cells without the formation of plasmodial stages. Sporogony is similar to the more typical development of microsporidia with sporoblast morphogenesis occurring after the last cell division. the development of cells within chambers of a septate, honeycomb-like, parasite-secreted fibrillar network and surrounded by a parasitophorous vacuole, however, is unique to this microsporidian, justifying the establishment of a new genus and species, Septata intestinalis n. g., n. sp.

Notes: Spores of the microsporidian parasitic protozoan Encephalitozoon hellem were purified and incubated at 37° C in a solution with an electrolyte composition similar to that of mammalian extracellular fluid, and in solution in which the calcium had been replaced with 0.2 mM EGTA. Polar filament extrusion (germination) was monitored by both scanning electron microscopy and light microscopy. Germination was pH-dependent, with optima at pH 7.4 and 9.5, and was significantly greater in the presence of medium calcium. Hydrogen peroxide caused a concentration-dependent increase in germination that was also reduced in a calcium-free medium. Four agents were found to inhibit spontaneous and H2O2-stimulated polar filament extrusion: the microfilament disrupter, cytochalasin D; the microtubule disrupter, demecolcine; the calcium channel blocker, nifedipine; and the antifungal agent, itraconazole. These results are consistent with the existence of a calcium-channel-mediated step, and requirements for an F-actin- and for a tubulin-containing element in the germination process of the spore of this parasite. Nifedipine, cytochalasin D and itraconazole all have different sites of action and were therefore able to potentiate one another when used in paired combination to inhibit germination.

Notes: Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was 〉 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (〈 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated 〉 99.5% purity. The total non-P. carinii protein in the final preparation (〈 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 108-109 organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80–95%.

Notes: Pneumocystis is a eukaryotic microbe that causes pneumocystosis, an AIDS-associated pneumonia. Pneumocystosis also occurs in many other mammalian species, and animal-derived organisms have been extensively utilized in Pneumocystis research. Pneumocystis from diverse hosts contain a large glycoprotein (gpA/MSG) on the surface. Antibodies elicited against gpA/MSG of Pneumocystis from humans sometimes cross-react with epitopes on proteins of similar size from Pneumocystis from other host species. Here we report the isolation and partial sequence of two presumptive gpA/MSG genes from human-derived Pneumocystis. The cloned human-derived Pneumocystis gpA/MSG genes and predicted peptides were different from those previously isolated from Pneumocystis from rats and ferrets. The genome of human-derived Pneumocystis contained multiple copies of sequences related to the two cloned gpA/MSG genes.

Notes: . Trypanosomatids isolated from corn seeds and from digestive tract and salivary glands of Leptoglossus zonatus (Hemiptera, Coreidae) were obtained in pure cultures. In experimental transmission, the flagellates present in naturally infected insects were able to infect laboratory-raised corn. A simplified liquid culture medium was established that increased parasite yield three- to five-fold. Cultured and cloned parasites, and forms found in insects and corn as well, were studied by light and electron microscopy. A remarkable finding was the observation that the cultured strain 163M bears a surface coat similar to that observed in naturally occurring African trypanosomes. but not observed in trypanosomes in vitro. Based on the biochemical characteristics of the arknine-ornithine cycle and on the presence of this cell coat, we propose that the strain 163M is a new species and name it Herpetomonas macgheei n. sp.

Notes: . Expression of a 21 kDa determinant (Pbs21), first detected on the surface of ookinetes, and of the circumsporozoite protein (CSP) was studied by immunofluorescence and Western blots during the developmental cycle of Plasmodium berghei in the mosquito A nopheles stephensi. The expression of Pbs21 was predominantly localised on the ookinete surface one day after the infectious blood meal, and thereafter reactivity declined to a minimum on days 2 and 3, the time of onset of oocyst development. A gradual increase in fluorescence was observed on the oocysts from day 6 that was retained until day 17 post-infection. In contrast, sporozoites released from oocysts or salivary glands showed little or no antibody labelling with anti-Pbs21. Circumsporozoite protein was not detectable in any rnidgut preparations until 5–6 days after feeding, when reactivity was observed against immature oocysts. Expression then continued and increased throughout oocyst and sporozoite development. Western blots confirmed that Pbs21 was expressed minimally during the oocyst development but was not detectable in sporozoites. Co-localisation of anti-Pbs21 and anti-CSP monoclonal antibodies to the 50 kDa and 60 kDa bands in Western blots of sporozoite suggests immunological cross-reactivity between the CSP and the anti-21 kDa antibodies.

Notes: . The amoebae, a phenotypically diverse, paraphyletic group of protists, have been largely neglected by molecular phy-logeneticists. To better understand the evolution of amoebae, we sequenced and analyzed the 16S-like ribosomal RNA genes of three vahlkampfiid amoebae: Paratetramitus jugosus, Tetramitus rostratus and Vahlkampfia lobospinosa. The Vahlkampfiidae lineage is monophyletic, branches early along the eukaryotic line of descent, and is not a close relative of the multicellular amoebae that also reversibly transform from amoebae to flagellates.

Notes: . The “flagellates” as a taxon has become obsolete and should be dispensed with. It is obsolete because it is not phylo-genetically coherent. Adopting an ultrastructural perspective, it is argued that the flagellates exemplify an extreme case of paraphyly. Meaningful discussion of the evolution or ecology of flagellates can only emerge after we identify the monophyletic constituents of the flagellates. There are over 40 known types of heterotrophic flagellates and these should form the basis of a new understanding of flagellate diversity.

Notes: . A soluble enzyme amylopectin synthase (UDP-glucose-α 1,4-glucan α-4-glucosyltransferase) which transfers glucose from uridine 5′-diphosphate glucose (UDP-glucose) to a primer to form α-I,4-glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella. UDP-glucose and not ADP-glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis-Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP-glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α-1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP-glucose-α-1,4-glucosetransferase EC 2.4.1.11).

Notes: . The recently established protozoan phylum Opalozoa Cavalier-Smith 1991 includes all those zooflagellates with tubular mitochondrial cristae that never have cortical alveoli or rigid tubular ciliary hairs (retronemes), and also the opalinids, proteomyxids sensu stricto, and plasmodiophorids. Opalozoa totally lack plastids but usually (though not invariably) have peroxisomes. They always have well-developed Golgi dictyosomes. The trophic phase is a unicellular ciliated phagotroph except in the only intracellular parasites, the plasmodiophorids, where it is a non-phagotrophic and non-ciliated microplasmodium, and in the proteomyxids where it is an amoeboflagellate (which may sometimes be nonciliated) or a multicellular meroplasmodium. Unlike the phagotrophic Mycetozoa, opalozoans do not form aerial fruiting bodies, but encystation is common. The first detailed classification of the phylum is presented here. It is divided into four subphyla (three new), eight classes (four new, one emended), three subclasses (all new), three superorders (all new) and 22 orders of which 12 are new and one is emended. Diagnoses of these taxa are given, as well as lists of the 31 families (11 new) and 62 genera included within them. Opalozoa, which include Cercomonas and Heterornita, the commonest soil flagellates, are ecologically and evolutionarily important.

Notes: . Two new hemimastigophoran flagellates are described using light and electron microscopy, and the family Spironemidae is revised. Spironema terricola n. sp. occurs in soil from the Grand Canyon (southwest USA). It moves in a conspicuously euglenoid manner and differs from S. multiciliatum Klebs by its vermiform shape and shorter kinetics. Spironema terricola is similar to Goodey's Spironema multiciliatum from soil in England. However, Goodey's vermiform species has a very elongate nucleus and is thus neither identical with S. terricola, which has a roundish nucleus, nor with Klebs' lanceolate S. multiciliatum; we consider it a new species, Spironema goodeyi n. sp, Stereonema geiseri n. g., n. sp. was discovered in the Aufwuchs (periphyton) of a river in Bavaria, Germany. the new genus differs from Spironema by its a contractility, and from Hemimastix by its shorter kinetics and less plicate cortex. the fine structure of Spironema and Stereonema is very similar to that of Hemimastix Foissner et al., viz., the cortex is composed of two plates having diagonal symmetry and the flagellated basal bodies have associated a short and a long microtubular ribbon. All species have unique extrusomes of the same type. the main differences between the three genera and five species recognized are contractility, length of kinetics, body size, shape of cell and nucleus, and particulars of the cortex and extrusomes. the phylogenetic relationships of the Hemimastigophora are still uncertain. However, the diagonal symmetry of the cortical plates and the pronounced euglenoid movement of Spironema spp. suggest a common ancestor with euglenids.

Notes: . The replication band is a large, migrating, macronuclear domain that is the site of DNA synthesis in hypotrichous ciliated protozoa. A number of agents that produce inactivation of this structure and its replicational activity are described here. These agents include heat shock, aphidicolin, cell crowding, various cAMP phosphodiesterase inhibitors and a calmodulin inhibitor. With the exception of aphidicolin, which has a direct inhibitory effect upon DNA polymerases, the mechanisms of inactivation are presently unknown. the inactivating properties of cAMP phosphodiesterase inhibitors suggest that intracellular cAMP levels may influence replication band structure and function.

Notes: . The ultrastructure of the oral region and the ultrastructural architecture of the basket of Prorodon aklitolophon and Prorodon teres are described. the oral region of Prorodon consists of: 1) A circumoral kinety at the anterior pole of the cell surrounding the typically slit-shaped cytostomial funnel. This kinety is composed of inversely oriented dikinetids in which both kinetosomes are ciliated and are associated with a very short postciliary microtubular ribbon and a few transverse microtubules; 2) Three brush rows aligned in parallel and extended meridionally in the anterior part of the cell. the individual brush rows consist of dikinetids, but in contrast to the dikinetids around the cytopharynx they are not inverted and only the anterior kinetosomes bear specialized short brush cilia and are associated with a divergent-tangential transverse microtubular ribbon. the posterior kinetosome is non-ciliated and bears a prominent convergent postciliary microtubular ribbon. Schematized dikinetid patterns of both oral regions of Prorodon are provided. In addition, a three-dimensional reconstruction of the basket of the genus Prorodon based on serial thin sections is presented. A phylogenetic tree, mainly based on stomatogenic data, is given to show the phylogenetic relationships of some prostomatid genera as well as the hypothesized sistergroup relationship of colpodid and prostomatid ciliates.

Notes: . This study describes the ultrastructure of the somatic cortex of Prorodon aklitolophon and Prorodon teres. the meridionally arranged somatic kineties of both species can be separated into two parts: a short anterior part, which consists of a few somatic dikinetids (in which both kinetosomes are ciliated), and a longer posterior consisting of monokinetids. the somatic monokinetids are associated with a convergent postciliary microtubular ribbon, a transverse microtubular ribbon flatly inserted in front of the kinetosome, a short and steeply extending kinetodesmal fibre attached to kinetosomal triplet 5 and 7, and a desmose anterior to triplet 3. From this desmose, two to five prekinetosomal microtubules originate and extend anteriorly. the posterior kinetosome of the somatic dikinetids is associated with the same microfibrillar and microtubular structures as the somatic monokinetid, except that no prekinetosomal microtubules originate from the desmose. the anterior kinetosome has a single postciliary microtubule and a tangentially oriented transverse microtubular ribbon. the permanent collecting canals of the unique contractile vacuole system extend parallel and adjacent to the somatic kinetics of Prorodon. the collecting canals are supported by the prekinetosomal microtubules. A similarly organized contractile vacuole system is not yet known from any other ciliate group. One of the most surprising results of this investigation was finding a significant similarity between the somatic dikinetid pattern of Prorodon and the colpodid dikinetid pattern. A hypothesis is presented to illustrate the evolution of the somatic kinetid patterns in colpodid and prostomatid ciliates.

Notes: . The morphological characteristics of two new species of Isospora observed in Emberiza cirlus (Cirl Bunting) from Italy are reported. the oocysts of Isospora normanlevinei n. sp. are spherical or sub-spherical, with a smooth double-layered wall, and measure 24.2 times 23.7 (21.0-26.5 times 21.5-25.5) μm; each oocyst contains 2 to 10 polar granules. No micropyle or residuum was observed. the piriform sporocysts measure 19.4 times 11.2 (17.0-21.0 times 10.0-12.5) μm and contain a dispersed residuum. the Stieda body is flat; the substiedal body, with scattered clear and dark granules, may be either symmetrical or asymmetrical. the oocysts of I. coluzzii n. sp. are asymmetrical and rounded shape and measure 28.6 times 24.2 (25.0-31.5 times 21.5-26.0) μm. the oocyst has a double-layered wall and 2 to 3 polar granules. Neither micropyle nor residuum is present. the sub-ellipsoidal sporocyst, measuring 18.2 times 10.0(16.5-20.0 times 9.0-11.0) μm, has a dispersed sporocyst residuum. the Stieda complex is symmetrical.

Notes: . Free-living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis). In a broad bacterial screening from soil and water we have isolated three strains (M-4, D-13 and A-12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp. and also against different Gram-positive and Gram-negative bacteria. Physical-chemical characteristics, partial purification and biological activities of a substance produced by the M-4 strain have been investigated. This substance (m-4) is stable at high temperature (up to 100°C) and extremes of pH (2.5-9.5) and also at -20°C for months. Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium. Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m-4.

Notes: . Rhodamine 123, a membrane potential-specific dye, has been evaluated as a probe to monitor the function of the mitochondrion in long slender bloodstream and procyclic trypomastigotes of several Trypanosoma brucei spp. By epifluorescence microscopy, mitochondrial development has been followed in long slender bloodstream and procyclic organisms stained with rhodamine 123. to photograph stained long slender bloodstream forms, it was necessary to develop a method to completely immobilize viable organisms. In both parasite forms, as the cell cycle progressed, the mitochondrion developed from a thread-like structure to a highly branched organelle. A dramatic reorganization occurred preceding cytokinesis to produce two progeny thread-like structures which were partitioned into newly formed daughter cells. the organelle within the long slender trypomastigote was found to stain optimally at 0.3 μ/ml of rhodamine 123, while the procyclic form required 3.0 μ/ml. the results suggest that the plasma membrane potential is higher in the long slender parasite than in the procyclic form. the effects of inhibitors that disrupt mitochondrial function were examined in long slender and procyclic parasites, and some of these agents were shown to affect rhodamine 123 accumulation and retention. In long slender trypomastigotes the trypanosome alternative oxidase does not appear to be coupled to proton pumping, whereas in procyclic organisms the effects of inhibitors indicate that this oxidase may be coupled to a pathway that is branched preceding an antimycin A1-sensitive site.

Notes: . The biochemical characterizations of lipophosphoglycans from various Leishmania species reported by other workers may or may not contain several types of lipophosphoglycan molecules. This is the first report in which a specific lipophosphoglycan has been defined by both its antigenie and electrophoretic properties. Furthermore, a purification procedure for this specific lipophosphoglycan is described and some biochemical characterizations are presented. Phospholipase C and the so-called phosphatidylinositol-specific phospholipase C of Bacillus cereus convert the amphipathic form of the lipophosphoglycan antigen to the hydrophilic form. Under equivalent incubation conditions, other phospholipases tested were not effective in conversion of the amphipathic to the hydrophilic form. Since the amphipathic form is present in conditioned media, antigen shedding cannot be explained by phospholipase C digestion of the amphipathic form, which would result in the release of only the hydrophilic form into the medium. Both the pellet and the supernatant fractions of conditioned media contained both forms of the antigen and did not differ in the relative amounts of the two. This observation rules out membrane blebbing as the major mechanism for the release of the amphipathic form.

Notes: . A myosin-like protein (Mr 175,000) was detected in the parasitic protozoan Gregarina blaberae, by both immunofluorescence and immunoblotting of one- and two-dimensional electrophoresis gels using anti-myosin antibodies. This protein was present in the trophozoite ghost but not in the cytoplasmic extract, nor in extract from the sexual stage, suggesting a protein-stage-dependent expression. the protein tightly bound to the cortical membranes was insoluble at low ionic strength, or in detergent solutions, but could be extracted from Gregarina ghosts by 6 M urea in high ionic strength solution (0.5 M NaCI) and in the presence of reducing agents (20 mM DTT). the protein was localized by indirect immunofluorescence in the cortex of the epimerite, in the fibrillar disc (the socalled septum) separating the proto- and the deutomerite segments, in the contractile ring or sphincter at the top of the protomerite, and as longitudinal lines underlying the G. blaberae epicyte folds. the presence of both actin-like and myosin-like proteins would be consistent with a role in gliding and other cell motility processes of this parasite.

Notes: . Explaining the low incidence of invasive disease (10%) in humans infected with Entamoeba histolytica has occupied the attention of generations of both clinical and nonclinical investigators. One possible explanation would be the existence of two morphologically identical species—one an invasive pathogen, the other noninvasive. This was first proposed by Brumpt in 1925, but his explanation was virtually ignored until 1978 when the first of several publications appeared suggesting that E. histolytica did indeed consist of two species. We have reexamined Brumpt's claim in light of recent biochemical, immunological and genetic studies and conclude that the data derived from these investigations provide unequivocal evidence supporting his hypothesis. With this in mind, we redescribe the invasive parasite retaining the name Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911), and set it apart from the noninvasive parasite described by Brumpt, Entamoeba dispar Brumpt, 1925.

Notes: . Vance Tartar (1911-1991) has made major discoveries concerning morphogenesis, patterning, and nucleocytoplasmic relations in the giant ciliate Stentor coeruleus, mostly by means of hand-grafting using glass microneedles. This article provides a chronological account of the major events of Vance Tartar's life, a brief description of some of his major scientific achievements, and a discussion of his distinctive personality and multifaceted interests. It concludes with a consideration of how his unique style of life and work contributed to his equally unique seientific contributions.

Notes: . Electron microsocpic investigations reveal that the Golgi apparatus of Tetrahymena thermophila consists of numerous tiny dictyosomes, each consisting of one or two cisternae. the dictyosomes are localized predominantly in the cell cortex closely associated with the mitochondria, arranged in meridians alternating with the ciliary meridians. We estimated about 300-400 of these dictyosomes in the periphery of a cell, a value corresponding to the number of somatic cilia per cell. Cytochemical assays of thiamine pyrophosphatase and acid phosphatase, both marker enzymes of trans Golgi cisternae, resulted in deposits of lead or cerium phosphate in the outermost cisternae of the dictyosomes. In addition, cisternae located at the bases of the basal body/parasomal sac arrangements are stained. This indicates that these cisternae may belong to the Golgi apparatus of the cell.

Notes: . The effects of different sulphonamides, dihydrofolate reductase inhibitors and other inhibitors of folate metabolism on growth of Acanthamoeba culbertsoni in a chemically defined medium are reported. Among the sulphonamides, sulphamethoxazole and sulphadiazine were most effective followed by sulphanilamide and sulphaguanidine. Inhibition by each sulphonamide was reversed by p-aminobenzoic acid as well as folic acid. 7-Methylguanosine, a pteridine synthesis-inhibitor, did not inhibit multiplication of A. culbertsoni. Among the dihydrofolate reductase inhibitors, pyrimethamine blocked the amoebic growth at 100 μg/ml, while trimethoprim and cycloguanil palmoate failed to cause significant inhibition of growth even at 250 μg/ml. Metoprine inhibited amoebic growth completely at 50 μg/ml. Methotrexate and a thymidylate synthetase inhibitor 5-fluorouracil inhibited growth strongly, with IC50 values (the concentration of the drug which causes 50% inhibition of the growth at 72 h) of 1.97 and 2.45 μg/ml, respectively. Inhibition by methotrexate, metoprine or 5-fluorouracil could not be reversed by folic acid, folinic acid, thymidine, or folinic acid plus thymidine. the results indicate unusual features in A. culbertsoni folate metabolism.

Notes: . Using monospecific antibodies, the presence and distribution of tubulin, actin, myosin, intermediate filaments, and lamins were examined in the exoerythrocytic liver schizont of Plasmodium berghei by conventional indirect fluorescent antibody methods and confocal laser scanning microscopy. the binding reactivity of the antibodies to parasite proteins was determined by Western blot analysis. the localisation of all antibodies in control host hepatocytes followed expected distributions in both uninfected and infected hepatocytes; by contrast, reactivity to the exoerythrocytic schizont was variable. the parasite reacted positively with selected anti-tubulin, -actin, and -myosin antibodies in both fluorescence and Western blot analysis. Anti-lamin antibodies were positive by confocal indirect fluorescent antibody labelling, but no labelling was detected with anti-intermediate filament antibody. Within the technical limits of resolution of the methods as applied to asynchronous parasite infections, not one of the antibodies reacting positively with the parasite by the indirect fluorescent antibody technique could be shown to identify unequivocally the classic architectural features associated with their respective target organelles, i.e. microtubules, stress-fibres or the nuclear envelope.

Notes: . Pyrimidine salvage enzyme activities in cell-free extracts of Toxoplasma gondii were assayed in order to determine which of these enzyme activities are present in these parasites. Enzyme activities that were detected included phosphoribosyltransferase activity towards uracil (but not cytosine or thymine), nucleoside phosphorylase activity towards uridine, deoxyuridine and thymidine (but not cytidine or deoxycytidine), deaminase activity towards cytidine and deoxycytidine (but not cytosine, cytidine 5′-monophosphate or deoxycytidine 5′-monophosphate), and nucleoside 5′-monophosphate phosphohydrolase activity towards all nucleotides tested. No nucleoside kinase or phosphotransferase activity was detected, indicating that T. gondii lack the ability to directly phosphorylate nucleosides. Toxoplasma gondii appear to have a single non-specific uridine phosphorylase enzyme which can catalyze the reversible phosphorolysis of uridine, deoxyuridine and thymidine, and a single cytidine deaminase activity which can deaminate both cytidine and deoxycytidine. These results indicate that pyrimidine salvage in T. gondii probably occurs via the following reactions: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively; uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase; and uracil is metabolized to uridine 5′-monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5′-monophosphate is the end-product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii.

Notes: Miamiensis avidus is a marine polymorphic scuticociliate that undergoes microstome to tomite transformation when nutrients are depleted. This transformation takes place as a result of two consecutive divisions and does not involve a cyst. Tomitogenesis was induced by nutrient deprivation. When returned to nutrient medium tomites transformed into microstomes. Changes in population were noted at one-hour intervals. Tomites of Miamiensis avidus Ma/2 are compared to dispersal stages in other scuticociliates and hymenostomes.

Notes: Leishmania donovani promastigotes in late-stationary phase incorporated label from [2-14C]acetate and [1-14C]laurate into the mannose residues of mannan, thus confirming the presence of a functional glyoxylate bypass in these parasitic protozoa. Isolated, washed calls also incorporated label from [2-14C]acetate and [1-14C]laurate into mannan during a 1-hr incubation in buffer. Glucose had no effect on label incorporation into mannan, but glutamate caused over a four-fold increase in incorporation from [2-14C]acetate and a 2.4-fold increase from [1-14C]laurate. Staurosporine, a protein kinase inhibitor that inhibits glutamate and alanine oxidation, did not inhibit label incorporation from [2-14C]acetate into mannan. Hyperosmolality caused about a 33% inhibition of label incorporation into mannan. These results show the glyoxylate cycle and/or the subsequent biosynthetic pathway from fructose-6-phosphate to mannan are subject to regulation.

Notes: Pneumocystis carinii from rats and from humans were compared with respect to electrophoretic karyotype, presence of DNA sequences known to be repeated in rat-derived P. carinii, overall DNA sequence homology, and the sequences at two genetic loci. The organisms from each host species were different in each respect. Neither of two repeated DNAs from rat-derived P. carinii was found in the genome of human-derived organisms, and total DNA from rat-derived P. carinii failed to hybridize to human-derived P. carinii DNA. The sequences of the α-tubulin genes from the two P. carinii were strikingly different and the base composition of the α-tubulin gene from rat-derived P. carinii was rich in adenine and thymine, while the base composition of this gene from human-derived P. carinii was rich in guanine and cytosine. The sequence from the 18S rRNA gene of human-derived P. carinii was twice as divergent from that of rat-derived P. carinii as the sequence from the corresponding region of Candida albicans was from that of Candida tropicalis. These data show that rats and humans can harbor distinct types of P. carinii that are sufficiently different to suggest that P. carinii from the two hosts could be different species.

Notes: Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme-linked immunotransfer blot technique and by the enzyme-linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free-living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal-monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb-based enzyme-linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool.

Notes: Marine sediments from 12 shallow water stations in Raritan Bay, New York were tested for the presence of Acanthamoeba. Eight stations were positive for one or more species of Acanthamoeba, A. castellanii, A. comandoni, A. hatchetti, A. lenticulata, A. polyphaga, A. rhysodes, and Acanthamoeba spp. Isolates that grew at 38–40° C were found at four stations (A. comandoni, A. lenticulata, and two unidentified strains). The two unknown strains were characterized on the basis of morphological features, isoenzyme profiles, and mouse pathogenicity tests. One of the two strains was determined to be a new species and is designated herein as Acanthamoeba stevensoni n. sp., ATCC 50388. Mature cysts were most similar to those of morphological Group II of Pussard & Pons (1977). Acanthamoeba stevensoni n. sp. was isolated from inshore coastal sediments where seawater ranged from 20–30%‰ (ppt.). The sediments supported commercially valuable populations of hard clams, Mercenaria mercenaria, that required depuration prior to sale because of contamination by sewage-associated bacteria.

Notes: A new Myxosporea, Zschokkella mugilis n. sp., was found in the gall bladder of several mugilids. It is distinguished from all previously reported Zschokkella from mugilids by the absence of valve ornamentation and from other species by the size of the spores. Prevalence of infection was 62.5% for Mugil capito, 70% for M. cephalus and 64.3% for Liza saliens. Plasmodia with rhizoids attached to epithelial cells appeared to compromise the host as evidenced in light and transmission electron micrographs presented herein. General ultrastructure and disporous sporogenesis of Z. mugilis resembled other Myxosporeans, but sporogonic cells did not seem to be formed in pansporoblasts.

Notes: Microtubular basal bodies and epiplasm (membrane skeleton) are the main components of the cortical skeleton of Tetrahymena. The aim of this report was to study functional interactions of basal bodies and epiplasm during the cell cycle. The cortex of Tetrahymena cells was stained with anti-epiplasm antibody. This staining produced a bright epiplasmic layer with a dark pattern of unstained microtubular structures. The fluorescence of the anti-epiplasm antibody disappeared at sites of newly formed microtubular structures, so the new basal body domains and epiplasmic layer could be followed throughout the cell cycle.Different patterns of deployment of new basal bodies were observed in early and advanced dividers. In advanced dividers the fluorescence of the epiplasmic layer diminished locally within the forming fission line where the polymerization of new basal bodies largely extincted. In wild type Tetrahymena, the completion of the micronuclear metaphase/anaphase transition was associated with a transition from the pattern of new basal body deployment and epiplasm staining of the early divider to the pattern of the advanced dividers. The signal for the fission line formation in Tetrahymena (absent in cdaA1 Tetrahymena mutationally arrested in cytokinesis) brings about 1) transition of patterns of deployment of basal bodies and epiplasmic layer on both sides of the fission line; and 2) coordination of cortical divisional morphogenesis with the micronuclear mitotic cycle.

Notes: Corticocolpoda kaneshiroae n. g., n. sp. was discovered in the bark of Ohia trees (Metrosideros polymorpha) from the Hawaiian Archipelago. Its morphology and infraciliature were studied in live cells, with the scanning electron microscope, as well as in specimens impregnated with protargol, silver nitrate, and silver carbonate. The new genus, Corticocolpoda, belongs to the family Colpodidae and is unique in having a left oral ciliary field (polykinetid) composed of 13–20 equidistantly spaced, paired rows of monokinetids. The new species, C. kaneshiroae, measures 100–300 × 60–250 μm and has a huge vestibulum used to capture large food items, mainly other ciliates. The oral ciliary fields are within the vestibulum and of equal length; the right field is a paroral membrane composed of a single row of dikinetids. Several micronuclei are attached to the ellipsoid macronucleus. The mucocysts are inconspicuous and arranged around the somatic dikinetids. The resting cyst is globular and has a 1–2 μm thin, firm, yellowish inner wall and a 20-μm-thick, fragile, hyaline outer wall. Convergent evolutionary paths and a possible polyphyly of the Colpodidae are discussed. An improved key to the genera of the family Colpodidae is provided. Twenty-three species of ciliates and one flagel***iate (Hemimastix amphikineta), which occurred together with C. kaneshiroae, are new for the fauna of Hawaii and listed in the ecology section.

Notes: A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent Km value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca2+ but not Mg2+ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (∼ 9%) and phosphatidylethanolamine (∼ 2%).

Notes: The microsporidian spore stage of the nerve parasite, Spraguea lophii, consists of outer envelope stabilized in part by keratins, including K4 and K13. The nonepidermal K4 and K13 keratins were found only in the spore envelope and were absent in the internal microsporidian sporoplasm. At the time of spore activation, the keratin-based outer spore envelope assemblage dissociated and became phosphorylated when the spores were placed in the presence of labeled ATP. Verapamil or lanthanum, agents which block S. lophii spore activation, also blocked spore envelope keratin disassembly and phosphorylation when the spores were incubated in activation medium with labeled ATP. However, after the removal of the verapamil or lanthanum, the spores regained the capacity to activate in discharge medium and the keratin analogues appeared to dissociate and phosphorylate.

Notes: Thus far the life cycle of Trypanosoma (Megatrypanum) theileri has not been studied. We collected tabanids during the mass hatching, when only few tabanids are infected with trypanosomes. Tabanids were caught immediately after attacking a bait cow to serve as controls or after they had been allowed to engorge on the Trypanosoma (M.) theileri-infected cow. Tabanids were kept in the laboratory and used to study the developmental cycle of T. (M.) theileri in the tabanid gut. From day 1 to day 10 the presumably unfed controls and the engorged tabanids were dissected and cytological smears made from the mid- and hindgut. In total 2.6% (1/38) of the controls and 39% (23/59) of the engorged tabanids were positive for trypanosomes in the 1991 season. From day 1 to day 4 after engorgement trypanosomes were found in the midgut. Epimastigotes with a length of 29 μm on day 1 after infection multiplied by inequal division to form smaller epimastigotes of 26 μm on day 3. On day 4 morphologically indistinguishable trypanosomes of 21 μm total length were found in both mid- and hindgut. From day 5 to day 10 trypanosomes were found only in the hindgut in which the transformation to metacyclics was demonstrated, i.e. epimastigotes transformed to amastigote stages of 5 μm in total length.

Notes: Possible covert damage from the use of the laser optical force trap (laser tweezers) to reposition micronuclei in Paramecium tetraurelia was assessed by measuring proliferation rates and postautogamous survival and mutation rates of cells after laser manipulations. No differences in subsequent daily proliferation rates among laser manipulated and various control classes of cells were seen. Similarly, the rates of postautogamous lethality and of “slow growth mutations” after repositioning of both micronuclei were not different from such rates in unmanipulated controls. In spite of extensive manipulations of micronuclei by the laser tweezers, there is no evidence of any damage induced by these manipulations. The laser tweezers therefore appears to be a tool of benign effect upon living cells, with tremendous potential use in many cell and developmental biological investigations.

Notes: When Paramecium calkinsi encounter hyperosmotic stress, intracellular free alanine increases. In vivo assays indicate that the reaction catalyzed by alanine aminotransferase contributes to the build up of alanine in response to hyperosmotic shock. 14C-pyruvate is converted to 14C-alanine in cells grown axenically at 200 mosm. When shifted to 600 mosm, the rate of conversion of pyruvate to alanine increases, and conversion at either 200 or 600 mosm is blocked by 1 mM aminooxyacetic acid (AOA), an inhibitor of aminotransferase. Intracellular free alanine increase is partially inhibited by AOA, and AOA prevents cells living in fresh water from acclimating to higher salinities, an indication that the increase in intracellular alanine is physiologically significant.

Notes: A pharmacological characterization has been performed of the opioid receptor involved in modulation of phagocytosis in the protozoan ciliate Tetrahymena. Studies on inhibition of phagocytosis by mammalian prototypic opioid agonists revealed that morphine and β-endorphin have the highest intrinsic activity, whereas all the other opioids tested can only be considered partial agonists. However, morphine (a mu-receptor agonist) is twice as potent as β-endorphin (a delta-receptor agonist). Furthermore, the sensitivity for the opioid antagonist naloxone, determined in the presence of morphine and β-endorphin, is very similar to the sensitivity exhibited by mammalian tissues rich in mu-opioid receptors. We suggest that the opioid receptor coupled to phagocytosis in Tetrahymena is mulike in some of its pharmacological characteristics and may serve as a model system for studies on opioid receptor function and evolution.

Notes: Efforts have been made to determine whether surface antigens could be used as biochemical markers to define strain differences in the parasitic ciliate Ichthyophthirius multifiliis. In previous studies, a wild-type isolate designated G1 was found to have surface proteins analogous to the immobilization antigens of Paramecium and Tetrahymena; rabbit antiserum against this strain immobilizes homologous cells in vitro. It has now been shown for two additional Ichthyophthirius isolates (designated G1.1 and G2) that immobilization antigens are both present and serologically distinct. Proteins of similar size, which cross-react in Western blots with rabbit antisera against immobilization antigens of the G1 strain, are nevertheless found in the G1.1 and G2 isolates. As shown by Southern blotting analysis, the G1.1 and G2 strains also contain genomic DNA sequences which hybridize with an immobilization antigen cDNA from G1 when probed under conditions of reduced stringency. The serotypic differences in immobilization between I. multifiliis isolates appear to be stable over time and provide a means of discriminating strains. In addition to providing a basis for comparative studies, the work described here has implications for the development of vaccines against this important fish parasite.

Notes: . Miamiensis avidus Ma/2 is a marine polymorphic scuticociliate. Eight strains of philasterine scuticociliates, currently in culture at the American Type Culture Collection (ATCC), were shown to produce a soluble factor that induces macrostomy in Ma/ 2: Miamlensis avidus (Ma and Ma/2), Paranophrys sp. (1–1), Parauronema acutum (110–3). Anophtyoides soldoi (116–1), Anophryoides sp. (33–8). Merunophrys sp. (113–8), and Paranophrys sp. (34–7). Of the strains tested, Paranophrys strain 1–1 showed the highest induction of transformation, although all eight strains showed statistically significant induction.

Notes: . A clinical isolate of Acunthamoeba polyphagu, associated with Acunthamoebu keratitis, was used for in vitro evaluation of antiamebic activity of selected azole compounds. Those antimicrobials tested included clotrimazole, bifonazole, fluconazole, itraconazole, and ketoconazole. The drugs were tested at varying concentrations on axenic trophic amebas and MgC12,-induced cysts. On the basis of their effects on the two stages in the ameba life-cycle, clotrimazole and bifonazole were the more promising of the azoles screened but, at concentrations tested, were amebastatic rather than amebicidal. Other strains of A. polyphaga and species of Acunthamoebu were used for comparing variation in response to these and other antimicrobial agents.

Notes: . We describe a new mass isolation procedure for both pure and stage-specific phagosomes from Tetrahymena thermophila. We prepared magnetic iron dextran particles about 1 μm in diameter to label the phagosomes. The oral apparatus of the cells concentrated these particles so readily that after 1 min the majority of the cells had formed a single phagosome. A short wash removed non-ingested particles, enabling us to follow the age-dependent changes of a single labeled phagosome through the cell. Phagosomes of different ages, including very young and nascent phagosomes, were removed easily from the non-magnetic cell debris of mechanically homogenized cells by means of a permanent magnet. The isolated phagosomes are pure as tested by enzymatic assays and light and electron microscopy. Since the yield of pure phagosomes of all ages is high (∼ 90%), this method could be generally applied for phagosome isolation from ciliates.

Notes: . Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent both in vitro and in vivo. This molecule, structurally related to S-adenosylmethionine, is a good competitive inhibitor of methyltransferases in vitro. The aim of this report is to analyze the impact of sinefungin on methylation pattern and the subcellular localisation of methyl groups and various methylases in promastigotes of Leishmania donovani. We have shown the presence of various methylated macromolecules in different subcellular fractions, with somewhat higher concentration in membrane fraction. In vitro, sinefungin inhibits the three main protein methylases, but in cells cultured in its presence the protein carboxylmethylations are specifically inhibited.

Notes: . The kinetics of lysosomal enzyme acquisition by newly formed phagosomes was studied by following the rate of digestion of radiolabeled yeast fed to Acanthamoeba. The distribution of hydrolases among phagosomes was assessed by electron microscopic acid phosphatase cytochemistry and by measurement of three glycosidases in isolated early and late phagosomes. The results show that compartmentalization of hydrolases limit the digestion of large phagocytic loads. The hydrolases appear to be sequestered into the early phagosomes and not to be distributed either by small vesicle transport or phagosome-phagosome fusion to those formed later. We infer from these results that newly internalized surface membrane in phagosomes is not rapidly randomized with internal pools, but is recycled to the surface as a function of the digestive process.

Notes: . We recently reported the isolation of a leptomyxid ameba from the brain of a mandrill baboon that died of meningo-encephalitis. Based on light and electron microscopic studies, animal pathogenicity tests, and immunofluorescence patterns, we conclude that our isolate differs fundamentally from the other two amebas (Leptomyxa and Gephyramoeba) included in the Order Leptomyxida. We therefore created a new genus, Balamuthia, to accommodate our isolate and described it as Balamuthia mandrillaris to reflect the origin of the type species. Briefly, B. mandrillaris is a pathogenic ameba that causes amebic encephalitis in humans and animals. It has trophic and cyst stages in its life cycle, and is uninucleate with a large vesicular nucleus and a central nucleolus. Mature cysts have a tripartite wall consisting of an outer loose ectocyst, an inner endocyst and a middle mesocyst. Unlike Acanthamoeba and Naegleria, the other two amebas that cause amebic encephalitis in humans, Balamuthia will not grow on agar plates seeded with enteric bacteria. However, Balamuthia grows on a variety of mammalian cell cultures and kills mice following intranasal or intraperitoneal inoculation. Based on immunofluorescence testing, 35 cases of amebic encephalitis in humans and three in other animals have been identified worldwide as being caused by Balamuthia.

Notes: . The proteolytic processing and secretion of a lysosomal enzyme, acid α-glucosidase, was studied by pulse-chase labeling with [35S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid α-glucosidase into the cultured medium during starvation. the secretion was found to be repressed by addition of ammonium chloride (NH4Cl). Acid α-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH4Cl-treated cells. NH4Cl did not affect the processing of the precursor acid α-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid a-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid α-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.

Notes: . Mitosis and cytokinesis in Katablepharis ovalis, a colorless flagellate, was investigated. Two new flagella are produced prior to prophase, resulting in a motile quadriflagellate cell during mitosis. the inner array of microtubules of the feeding apparatus disappears before prophase begins. the nuclear envelope disperses during prophase, apparently being converted into rough endoplasmic reticulum. the chromatin condenses and the nucleolus disperses with spindle microtubules appearing oriented perpendicular to the longitudinal axis of the cell. At metaphase, the chromatin is condensed as a single disc-shaped mass and rough endoplasmic reticulum flanks the chromatin mass on each side. Groups of spindle microtubules pass through tunnels in the rough endoplasmic reticulum and through electron-translucent areas of the chromatin. the spindle microtubules end at a number of minipoles in the cytoplasm. Vesicles, ribosomes, mitochondria and endoplasmic reticulum migrate among the spindle microtubules. There is no polar body or any electrondense area associated with the spindle poles. the basal bodies of the flagella remain attached to the axonemes and do not participate in mitosis. In anaphase, the chromatin separates and migrates to the poles. During telophase, the nuclear envelope reforms from the rough endoplasmic reticulum and the nucleoli reappear. the spindle microtubules are persistent during telophase. Cytokinesis occurs by longitudinal fission, starting at the anterior end and progressing posteriorly. Cytokinesis may be driven by elongation of the spindle microtubules since there is no visible structure associated with the furrowing.

Notes: . Development of young gamelocytes of Leucocytozoon smithi into morphologically mature forms was studied using electron microscopy. Gametocytogenesis began on day seven post inoculation when merozoites, released from ruptured hepatic schizonts, developed into gametocytes within mononuclear phagocytes or leukocytes (monocytes or lymphocytes). No gametocytes were observed in any erythrocytes or polymorphonuclear leukocytes. Two gametocyte forms, round and elongate, were observed. Immature round gametocytes occurred on days 7-10 post inoculation in the deep vasculature of liver, lung and spleen. Mature elongate gametocytes were observed beginning on day 12 post inoculation in both the deep tissue vasculature and peripheral circulation of the turkey host. Growth and elongation of the gametocyte resulted in distortion of the host cell and its nucleus. the host cell nucleus initially was elongated and displaced to one side or indented by the growing parasite. Eventually, the nucleus was laterally compressed or split into two or three fragments. the compressed host cell cytoplasm was displaced longitudinally and stretched over the parasite to form hornlike cytoplasmic extensions from each end. the potential role of microtubules in the elongation of the gametocyte and its host cell, and possibly in the indentation and splitting of the host cell nucleus, is discussed.

Notes: . Using the polymerase chain reaction and arbitrarily selected oligonucleotide primers of 10 or 11 bases, we have amplified DNA sequences from Trypanosoma vivax parasites isolated from South America and Africa. On the basis of polymorphisms in the DNA fingerprints generated by three of the primers, the parasites could be separated into two major groups, one comprising T. vivax isolates from Kenya and the second including all the other T. vivax parasites (from Colombia, The Gambia, Nigeria and Uganda). One of these three primers (ILo 525) also gave isolate-specific DNA fingerprints for the parasites for the parasites tested, which will allow the use of this technique both in the species identification and discrimination of T. vivax parasites.

Notes: . The fine structure, elastic properties, and distribution of the fibrous, meshlike cement (bioadhesive) were studied for the test of the antarctic agglutinated foraminiferan Astrammina rara. Grain-size analysis of particles incorporated into the test compared with adjacent sediment indicates that A. rara is grain-size selective. Fractured tests curl inward, suggesting that the test is under tension—an impression substantiated by micromanipulation observations. Changes in test appearance were examined by scanning electron microscopy after sequential chemical treatments combined with ultrasonication. Organic fibrils securing fine-grained particulates on the test exterior were removed during initial sonication. A veil of fibrous organic material lining the test interior (i.e. inner organic lining) was removed by treatment with a nonionic detergent, revealing ligamentous cables of bioadhesive securely joining large grains. These cables are partially disrupted by treatment with sodium dodecyl sulfate, and further disrupted by disulfide reducing agents, suggesting that protein is an integral adhesive component. The large detrital grains incorporated into the test are arranged in an interlocked, optimally packed fashion. Together, these observations indicate that the seemingly simple spherical architecture of A. rara's test is in fact quite complex, consisting of large grains compressed by tensile cables of a proteinaceous bioadhesive, with additional rigidity supplied by fine particulate “mortar” deposited externally.