ALBERT

Notes: Books reviewed in this article: Perspectives on Biodiversity: Case Studies of Genetic Resource Conservation and Development. Christopher S. Potter, foci 1. Cohen, and Dianne Janczewski, editors

Notes: To evaluate reclamation success on the Wooley Valley phosphate mine in southeastern Idaho, we compared vegetation structure and soil physical, chemical, and elemental properties of several different reclamation treatments with those of a nearby reference area (a native Artemisia tridentata vaseyana/Festuca idahoensis association) after 14 years. Vegetation data had been collected four years after reclamation, and we were able to compare differences in biomass and species composition between dates on the reclaimed area. Four years after reclamation there were no differences in total biomass between topsoil or spoil or between seed only, seed + mulch, or control treatments on the different soil types. Most treatments were dominated by seeded perennial grasses. Fourteen years after reclamation there were no differences in biomass or cover between spoil and topsoil plots, but on spoil plots the seeded and mulched treatment had higher total biomass and vegetation cover than on control or seed-only treatments. The seeded perennial legume Medicago sativa was codominant with the seeded forage grasses on all of the treatments. High initial fertilization rates probably facilitated the early establishment and dominance of the forage grasses; once nutrient levels, especially nitrogen, began to decline, the legume increased in abundance. Similarity between the reclaimed area and the reference or native area was low. Reclaimed treatments had higher biomass but lower species richness. The topsoil and spoil plots had similar soil texture, bulk density, pH, cation exchange capacity, electrical conductivity, and phosphorus. Differences in organic carbon, total nitrogen, carbon: nitrogen ratios, and available moisture were related more to treatments than to soil type. High biomass and, thus, litter input on the seed + mulch treatment on spoil plots resulted in both higher OC and TN than any on other soil/treatment combination. The reclaimed area had lower OC, TN, and available moisture than did the reference area on all but seed + mulch spoil plots. Bulk density was higher on reclaimed plots. The long-term differences observed between the reclaimed and reference areas parallel those obtained for other western reclamation sites. Although successional trajectories depend on the attribute measured, similarity to native reference areas depends on the initial reclamation methods. We discuss reclamation methods that would increase the structural and functional similarity of reclaimed and reference areas on the Wooley Valley phosphate mine.

Notes: In land restoration it is imperative to study the potential role of disturbances, biotic or abiotic, that may provide sites for colonization by specific plants. Disturbances can alter community composition by removing species or allowing others to become established. In communities where animal-generated disturbances open sites for seedling establishment, animals may have important indirect effects on several aspects of plant community structure. Animal disturbances in Quercus havardii communities of western Texas appear to open sites for colonization by herbaceous species. These animal disturbances vary in spatial distribution, density, and abiotic and biotic characteristics. The abundance of herbaceous plant seedlings is positively related to bare ground and the number of distinct disturbances. Thus, the density and the spatial distribution of these disturbances may be expected to have an important influence on the abundance and dispersion of plant species. Therefore, successful restoration efforts of sand shinnery oak communities and other similar habitats must consider the effects of animal disturbances and the role of plant-animal and plant-soil microbe interactions on plant community composition and the maintenance of plant species diversity.

Notes: The rates of seston elimination by zooplankton and primary production were measured in Funada-ike Pond, typical of human-made impoundments in Japan, from April to September in order to evaluate various treatments of the pond aimed at improving water quality by reducing seston abundance. The treatments included draining the pond water, dredging the bottom mud, eliminating the wastewater inflow, and biomanipulation through removal of all fish. After the treatment, seston abundance was reduced from more than 10 to 0.4–2.5 mg C/liter, and large daphnid species, Daphnia similis and D. magna, occurred and predominated in the zooplankton community. Seston abundance remained at a relatively low level from June to August but increased markedly in late August, while the biomass of zooplankton became high from June to mid-August and then decreased. A decrease in seston abundance was found when the elimination rate exceeded the primary production rate. The results indicate that the development of daphnid populations was effective in keeping seston abundance at a low level. The relationship between the rate of primary production and the zoo-plankton biomass required to offset this rate, however, suggests that biomanipulation aimed at increasing zooplankton biomass alone is less effective in a pond with a high primary production. The success in improving water quality in this pond seems to depend not only on the increase in biomass of large daphnid species that resulted mainly from the removal of fish, but also on the decrease in nutrient load that was realized by the other treatments.

Notes: This essay reviews the recent attempts by the Northwest Power Planning and Conservation Council (NPPC) to conserve and restore wild salmon lost to hydroelectric development along the Columbia River and its tributaries. The restoration of the wild salmon is predicated on cooperation between myriad stakeholders in a planning process that includes the NPPC, 11 state and federal agencies, 13 Indian tribes, 8 utilities, and numerous interest groups. The two goals of the essay are (1) to review the recent amendments to the NPPC's fish and wildlife program, and (2) to describe the political barriers to restoration versus restocking of wild salmon in the Columbia River. The failure of political and administrative entities to deal with the problem of restoring wild salmon may result in drastic requirements being imposed by the imperatives of the Endangered Species Act.

Notes: An environmental revolution is urgently needed that will lead to a post-industrial symbiosis between man and nature. This can be realized only if the present unrestrained biological impoverishment and neotechnological landscape degradation are replaced by the creation of healthy and attractive landscapes. Restorationists can fulfill a vital role in this process. They must broaden their scales from biodiversity restoration in small, protected nature islands to the large-scale restoration of natural and cultural landscapes. To achieve this they must restore not only the patterns of vegetation but also the processes that create these patterns, including human land uses. Their goal should be to restore the total biological, ecological, and cultural landscape diversity, or “ecodiversity,” and its intrinsic and instrumental values of highly valuable, endangered seminatural, agricultural and rural landscapes. For this purpose it is essential to maintain and restore the dynamic flow equilibrium between biodiversity, ecological, and cultural landscape heterogeneity, as influenced by human land uses, which occur at different spatial and temporal scales and intensities. Recent advances in landscape ecology should be utilized for broader assessment of ecodiversity, including proposed indices of ecodiversity, new techniques such as Intelligent Geographical Information Systems (IGIS), and Green Books for the holistic conservation and restoration of valuable endangered landscapes. Restoration ecology can make an important contribution to an urgently needed environmental revolution. This revolution should lead to a new symbiosis between man and nature by broadening the goal of vegetation restoration to ecological and cultural landscape restoration, and thereby to total landscape ecodiversity.

Notes: A study of spider (Araneae) communities was conducted in rehabilitated bauxite mines at the Jarrahdale mine site of Alcoa of Australia Ltd. and in the nearby native jarrah (Eucalyptus marginata) forest in southwest Western Australia. The study was conducted from March to August 1993 in five rehabilitated sites of different age and method of rehabilitation and in two forest sites. A variety of collection methods was used, including pitfall trapping, litter sampling, sweep netting, tree beating, and visual searching. These methods were the same as those carried out in a previous study of some of these areas in 1983. We collected 151 spider species belonging to 102 genera and 34 families. We examined the relationship between various habitat features, including the age and method of rehabilitation, of the spider communities present. It was found that leaf litter depth and cover and vegetation density had a significant positive influence on recolonization by the various spider guilds. The age and method of rehabilitation were found to influence different vegetational and habitat features; these, in turn, influenced the spider communities. Thus, the older a rehabilitated site the greater the species richness of both plants and spiders. We compared these results with those of the 1983 study to determine the spider succession of the aging rehabilitation. The spider communities and guild composition were found to change as the vegetation matured, from a dominance of pioneer species to a community of species requiring less harsh conditions. By comparison with the pre-1983 rehabilitation, the latest method of rehabilitation increased the rate of recolonization by both plants and spiders.

Notes: Peak flowering activity among woody species in the tropical dry forests of St. John, U.S. Virgin Islands, coincided with the brief spring rainy season but continued at moderate levels for six months, abating with the autumn rains. Fruit maturation showed a major peak in the long winter dry season and a minor crest during the summer dry season. Seeds of wind-dispersed species disseminated mainly during the winter dry season, while animal dispersal of seeds (74% of all woody species) followed the bimodal pattern (for wet and dry seasons) described for the community as a whole. Under shadehouse conditions, most dry forest tree species germinated well (〉 80%) and emerged promptly (within four weeks of planting) and synchronously (90% emergence within a four-week interval). Nine of 29 species tested in the shadehouse manifested dormancy of at least six weeks. Seed germinability varied among tree species, and the viability of most species began to decline following six months of dry storage. Few species retained high germinability after nine months of dry storage. The species composition of soil seed banks did not correspond closely with above-ground communities on three forested sites of varying stand age. In the youngest stand (35 years old), dominated by the weedy, arborescent legume Leucaena leucocephala, the soil seed bank was also dominated by this species, but no seeds of any other tree species were found in the soil samples. Seeds of native trees were scarcely encountered (only one indigenous species) in soil seed bank samples of three forest sites. Local seed rain from less disturbed forest may not be sufficient for prompt recovery of the dry forest community on degraded sites.

Notes: Fire is a common but poorly understood disturbance in the forested ecosystems of the Sierra Madre Occidental of Mexico. In this study, fire history, forest structure (density, species composition, regeneration, forest floor fuels, herbaceous cover, and age of pines), and the dendrochronological tree-ring record were measured at two unharvested 70-ha pine-oak sites near Ojito de Camellones, Durango, Mexico. Study sites were matched in slope, aspect, elevation, slope position, and plant composition, but they differed in fire history since 1945 and in forest structure. The long-term mean fire intervals (MFI) for all fires at both sites up to 1945 were similar—4.0 years at Site 1 (1744–1945) and 4.1 years at Site 2 (1815–1945)—but Site 1 burned only three times at the site margins since 1945 while Site 2 had 9 fires that scarred two or more sample trees and 15 total fires since 1945. Density measurements and age and diameter distributions showed that Site 1 was dominated by numerous, younger, smaller trees (mean total basal area of 23.4 m2/ha and 2730 trees/ha), while Site 2 had fewer, older, larger trees (basal area of 37.2 m2/ha, 647 trees/ha). Large, rotten fuel loading and duff depth were also greater at Site 1. Because regeneration averaged 6200 stems/ha at Site 1 and 8730 stems/ha at Site 2 (no significant difference), forest density at Site 2 was not limited by regeneration capability. The distributions of overstory diameter and pine age at both sites indicate that tree establishment occurred in pulses, with the largest cohort of trees establishing at Site 1 following the 1945 fire. The dense regeneration and heavy fuel accumulation at Site 1 are likely to support a switch from the former low-intensity fire regime to a high-intensity, stand-replacing fire across the site when the next suitable combination of ignition and weather occurs. Baseline quantitative information on fire frequency and ecological effects is essential to guide conservation or restoration of Madrean forests and may prove valuable for restoration of related fire-dependent ecosystems that have experienced extended fire exclusion elsewhere in North America.

Notes: The objective of this study was to evaluate seven woody plant species on four growth media for their potential contribution to moose habitat and establishment of viable plant communities on a proposed mine site in southcentral Alaska. Populus balsamifera (balsam poplar), Salix alaxensis (feltleaf willow), S. barclayi (Barclay willow), S. bebbiana (Bebb willow), Alnus tenuifolia (thinleaf alder), Betula papyrifera (paper birch), and Picea glauca (white spruce) were selected for their functions in moose habitat, ease of propagation, and presence in the existing native vegetation. Three native soils were selected for biological characteristics such as different potential to form mycorrhizae and to regenerate local plant species, both of which are governed partly by existing vegetation. The fourth growth medium, glacial till or overburden, was expected to have little or no biological activity. A mining disturbance was simulated on three sites by removing existing vegetation from the plots, stripping the native soils, and then spreading these soil materials over the respective study plots. Rooted cuttings of the Salicaceae and nursery seedlings of the other species were planted in each of the four growth media. Height and survival of all plant species were greater on the three soil media than on the glacial till during the second and third years. Percentage of ectomycorrhizal infection on transplants was similar among growth media, although lower ectomycorrhizal infection occurred on volunteers in grassland soils than in the other growth media. Browsable plants were produced within three years on the disturbed native soils but not on the glacial till.

Notes: Seagrass transplanting experiments were conducted in Back Sound, Carteret County, North Carolina, and Tampa Bay, Pinellas County, Florida. In Florida, we compared three planting methods (cores, stapled bare root, and peat-pot plugs) for shoot addition rate coverage, and labor cost (harvest, fabrication, and deployment) using Halodule wrightii. Only planting methods and development rates were recorded for Syringodium filiforme. Fertilizer additions were made to peat-pot plantings of H. wrightii and Zostera marina in both North Carolina and Florida. Exclosure cages were tested to attempt to minimize bioturbation of H. wrightii and Z. marina in both North Carolina and Florida. Recovery from harvesting impacts to existing, natural beds of S. filiforme and H. wrightii were assessed in Florida. The peat-pot method was about 35% and 63% less expensive in work time than staples and core tubes, respectively. Response to fertilizer additions was masked by inconsistent release properties of the fertilizer, although some indication of positive response to phosphorus fertilizer in sediments with low carbonate content, and nitrogen in general, was detected. Complete loss of peat pots, largely ascribed to bioturbation, occurred in a large planting (Tampa Bay) but not in nearby smaller ones where exclosure cages were used. Cages did not affect planting unit survival in North Carolina but did improve number of shoots per planting unit in one of three experiments. No detrimental effects of cages were noted. Existing natura beds used to harvest transplanting stock in Tampa Bay recovered from excavations as large as 0.5 m2 in one year. Significant cost savings were found to be possible through methodological improvement, including planting techniques, bioturbation exclusion, and possibly fertilizer additions.

Notes: Land rehabilitation is proposed as a management strategy to reverse the negative consequences of tropical deforestation and land degradation. We first define the concepts associated with ecosystem modification—conversion, damage, and degradation—and those associated with ecosystem repair—restoration, rehabilitation, and reclamation. We then present a scheme of sustainable land use in the tropics, with illustrations of how rehabilitation and restoration activities fit into the overall scheme of the use of land. Because damaged lands cannot contribute effectively to sustained economic development, land rehabilitation is a necessary step for increasing the chances of attaining sustainability. Approaches for rehabilitating ecosystems are discussed, including the management of stressors and subsidies in relation to their point of interaction in the ecosystem. Finally, we illustrate the concepts of ecosystem rehabilitation of damaged, degraded, and derelict lands with examples of case studies from dry to humid life zones in island and continental situations throughout the tropics. The case studies demonstrate that opportunities for success exist, even with severely degraded lands, but a considerable amount of research remains to be done before we have a full understanding of the complexity of the task facing us.

Notes: The foundation of a successful revegetation or restoration program is quality native seed. This requires careful collection, processing, and storage. Mature seed should be collected from healthy, local stands with a sufficiently broad genetic base. Careful identification of the site characteristics and seed-lot tracking are essential. Yearly variation in seed production and seed quality can be very high, and an early determination of seed quality can prevent expensive failures. Nondestructive evaluation using X-rays is effective and economical, but techniques such as staining, inspection, and germination tests can also be helpful. Cleaning, dewinging, and upgrading seed before storage can (1) reduce weight and bulk, (2) improve storage life, (3) increase germination, and (4) make greenhouse production and field planting easier and more economical. The seeds of many native plants can lose their viability quickly if they are not stored under controlled conditions. Seeds in storage must also be protected from rodents, pests, and disease. Dormancy is common in the seeds of many native species, and experimentation is often necessary to determine the best way to break seed dormancy. This can be complicated by year-to-year and plant-to-plant variation.

Notes: The performance of woody plants was analyzed in 15 successional seres starting at bare ground in central European manmade habitats. The total cover of woody species after 10 years of succession was significantly related neither to initial soil moisture nor to nitrogen (expressed using Ellenberg indicator values). But the comparison of seres indicates that establishment of woody plants was easier under moderate environmental conditions and retarded in extreme habitats (dry, nutrient-poor, or acid). The arrival of the first woody plants was delayed in dry sites. No significant differences were found between primary and secondary seres, either with respect to the total cover of woody plants reached after 10 years of succession or considering the time of their arrival. In total, 24 woody species (10 shrubs and 14 trees) appeared in the series investigated. Their successional performance (in terms of the number of seres in which the species occurred and maximum cover reached in any sere) was not related to species traits (life strategy, type of mycorrhizae, mode of dispersal, diaspore weight), except for the regeneration strategy, species with seasonal regeneration by seeds were capable of creating higher cover. Betula pendula (European birch) was the most successful species in spontaneous succession, especially on moist sites. Practical suggestions for the management of particular habitats (sites disturbed by mining, sites reclaimed after acid rain deforestation, urban sites, abandoned fields) are provided regarding the establishment of woody plants.

Notes: Books reviewed in this article: Semiarid Lands and Deserts: Soil Resources and Reclamation J. Skujins, editor

Notes: Several landscape restoration alternatives were evaluated in the Gorecht area, a cultivated river plain in the northern part of the Netherlands. A landscape analysis was performed to investigate the hydrological functioning of this area. Groundwater composition in the area was assessed by using distribution patterns of indicative plant species. Results proved to be consistent with an interpolation of actual data of the groundwater composition. Groundwater flow was simulated with hydrological models to explain the observed patterns in water chemistry. It appeared that upwelling Ca-rich groundwater is now absent in the area, contrary to the past situation. Because a constant supply of Ca-rich water is an essential condition for mesotraphent fen vegetation, we concluded that under the present conditions the regeneration prospects for these vegetation types are poor. We suggest that the plan to regenerate groundwater-fed fens be abandoned for a plan to create surface-water-fed marshes.

Notes: Restoration of an abandoned 16-ha Nike missile base site located on a former wetland in the lower Detroit River (Michigan, U.S.A.) was investigated with an emphasis on wetland restoration. The site included a 2.7-ha abandoned missile base, a 1.3-ha lake, 10.4 ha of emergent and submersed wetlands, and 1.8 ha of uplands. Aquatic beds in the shallow bay connected to the river supported floating leaved and submersed aquatics including Nymphaea tiiberosa, Vallisneria antericana, Elodea canadensis, and Heteranthera dubia, with mats of green, filamentous algae. A 10-ha diked wetland adjacent to the site was dominated by Typha spp., Salix spp., Nymphaea tuberosa, Myriophyllum spicatum, Elodea canadensis, Chara sp., and Juncus effusus. Restoration objectives included a nature preserve, an outdoor recreation area, an experimental wetland complex, and an environmental education center. Ten alternative designs were suggested, including four with wetlands open to the bay, three with diked wetlands, and the rest involving island construction, a reconstructed upland site, or a wetland research facility. Alternatives were evaluated for their contribution to local ecology, research, education and wildlife, ease of maintenance, and probability of success. Construction of a wetland resembling conditions before construction of the base was recommended for its low maintenance and opportunity for research on non diked wetland design and construction in protected bays in the Laurentian Great Lakes. Recreational and educational opportunities were also recommended as part of the site restoration.

Notes: The feasibility of measuring soil salinity with electromagnetic induction (EM) for determining riparian restoration potential was investigated on a 28-hectare plot at the Bosque del Apache National Wildlife Refuge in central New Mexico. The plot was cleared of exotic Tamarix chinensis (saltcedar), surveyed and gridded into 1370.2 hectare sections. Soil samples and EM measurements were taken at each section. We compared laboratory-determined ECe values from the soil samples with ECa values calculated from the EM measurements using a model developed by Rhoades et al. (1990). Direct comparison of ECe values determined from the two methods yields a low correlation due to sample-size differences but the calculated ECa was able to accurately predict whether the measured ECe would lie above or below some threshold value. An assessment of general site suitability for riparian restoration with electromagnetic induction has proven to be a rapid, accurate, and cost-effective alternative to intensive soil sampling.

Notes: Extensive areas of the tropics have been converted into pasture for cattle ranching. Frequently, abandoned pasture does not revert to forest. The goal of this project was to identify barriers to lowland moist forest regeneration in highly degraded grasslands in the Sierra Nevada de Santa Marta, Colombia. The barriers we considered were seed source, seed predation, competition with grasses, microclimate and soil limitations on plant growth, and fire. Seed dispersal into the grasslands is limited to within 10 meters of forest fragments, but this barrier can be overcome by sowing seeds and planting seedlings and by establishing perches to attract dispersers. In these degraded grasslands, seed predation was lower than in the adjacent forest patches, and there was no evidence that grasses inhibited the establishment of woody species. The most important barrier was the severe degradation of the soils. In much of the area, the A and B horizons have been eroded away, leaving saprolite at the soil surface. Seedlings of two fast-growing pioneer species, Ochroma pyramidale and Cochlospermum vitifolium, grew to a maximum height of only 2.5 and 12 cm, respectively, during the first eight months. The slow plant growth in the degraded grassland soils compared to forest soils was associated with lower levels of cation-exchange capacity, calcium, magnesium, and potassium. Even if these barriers could be overcome, the frequent and extensive use of fire in the region must be controlled to avoid killing established woody plants.

Notes: Biological resources can be more usefully incorporated into many aspects of restoration ecology. During the planning and design stage, the wide genotypic variation in natural plant populations must be recognized and exploited. This will ensure that genotypes used on a site are best adapted to local conditions and have a greater probability of survivorship than arbitrarily chosen material. Also, certain unusual genotypes can be located using the principles of evolutionary ecology and can be installed in areas with extreme conditions, such as soils contaminated with heavy metals, in areas where rapid colonizing ability (high seed set and/or clonal growth) is particularly advantageous, or where soils are of poor quality. Similarly, where high herbivore pressure is a threat to restoration, genotypes that are well defended, chemically or mechanically, against animal enemies should be selected to initiate the restoration process. The nursery industry can be encouraged to supply an ecologically wider selection of material for restoration, originating from local biological reserves and natural habitats. During the management phase of a restoration, local natural habitats are critical as reservoirs of biological control agents, seed sources for plant species, and members of higher trophic levels and additional plant species needed during succession. Mutualists such as pollinators, seed dispersers, and mycorrhizal fungi are vital to the success of a restoration project, and these must invade from nearby natural habitats or must be deliberately introduced. During the evaluation phase of restoration, local natural areas should be used as templates of community composition and structure from which one measures success. A functioning restoration project will interact biologically with surrounding areas, the exchange of species and genes being particularly important. Analysis of the microbial and invertebrate communities that have invaded the installed plant community may be useful and accurate determinants of ecological function. For these latter stages of the restoration process, the value of preserving local habitat remnants is high and complements their usefulness as a source of ecologically precise material for installation.

Notes: Community classification has emerged from ecology textbooks into the arena of environmental impact evaluation and mitigation. This development is especially apparent in southern California, where the fate of a community called coastal sage scrub is being decided. Regional plans for development, mitigation, preservation, and restoration are being formulated that will permanently affect the natural landscape. This paper demonstrates the potential for the name of a plant community to affect tradeoffs in planning processes that are intended to offset removal of habitat by development. The discussion focuses on two types of classification systems. One system is hierarchical, with established nomenclatorial rules that allow natural variation to refine the community definitions. Names of plant associations directly reflect the dominant species in the association, as in Artemisia californica—Eriogonum fasciculatum (California sagebrush—California buckwheat). The second system is nonhierarchical, in which observations of natural landscapes are fit into established definitions of community types, such as Diegan coastal sage scrub. Advantages and disadvantages of both types of systems are discussed and illustrated by two examples of problems in classifying coastal sage scrub. The discussion concludes with the point that the name of a community has the potential to significantly affect the extent to which equal tradeoffs between community types are actually achieved in mitigation and restoration efforts.

Notes: This article contributes to the worldwide trend of using microbiological methods for bioindication of the influence of abiotic, biotic, and anthropogenic factors on the soil environment, especially on the plough layer of soils. It compares two approaches to assess microbial biomass-C: (1) chemical treatment of soil samples by CHCl3 vapor, followed by extraction, and (2) heat treatment in the range of 62–65°C for 24 hours, followed by extraction. It recommends the analysis of different forms of organic carbon, the total carbon of soil organic mass, and microbial biomass-C and other forms of organic carbon extractable with 0.5 M K2SO4 and 0.5 M (NH4)2SO4 respectively. It presents a simple mathematical model suitable for assessment of microbial recuperation of the topsoil (0–15 cm) after restoration. The relation between the measured values and the values calculated with the model expressed as percentages expresses the quality of restoration achieved and the stage of disturbance of the living microbial part of the topsoil. It indicates not only the ecological impact but also the deficit and stress situations, to which the living microbial part of the topsoil is exposed in the long term.

Notes: Books reviewed in this article: Ecological Principles of Nature Conservation. L. Hansson, editor Saltmarsh Ecology. P. Adam

Notes: Within an urban park in southern California, the relationship between the structure and floristics of vegetation and the distribution, abundance, and behavior of wildlife was studied in relatively undisturbed areas (San Luis Rey) and in contiguous areas (Guajome Park) in need of restoration. These data were used to develop recommendations for the enhancement of native animal species in the park. The abundance of amphibians and reptiles was highest in native upland scrub and willow (Salix)-riparian vegetation types, and lowest in dry, disturbed sites. Western fence lizards (Sceloporus occidentalis) were the most abundant reptile throughout both study areas. Overall, bullfrogs (Rana catesbiana), an exotic species, were the dominant amphibians; the native Pacific treefrog (Hyla regilla) was rare throughout. At both study areas, the small mammal community was dominated by western harvest mice (Reithrodontomys megalotis) and deer mice (Peromyscus maniculatus) and, to a lesser extent, by brush mice (P. boylii) and exotic house mice (Mus musculus). Negative correlations in abundance existed between house mice and harvest mice, and between house mice abundance and overall small mammal abundance. In riparian sites, cottonwood (Populus fremontii) and various height classes of willow were the dominant factors in the majority of bird abundance–habitat‘correlations and where foraging activities were concentrated. Recommendations for enhancing native animal species include reduction of marsh sedimentation, removal of feral species, and development of connections between the park and nearby natural areas. A corridor of native riparian vegetation (primarily cottonwood-willow) should be developed to replace the existing agricultural fields, thereby linking Guajome with the San Luis Rey River.

Notes: Eight individual rock iguanas (Iguana pinguis) from Anegada Island were relocated to Guana Island by Lazell, 1984–1987, in order to establish a second population reservoir for this endangered species. The species may have originally occupied the entire Puerto Rico Bank. The relocation has been successful and, in the area currently providing the best habitat, we estimate a density of 9 or 12 animals of various age classes per 19 ha. The optimal area contains a sheep exclosure with relatively dense understory vegetation and numerous exotic as well as native species of plants. Iguana activity is concentrated on east facing slopes and ridge-tops that get morning sun. Outside the exclosure most edible ground cover and shrubs have been eaten by sheep, leaving toxic or noxious species, such as Croton or Lantana, in the understory where I. pinguis adults generally forage. Removal of sheep may be critical to continued population growth of these reptiles. Views on relocation or repatriation of other endangered Antillean Iguana species are advanced, with some ideas on minimum viable population sizes and a possible explanation for the extirpation of I. pinguis from much of its former range.

Notes: Books reviewed in this article: Functions of Nature Evaluation of Nature in Environmental Planning, Management and Decision Making. Rudolf S. de Groot Landscape Restorastion Handbook D. Harker, S. Evans, M. Evans, and K. Harker

Notes: The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.

Notes: Descriptive information is provided for five ciliates collected from marine coastal British Columbian surface waters. In this paper we identify three new species, Strobilidium neptuni n. sp., Strobilidium veniliae n. sp., and Strombidinopsis multiauris n. sp.; provide a new name and description for one previously described species, Strombidium siculum nom. nov. and provide more taxonomic data for Strombidinopsis cheshiri Snyder and Ohman, 1991.

Notes: Giardia lamblia trophozoites contain acetylated α-tubulin but lack detectable levels of tyrosinolated α-tubulin, as demonstrated in immunoblots with monoclonal antibodies specific for these tubulin forms. By immunofluorescence microscopy, acetylated α-tubulin is localized in axonemes, median bodies and in the adhesive disk. Post-embeddment immunogold labeling of thin sections of cells was used to evaluate acetylation at the level of individual microtubules by electron microscopy. Cells were fixed with glutaraldehyde and embedded in the acrylic resin LR Gold. Results indicate all microtubules in adhesive disk, axonemes, basal bodies, funis and the median bodies contain acetylated α-tubulin. Unlike immunofluorescence labeling, all microtubules of the adhesive disk and the funis could be gold labeled. No nonspecific labeling of the cytoplasm or of structures other than microtubules was observed. Acetylated microtubules in G. lamblia do not appear to be a subset of microtubules and acetylation appears uniform along the entire length of individual microtubules. Acetylation and the tyrosinolation state of microtubules in Giardia are discussed in the context of microtubule stability and crosslinked features of the cytoskeleton.

Notes: A model for the in vivo evaluation of antipneumocystis drugs has been developed in SCID mice infected intratracheally with cryopreserved mouse-derived Pneumocystis carinii. The development of a highly reproducible fatal P. carinii pneumonia occured within 10 weeks (mean survival time ± SEM = 72.2 ± 1.2 days). Continuous administration of dexamethasone (2 mg/liter in the drinking water) exacerbated the rate of onset of severe P. carinii pneumonia (mean survival time ± SEM = 63 ± 1.3 days) in SCID mice. The number of cysts per g of lung homogenate (homogenate counts) were maximal with an inoculum of 20,000 cysts at 6 weeks post infection. Homogenate counts correlated with infection scores (graded assessments of immunofluorescent cysts on lung impression smears) suggesting that infection scoring accurately and rapidly reflects the severity of P. carinii pneumonia in SCID mice. These studies led to the development of a drug screening protocol in which Pneumocystis-free female SCID mice (20–25 g) were started on dexamethasone 7 days prior to IT inoculation with a single dose of 20,000 cysts. Drugs were evaluated either for: a) prophylaxis (continuously from day 1 post infection) or b) treatment (from day 21 post infection) until day 42 post infection, when all mice were killed and infection scores determined. Co-trimoxazole (at 250 mg sulfamethoxazole + 50 mg trimethoprim/kg/day) given in the drinking water was found to be highly effective in both the prophylaxis and treatment of mouse P. carinii pneumonia. Co-trimoxazole remained very effective in the prophylaxis P. carinii pneumonia in the SCID mouse at 125 mg sulfamethoxazole + 25 mg trimethoprim/kg/day p.o. and showed some enhancement of efficacy over sulfamethoxazole alone at 125 mg/kg/day p.o., suggesting limited synergy between sulfamethoxazole and trimethoprim. The results presented provide confirmation of the usefulness and predictability of the model.

Notes: Immunofluorescence was used to reveal the presence of a rhodopsin-like molecule on the plasma membrane of the photoresponsive ciliate Fabrea salina. The ciliate, fixed on a glass support, showed a spontaneous red-orange fluorescence. Cells incubated with a fluoresceinated, anti-bovine opsin, sheep polyclonal antiserum showed instead a green fluorescence typical of fluorochrome excited at 436 nm, whereas cells incubated with a fluoresceinated, nonimmune sheep serum kept their original red fluorescence. Excess bovine opsin did not inhibit the antibody reaction, but detergent-coated purified bovine opsin was proven able to bind to the cell membrane. Since rhodopsin belongs to a gene-related protein family, which includes adrenergic or cholinergic receptors, the cell response to acute exposure to muscarinic and adrenergic agonist and antagonist drugs was investigated. Acetylcholine, atropine, hexamethonium, noradrenaline, or phenoxybenzamine in the culture medium (10-4 M) did not influence motion behaviour nor phototaxis of Fabrea salina. These findings raise the possibility that a rhodopsin-like molecule is present on the surface of Fabrea salina. Nevertheless, they do not prove that this surface antigen is a rhodopsin, or that this molecule is responsible for photoresponsiveness in this organism.

Notes: The life cycle of malaria parasites in the mosquito vector is completed when the sporozoites infect the salivary gland and are ready to be injected into the vertebrate host. This paper describes the fine structure of the invasive process of mosquito salivary glands by malaria parasites. Plasmodium gallinaceum sporozoites start the invasion process by attaching to and crossing the basal lamina and then penetrating the host plasma membrane of the salivary cells. The penetration process appears to involve the formation of membrane junctions. Once inside the host cells, the sporozoites are seen within vacuoles attached by their anterior end to the vacuolar membrane. Mitochondria surround, and are closely associated with, the invading sporozoites. After the disruption of the membrane vacuole, the parasites traverse the cytoplasm, attach to, and invade the secretory cavity through the apical plasma membrane of the cells. Inside the secretory cavity, sporozoites are seen again inside vacuoles. Upon escaping from these vacuoles, sporozoites are positioned in parallel arrays forming large bundles attached by multilammelar membrane junctions. Several sporozoites are seen around and inside the secretory duct. Except for the penetration of the chitinous salivary duct, our observations have morphologically characterized the entire process of sporozoite passage through the salivary gland.

Notes: CLARKE BRADBURY, Department of Zoology, North Carolina State University, Raleigh, North Carolina 27695-7617.

Notes: Hybridization using kDNA and rDNA sequences as probes was performed to study phylogenetic relatedness of different species of trypanosomatids. Using this approach, we identified five organisms which had been classified as Phytomonas and Herpetomonas that were more closely correlated to each other phylogenetically than to any other species or isolates from either genera. These findings raise doubts about the validity of the current classification of Trypanosomatidae. Finally, we demonstrated the usefulness of kDNA sequences as an alternative to genomic sequences in obtaining phylogenetic information on trypanosomatids.

Notes: The relative capacity of Na+, K+ and Cl- to stimulate germination of spores of the microsporidian Nosema algerae, a pathogen of mosquitoes, was examined by ion substitution experiments. Sodium at 0.1 M was ineffective to produce the high percentage of germination that typically occurs with 0.1 M NaCl (the normal stimulation solution) if Cl- was substituted with the usually impermeant anions SO42-, HPO42-, or the organic acids oxalate, cacodylate, EGTA, MES and HEPES. However, substantial concentration- and pH-dependent germination was seen with Na2SO4 in the 0.2-0.8 M Na+ range. Similar results were obtained with solutions of K+ accompanied by impermeant anions. In contrast, the chloride salts of usually impermeant cations, like choline and triethanolamine, failed to germinate spores even at 0.8 M unless Na+ or K+ was independently added. The presence of 0.5 M choline chloride in the medium reduced the levels of Na2SO4 required to produce germination down to equivalence with those of Na+ in the normal stimulation solution. Monensin, a Na+ ionophore, facilitated the germination induced by a medium-level stimulus (0.04 M NaCl) in sonicated samples. These findings indicate that N. algerae spores germinate in response to the alkali metal cations, while CI- plays a passive role by diffusing to maintain internal electroneutrality during cation influx. A possible mechanism of cation action in spore germination is suggested on the basis of these results and observations on other systems of intracellular motility.

Notes: Certain monoclonal antibodies interact with proteins of Tetrahymena thermophila found in the conjugation junction as well as around the gametic nuclei (pronuclei) of conjugating cells; they also react with the oral primordium and fission zone of vegetative cells and with the cytoproct and contractile vacuole pores of all cells. One of these (FXIX-3A7) was investigated in detail. Immunogold labelling suggests that the material labelled by the 3A7 monoclonal antibody, which we call “fenestrin,” is located beneath the epiplasm (membrane skeleton). Immunoblots reveal that the major and perhaps sole antigen is a 64 kDa polypeptide, found in two isoelectric variants. Developmental studies implicate fenestrin in two processes involved in conjugation. The first is “tip transformation.” During preliminary starvation (“initiation”), labelling of fenestrin first appeared as a spot at the anterior end of starved mature cells, then after mixing of different mating types (“costimulation”) it extended posteriorly along the anterior suture. After pairing, this region spread to form a widened plate. The second process is pronuclear transfer. Fenestrations representing channels between the conjugating cells began to appear 0.5 to 1 h after the conjugants united, and eventually merged to form a small number of temporary large holes during exchange of the transfer pronuclei. A fenestrin envelope also enclosed both the transfer and resident pronuclei; a strand of fenestrin connected the two. Shortly after pronuclear transfer, both transfer and resident pronuclei were released from fenestrin caps and fused to produce a zygotic nucleus (synkaryon) not associated with fenestrin. Fenestrin thus appears to be intimately involved in the process of pronuclear exchange.

Notes: . The ultrastructure of meiospores of six different species of Amblyospora found infecting larval mosquitoes of Aedes abserratus, Aedes aurifer, Aedes cinereus, Aedes excruciates, Aedes sticticus, and Aedes stimulans are described. Meiospores of all species exhibited characteristics typical for the genus Amblyospora including: a single nucleus, a large posterior vacuole, a thick undulating exospore and thinner endospore, a bipartite and lamellate polaroplast with more tightly stacked membranes in the proximal region, and an anisofilar polar filament with distal coils reduced in thickness. Distinct differences were found in the arrangement and number/ ratio of coils formed by the broad basal and narrow distal portions of the polar filament. These differences, when quantified and averaged, were unique from all other mosquito-parasitic species that have been examined ultrastructurally. Information on parasite development, natural field prevalence and transmission to suspected intermediate copepod hosts is presented. The creation of six new species, Amblyospora abserrati, Amblyospora auriferi, Amblyospora cinerei, Amblyospora excrucii, Amblyospora stictici, and Amblyospora stimuli is proposed. Synonymies and complete host lists of all forms and species of Amblyospora described from mosquitoes are given.

Notes: . The α- and the β-tubulin genes of the hypotrichous ciliate Euplotes octocarinatus were isolated from a size-selected macronuclear DNA library. The α-tubulin gene is located on a 1,587 bp macronuclear DNA molecule and the β-tubulin gene on a 1,524 bp macronuclear DNA molecule. Sequencing revealed that all the cysteine residues of the two genes are encoded by the common cysteine codons UGU and UGC and none by an UGA codon. This is in contrast to the genes of E. octocarinatus sequenced so far, where some of the cysteines are encoded by the opal codon UGA. The tubulin genes end like other Euplotes genes with a TAA. They do not contain introns. The last codon for an amino acid in the α-tubulin gene is a GAA which codes for glutamic acid. This is in contrast to what has been reported for most α-tubulin genes, but it supports findings for other hypotrichous ciliates. No evidence for the existence of more than one type of α- and one type of β-tubulin genes could be obtained.

Notes: Intrachromosomal variant surface glycoprotein (VSG) genes in Trypanosoma brucei are expressed by a mechanism involving gene conversion. The 3’boundary of gene conversion is usually within the last 130 bp of the VSG gene, a region of partially conserved sequences. We report here the loss of the predominant telomeric A VSG gene in the cloned variant antigenic type (VAT) 5A3, leaving only an intrachromosomal A VSG gene (the A-B gene). The nucleotide sequence of the A-B VSG gene reveals that it lacks the normal VSG 3′ sequence. Surprisingly, we find cells expressing this A-B VSG gene in relapse populations arising from VAT 5A3. Since the A VSG mRNAs from these cells have a normal 3′ sequence, the incomplete A-B VSG gene must be expressed via a partial gene conversion that supplies the functional 3’end. Although the A-B VSG gene is no longer predominant like the telomeric A VSG gene, it is still expressed more frequently than other intrachromosomal VSG genes, suggesting that factors other than a telomeric location determine whether a VSG gene is expressed early in a serodeme.

Notes: Mixed life cycle stages of rat-derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas-liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii, respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas-liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost-5-en-3-ol), cholest-5-en-3-one, and β-sitosterol (stigmast-5-en-3-ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C28 and C29 sterols with one or two double bonds, and a hydroxyl group at C-3 (ergost-5-en-3-ol, ergost-7-en-3-ol, ergosta-dien-3-ol, stigmast-5-en-3-ol, stigmast-7-en-3-ol and stigmasta-dien-3-ol). Steryl esters of P. carinii, probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3-hydroxy-3-methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own “metabolic sterols”

Notes: Cultured P. carinii rapidly took up a variety of fatty acids. The relative rates of uptake for four fatty acids were 18:1 〉〉 16:0 ≅ 18:0 ≅ 18:2. Fatty acids were primarily incorporated into phospholipids and the uptake process was specifically inhibited by 2.2 and 22 μM primaquine, a concentration having no effect on host cells. Amino acids were also taken up by cultured P. carinii in a primaquine sensitive process. Radiolabeled leucine was incorporated into the major surface glycoprotein of P. carinii. The formation of radioactive P. carinii-specific proteins indicated that the cultured organisms carried out transcription and translation and that the incorporation of amino acids was dependent upon P. carinii rather than rare HEL human embryonic lung cells. The spinner flask culture system provides convenient access to P. carinii for metabolic studies in defined medium for a period of 5–14 days after inoculation.

Notes: The roles of the myosin I class of mechanoenzymes have been investigated by single and double gene knockout studies in the amoeba Dictyostelium discoideum. Cells lacking different myosin I pairs (myoA-/myoB-, myoB-/myoC-, and myoA-/myoC-) were examined with respect to their cytoskeletal organization. F-actin localization by rhodamine-phalloidin staining of cells indicates that the myoA-/myoB-, myoB-/myoC-, and myoA-/myoC- cells appear to redistribute their F-actin more slowly than wild type cells upon adhesion to a substrate. These studies suggest that Dictyostelium myoA, myoB, and myoC may have overlapping roles in maintaining the integrity or organization of the cortical membrane cytoskeleton.

Notes: The new microsporidium, Napamichum cellatum, a parasite of the adipose tissue of midge larva of the genus Endochironomus in Sweden, is described based on light microscopic and ultrastructural characteristics. Plurinucleate Plasmodia with nuclei arranged as diplokarya divide, probably by plasmotomy, producing a small number of diplokaryotic merozoites. The number of merogonial cycles is unknown. Each diplokaryotic sporont yields eight monokaryotic sporoblasts in a thin-walled, more or less fusiform sporophorous vesicle. A small number of multisporoblastic sporophorous vesicles were observed, in which a part of the sporoblasts were anomalous. The sporogony probably begins with a meiotic division. The mature spores are slightly pyriform. Fixed and stained spores measure 2.1-2.4 × 3.7-4.5 μm. The five-layered spore wall is of the Napamichum type. The polar filament is anisofilar with seven to eight coils (142-156 and 120 nm wide). The angle of tilt is 55-65°. The polaroplast has an anterior lamellar and a posterior tubular part. The granular, tubular and crystal-like inclusions of the episporontal space disappear more or less completely when the spores mature. The crystal-like inclusions are prominent in haematoxylin staining, but not visible with the Giemsa technique. The microsporidium is compared to other octosporoblastic microsporidia of midge larva and to the species of the genera Chapmanium and Napamichum.

Notes: CB1-glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1-glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1-glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1-glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.

Notes: Trichocyst discharge is an effective defense of Paramecium against Dileptus margaritifer. The possible defensive function of backward swimming, which often follows trichocyst discharge upon Paramecium-Dileptus encounters was studied. Mutants incapable of backward swimming (pawnA in P. tetraurelia, cnrA in P. caudatum) escaped from dilepti nearly as frequently as wild-type cells. Double mutants (pawnA-nd7, cnrA-tnd2) were eaten nearly as frequently as mutants incapable of trichocyst discharge. Thus, in the defense of Paramecium against D. margaritifer, the role of backward swimming is minor, if any, compared to trichocyst discharge. Among escaped cells, about a half of wild-type and essentially none of pawnA (cnrA) cells showed backward swimming. Paramecium behavior during the encounter can be mimicked by the local, not global, application of lysozyme which is a strong secretagogue of trichocyst.

Notes: A total of 4.1% infestation with Chilodonella spp. was found among fish studied in 144 tanks in 1987–1989, representing 14.0% of the tanks in which fish are reared at four salmonid farms in northern Finland. Two species were found, C. hexasticha and C. piscicola, and both occurred on salmon (Salmo salar L.), sea trout [S. trutta m. trutta (L.)] and brown trout [S. t. m. lacustris (L.)]. Variability was observed in the length and width of the C. piscicola specimens and the number of ciliary rows or kineties. Large specimens which had more kinetics than average for C. piscicola were found mainly on the skin of salmon aged 1–2 years. The number of kinetics in the right ciliary band was found in stepwise logistic regression analysis to be of importance when typing C. piscicola specimens. Fingerlings were found to be more susceptible to Chilodonella infestation than older fish, and mortality varied in the range 2–10% in the course of the epizootics in the three fish species. Most mortality cases were caused by C. hexasticha, occurring mainly on the gills of the fish. Chilodonella piscicola was most often found in salmon and occurred at lower water temperatures than C. hexasticha (mean water temperature when found for the first time being 13° C and 16° C, respectively).

Notes: Chlamydomonas reinhardtii exhibits photophobic and positive and negative phototactic responses that can be defined for cell populations using computerized cell tracking and motion analysis. Mutants CC-2359 and FN68 are pigment deficient mutants that are blocked in carotenoid synthesis and lack these photo responses. In particular, neither mutant exhibits flash-induced photophobic responses to visible light stimuli to which wild-type gametic cells exhibit a strong response, with several behavioral stages. Upon addition of all-trans retinal to these mutants, the photophobic responses are restored with minor quantitative differences from wild-type populations. Using both light and electron microscopy, we have compared the ultrastructural characteristics of wild-type C. reinhardtii to those of both mutants. As previously described, wild-type cells contain an eyespot consisting of 2–4 layers of pigmented granules encased within thylakoid membranes, located between the distal extremities of the flagellar root. This structure is also visible as an orange-red spot in light microscopy. The photoreceptor is thought to be concentrated in the plasma membrane above the eyespot. The mutant, CC-2359, lacks this eyespot as seen by both light and electron microscopy, even when the photophobic response has been restored. FN68-like mutants studied earlier by Morel-Laurens and Feinlieb and others contain an eyespot which can be seen only by electron microscopy. In FN-68, the eyespot generally has the same dimensions as in wt cells, differing mainly in pigment granule appearance. Consistent with these findings, several laboratories have shown that the full range of phototactic responses can be reconstituted in FN68 and CC-2359, but that negative phototaxis requires a significantly stronger light stimulus in the latter strain. We confirm the suggestion that the eyespot is not necessary for the photophobic response, and is not critical for the appropriate assembly and function of the photophobic response receptor in the membrane. Furthermore, the locus of reconstitution of the functional receptor is not the eyespot. Because of the definitive demonstration of the absence of the eyespot in CC-2359, however, the eyespot may play a role in negative phototaxis.

Notes: The rDNA repeat unit from a new human Giardia duodenalis strain shows significant differences from the previously reported G. duodenalis rDNA repeat. Twelve base-pair changes occurred in 490 bp of the SSrRNA gene and new restriction enzyme sites occurred in the LSrRNA gene. The overall length of the rRNA genes is the same but the spacer is 76 bp longer than previously reported. A boundary within the spacers of the two different rDNA units divides a region of 50% homology near the LSrRNA gene from a region of 80% homology toward the SSrRNA gene. This boundary region includes two copies of a 78 bp repeat.

Notes: Twenty-one different strains of Euplotes of the cirrotype 10, double dargyrome morphotype were collected in marine and brackish habitats from widely varying geographic locations. The strains were cultured under similar conditions prior to fixation and staining. The morphological features considered included the cell lengths and widths, the number and position of all the ventral cirri, adoral membranelles and dorsal cilia, and the position of the peristomal shelf. Univariate and multivariate analyses revealed continua in these measurements among the strains, as well as significant correlations between cell length and the number of adoral membranelles, the number of dorsal cilia, and the length, width and area of the oral cavity. Based on data from this study and from previous studies, we conclude that the traditional criteria used for species descriptions within this morphotype are insufficient to provide robust distinctions. Therefore, we submerge all of the descriptions within this morphotype under the nomenclaturally superior Euplotes charon Müller, 1786.

Notes: The Hartmannella vermiformis small-subunit rRNA coding region was amplified, and the amplified DNA was cloned and sequenced. The coding region is 1,840 nucleotides long, and is typical of eukaryotic rRNA genes in both size and composition. Different clones contained different nucleotides at three positions.

Notes: The micronuclear version of the gene encoding β-telomere binding protein (β-TBP) in Oxytricha nova has been sequenced and compared to the macronuclear β-TBP gene, previously described. The micronuclear gene contains three AT-rich internal eliminated sequences (IES) of 37, 40, and 43 bp and four macronuclear destined sequences (MDS). The IES interrupt the gene once near the 5′ end of the coding region and twice in the 3′ trailer downstream from the TGA stop codon. The sequences of the micronuclear and macronuclear genes are colinear. Thus, the micronuclear β-TBP gene is not scrambled, which contrasts with the highly scrambled state among the 14 MDS in the micronuclear α;-TBP gene.

Notes: A new species of marine interstitial ciliate, Tracheloraphis primitarum sp. n., is described from intertidal sands (160-200-μm medium grain size) along the Northwest Atlantic coast. Living specimens are not transparent under incident light, 0.4-0.9 mm in length, and are characterized by an elongated body, bulbous anterior and rounded posterior regions. Cytostome is simple without a cleft on the lip. Protargol silver impregnation shows 18-29 longitudinal kineties. The glabrous zone is very narrow, 6-7 μm wide, and corresponds to the area occupied by one kinety and two interkinetal spaces. Ten to fifteen kineties terminate against the glabrous zone. Interkinetal extrusomes are present. The nuclear apparatus is a single centrally-placed nuclear group that consists of three large irregular macronuclei and 2-3 micronuclei; these are either clustered or encapsulated.

Notes: The most commonly encountered protostelids are nominal members of the genus Protostelium Olive & Stoianovitch. These are Protostelium mycophaga Olive & Stoianovitch and P. irregularis Olive & Stoianovitch. Both species share the common features of long-stalked fruiting bodies with single, uninucleate, deciduous spores and trophic states that consist solely of uninucleate amoebae, but they are quite different with respect to their detailed structure at both the light and electron microscopic levels. Based on this evidence, it seems unlikely that the species are congeneric, and it is proposed that P. irregularis be assigned to Soliformovum n. g. Examination of other species of Protostelium indicates that P. nocturnum Spiegel should be retained in the genus and that P. expulsum Olive & Stoianovitch should be reassigned to Soliformovum. The group most closely related to Protostelium, under this new concept, is the genus Planoprotostelium Olive & Stoianovitch, while Soliformovum is best treated as a genus of Eumycetozoa incertae sedis, though it does share some similarities with the group that includes the genera Ceratiomyxella Olive & Stoianovitch, Nematostelium Olive & Stoianovitch, and Schizoplasmodium Olive & Stoianovitch.

Notes: Dyer, B. D. & Obar, R. A. 1994. Tracing the History of Eukaryotic Cells. The Enigmatic Smile. Dyer, B. D. & Obar, R. A. 1994. Tracing the History of Eukaryotic Cells. The Enigmatic Smile. Plattner, H. 1993. Advances in Cell and Molecular Biology of Membranes.

Notes: A monoclonal antibody (mAb) IR-2-1 was raised against a 67-kDa protein purified from the macronucleus-specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67-kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR-2-1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa-free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air-dried P. caudatum. These results suggest that the 67-kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule-specific mAb, DS-1, the antigens for IR-2-1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium. Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.

Notes: By adding the protein synthesis inhibitor, emetine (10-4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2-Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non-dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non-dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.

Notes: . Classical genetic techniques were applied to clonal cultures of the Euplotes vannus-crassus-minuta sibling species complex in an effort to provide some resolution to the species problem among these hypotrichs. Complex mating interactions were observed among clonal stock cultures derived from samples collected from sympatric and allopatric populations in a wide geographic survey. These results suggested that the classical model for the mating type inheritance and determination in these Euplotes is necessary but not sufficient to describe the mating mating interactions among populations of these ciliates. Successful conjugation between the nominal species E. crassus and E. vannus was observed routinely, and crosses between these two nominal species did not differ significantly from those among the other clonal stock cultures with respect to mating intensity and exconjugant survival. Data from backcrosses suggests that E. vannus and E. crassus can and do exchange genes. Based upon these data, we conclude that E. vannus and E. crassus comprise a single, highly polymorphic species with countless small populations, among which incomplete genetic exchange takes place.

Notes: . Twenty different clonal strains of marine and brackish Euplotes, representing four morphotypes, were tested for hyposalinity tolerance by a method which gradually acclimated the cells to lower salinity medium. The lowest salinities in which the strains could thrive ranged from 60% of normal seawater to complete freshwater. The morphological effects of culture medium salinity were also examined for two strains of a small “Euplotes charon” morphotype, as well as for two mating compatible “Euplotes vannus” strains and several of their exconjugates. There were no differences between the euryhaline strains grown in fresh or saltwater, except for a slight increase in overall cell size in one strain when cultured in freshwater medium. E. vannus strains increased in overall cell size with decreased salinity; also, the dorsal surface of the cells can become disorganized when the cells are cultured in 30% normal seawater.

Notes: . A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5′ and 3′ termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (〉 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.

Notes: . Highly-pathogenic, mouse-passaged Naegleria fowleri amoebae are complement resistant. The present study evaluates the effect of complement on N. fowleri and the virulence of the amoebae after animal passage and growth in two different axenic media. Pathogenic N. fowleri maintained in “enriched” Cline medium are virulent for mice and resistant to complement lysis. A rapid decline in resistance to complement and virulence for mice is observed when highly-pathogenic N. fowleri are grown in Nelson medium lacking hemin. N. fowleri maintained in Nelson medium can be rendered complement-resistant by shifting the amoebae to growth in Cline medium for 2 h prior to the addition of complement. Cycloheximide treatment of N. fowleri maintained in Nelson medium blocks the transition to a complement-resistant phenotype following a shift in growth medium. Proteins were radiolabeled with [35S] during a shift from Nelson to Cline medium to identify specific polypeptides which may be associated with the functional activities related to virulence and resistance to complement.

Notes: . Large percentages of Toxoplasma gondii tachyzoites could be induced to display two types of movement associated with active invasive behavior by exposing them for 1 min to 0.002% trypsin in phosphate-buffered saline (PBS). The motile activity, consisting of clockwise rotation around the posterior end (about 20 revolutions per min) and twirling-gliding over a poly-L-lysine substrate (1.2 ± 0.2 μm/s standard deviation), was observed and recorded by video-enhanced contrast microscopy. The number of active tachyzoites reached a maximum 1 min after trypsinization; the motile response of the population lasted for about 5 min. Activation was prevented by soybean trypsin-inhibitor, and could not be induced again in previously treated specimens. Electron-microscopy of trypsinized tachyzoites fixed in the presence of ruthenium-red revealed discrete discontinuities of the plasma membrane, which sealed within 90 min after washing with PBS. Treated tachyzoites were able to invade cultured epithelial cells with a higher relative infectivity than that of untreated parasites. Perfusion of trypsinized tachyzoites with 1 mM of either CaCl2 or MgCl2 and 1 mM ATP increased the number of activated parasites to over 60%; on the other hand, all induced motility was inhibited or blocked by agents that chelate divalent cations. The present preparation, which provided the first serial illustrations of T. gondii movements induced by a defined chemical stimulus, may offer a useful experimental model for the study of motility in this parasite.

Notes: Lagenophrys novazealandae n. sp. occurs on the gills of Paranephrops zealandicus, a parastacid crayfish from New Zealand. The new species has the hemispheroidal lorica most common among members of its genus and is distinguished by its possession of large tubercles on the thickened edge of the anterior lip of the lorica aperture, a deep cleft in the left side of the lip's edge, and a ovoid to reniform macronucleus located in the right-hand part of the body. It is probable that an as yet unnamed species of Lagenophrys known to occur on another species of Paranephrops in New Zealand is distinct from L. novazealandae but phylogenetically related to it. Lagenophrys petila n. sp. occurs on setae of Parastacoides tasmanicus, a parastacid from Tasmania. The new species has an ovoid lorica tapering to a slender pseudostalk at the posterior end, a type of lorica possessed by only two other members of its genus that also attach to their host's setae. It is distinguished from the other ovoid species by the proportions of the lorica, the extreme shortness of the lips of the lorica aperture, and an ovoid macronucleus located in the right, anterior part of the body. Clefts in the lips of L. novazealandae and other members of Lagenophrys may function as points of flexure to allow the lips to bend in ways that accommodate interspecific differences in the size of the epistomial disk and its operation during suspension feeding.

Notes: . Parasitic amebas propagate among hosts through cysts, the resistant forms in their life cycle. In spite of their key role in infection, little is known about the encystation process and the mechanisms involved in reaching this stage. Two features drastically affected by encystation are motility and cell shape, both of which are determined by the cytoskeleton, composed mainly of actin in these organisms. Therefore, we studied the occurrence and relative levels of actin and actin synthesis during encystation of Entamoeba invadens. Using a cDNA actin probe obtained from a library of E. histolytica and a monoclonal antibody against actin, we found that, while the total actin levels sharply decrease as encystation proceeds, the levels of actin mRNA are reduced only in mature cysts. Moreover, actin synthesis does not take place in precysts and the later stages of cyst formation. In contrast, the levels of other proteins remain stable in trophozoites, precysts and cysts, and stage specific peptides are actively synthesized in precysts. The results indicate that encystation is accompanied by a preferential down-regulation of actin synthesis and a decrease in actin levels. The reorganization of the cytoskeleton occurring as trophozoites transform into round, quiescent cells, could be a regulatory factor in the observed changes.

Notes: . Metanophrys diminuta n. sp., a marine philasterine scuticociliate collected from the south Florida coast, was preserved for transmission electron microscopy using a mixed fixation technique. Rough endoplasmic reticulum profiles were found proximal to mitochondria and within mitochondrial fenestrations (or deep pockets). In longitudinal sections mitochondrial profiles appear to be very long, occupying almost the entire length of the cell. The micronucleus is roughly spherical to ovoid, semi-enclosed in the macronucleus. The oral polykinetid #1 is composed of two files of kinetosomes. The c segment of the oral dikinetid is composed of three to four kinetosomes. This species is the smallest within the genus Metanophrys.

Notes: . The microsporidium Nadelspora canceri n. g., n. sp., is described from the striated musculature of the Dungeness crab (Cancer magister) in Oregon, USA. The needle-shaped spores were rounded anteriorly, tapered to a posterior point and measured 7.1–11.8 × 0.2–0.3 μm in fixed preparations. The extremely narrow spore diameter prevented observation of morphological details at the light microscopic level and ultrastructural details of mature spores were difficult to resolve. Meronts were not observed and the monokaryotic merozoites and sporonts were not contained within either parasitophorous or sporophorous vesicles. Sporonts were disporoblastic and gave rise to monokaryotic sporoblasts that became narrow and elongate as they developed into immature spores with a developing polar filament. The nucleus was not clearly resolved in mature spores and may have been surrounded by the lamellar polaroplast. The polar filament was of nearly uniform diameter throughout most of its length and ended abruptly about three-fourths of the distance from the anterior end of the spore. Unusual spherical non-membrane bound granules surrounded the polar filament in a spiral arrangement. The new microsporidium resembles members of the family Mrazekiidiae, but differs in lacking a diplokaryon at any stage. It is probably most closely related to Baculea daphniae from which it differs primarily by spore shape and size. The familial relationships of the genus Baculea have not been determined and it is proposed to include it with Nadelspora in the new family Nadelsporidae.

Notes: . The clonal life history of ciliated protists is characterized by a sequence of phenotypes; sexual immaturity, maturity, and senescence. The distinctiveness of immaturity and maturity has been investigated. Standard assays of the onset of maturity of progeny clones from a cross between stocks EC1 and EC2 of Euplotes crassus demonstrated significant differences among clones and among testers within clones. They also revealed that the first positive test(s) of a progeny subclone were typically followed by at least one negative test. Special protocols were devised to investigate if maturity was reversible at the cellular level. In these experiments, the first mating pair of a progeny subclone was split before the consummation of mating. From these two cells as well as from control progeny and tester cells, subclones were established and every leftover cell was tested for maturity after each transfer. Both standard and split-pair progeny subclones had immature and slow- to-mate cells. The number of fissions before progeny exhibited sexual behavior indistinguishable from the testers was more than twice that to the first mating reaction of a subclone. At the first sign of maturity, progeny lines are a heterogeneous population of cells able and not able to mate, but remarkably, clonal descendants of those able to mate may become unable to mate. The development of maturity is progressive, quantitative and non-monotonic rather than an instantaneous switch.

Notes: . Cell division in higher eukaryotes is mainly controlled by p34cdc2 or related kinases and by other components of these kinase complexes. We present evidence that cdc2-like kinases also occur in Paramecium. Two polypeptides reacted with an antibody directed against the perfectly conserved PSTAIR region found in cdc2 kinases in other eukaryotes. Only the less abundant peptide bound to p13suc1 from Schizosaccharomyces pombe. Using centrifugal elutriation to select cells on the basis of size, we isolated highly synchronous Paramecium G1 cells. With this procedure, we demonstrated that the p13suc1-associated cdc2-like histone H1 kinase was activated before cell division at the point of commitment to division in Paramecium. Further, we show that Paramecium cdc2-like proteins occurred principally as monomers and that these monomers were active as histone H1 kinases in vitro.

Notes: . Strombidium purpureum Kahl, is an anaerobic oligotrichous ciliate with endosymbiotic phototrophic bacteria. Like other anaerobic ciliates, S. purpureum reacts to O2-pressure. In the light, the ciliates avoid even traces of O2 (〈 1% atmospheric saturation). In the dark, however, the ciliates accumulate in water with a pO2 of 1–4% atmospheric saturation. Experiments show that ciliates which have accumulated in the dark under microaerophilic conditions react when the light is turned on, and their ability to escape is enhanced by steep O2-gradients. The ciliates orient themselves in O2-gradients by a series of phobic responses. They tumble whenever they swim towards a higher O2-tension and thus they eventually all swim in the direction of lower pO2. The ecological implications of such behaviour are discussed.

Notes: . The application of an immunocytochemical method to identify precystic stages and to analyse the encystment kinetics, by using a polyclonal antiserum against isolated cyst walls from the ciliate Colpoda inflata, is reported for the first time. Three different precystic phases were chosen on the basis of morphological changes and degree of cyst wall formation. By using this procedure a better identification of mature resting cysts with regard to precystic cells or young cysts is provided. An average consensus encystment kinetics of C. inflata, by using an accumulated class frequency analysis, is reported.

Notes: . Using RNA: RNA in situ hybridization, the intracellular location of a transcript encoded by and spanning the entire length of a Trypanosoma cruzi kinetoplast DNA minicircle was determined. In axenically cultured T. cruzi epimastigotes, the hybridization signal was restricted to the kinetoplast, which was situated in the perinuclear region of the cell. Following conversion of epimastigotes to culture-derived metacyclic trypomastigotes, the kinetoplast moved to an acentric position in the metacyclic trypomastigote. Again, the hybridization signal co-localized with the position of the kinetoplast. These results suggested that the transcript remained closely associated with the T. cruzi kinetoplast within the mitochondrion in each of the morphological forms. Using specific oligonucleotide probes derived from a cDNA encoding the transcript, the entire native kDNA minicircle encoding the transcript was cloned and its nucleotide sequence was determined. The nucleotide sequence of the intact native minicircle was identical to that of the full-length cDNA corresponding to the minicircle transcript, indicating that the transcript was not modified prior to the time of cDNA synthesis and cloning.

Notes: . Eukaryotic mitotic cell cycles have been extensively studied in yeasts and vertebrate cells but little is known about cell cycle mechanisms in early branches of the eukaryotic lineage. Trichomonas vaginalis represents one of the earliest branching eukaryotic lineages available for study. In contrast with most yeasts and vertebrate cells, the T. vaginalis G2 period was prolonged, comprising 50 to 58% of the cell population. Hydroxyurea, aphidicolin, and excess thymidine, all of which arrest yeasts and vertebrate cells at the G1/S phase boundary, had no effect on the T. vaginalis cell cycle, probably due to the known absence of synthetic pathways. The antimicrotubule mitotic inhibitors, colchicine and nocodazole, induced G2 phase synchrony. Metronidazole, a therapeutic reagent, also caused G2 phase arrest. These observations suggest that T. vaginalis is similar to yeasts and vertebrate cells in G2 and M phases, but the parasite's G1/S phase transition is distinctive. The results also suggest potentially therapeutic, anti-trichomonad activity of microtubule inhibitors such as nocodazole. The cultured parasite may prove useful as a model for the mitotic cell cycle in the absence of G1/S phase transitional activities universal in yeasts and vertebrate cells.

Notes: . Reversible changes in kinetoplast DNA (kDNA) minicircles sequences were observed in clones of Trypanosoma cruzi strain Y, following a number of passages during exponential growth phase or after subcloning in blood-free medium. kDNA restriction patterns of clones were similar to those of the original uncloned strain, while subclones presented distinct kDNA restriction patterns. Homology experiments demonstrated strong hybridization between kDNA with the same electrophoretic mobility patterns while only weak signals were observed with kDNA of different patterns. The changes observed, which are unprecedented in T. cruzi clones, characterize transkinetoplastidy, and seem to be associated with similarly reversible changes both in zymodeme and in infectivity.

Notes: . Growth of Acanthamoeba castellanii in batch culture at 30° C was associated with marked changes in cellular fatty acid composition. The largest change occurred in the linoleate to oleate ratio, which was maximal in early- to mid-exponential phase cultures but decreased approximately 10-fold as cells approached stationary phase. The higher degree of lipid unsaturation in young cultures was accentuated by a greater proportion of 20-carbon polyunsaturated fatty acids than in stationary phase cultures. The unsaturation index (average number of double bonds per fatty acid) was maximal in mid-exponential phase cultures after 24 hours growth. Incorporation of [1-14C]acetate into polyunsaturated fatty acids in short-term (2 hour) experiments was high in 12 and 24 hour old cultures, where linoleate and eicosadienoate accounted for up to 26% of total labelled fatty acids. Incorporation of [1-14CJacetate into these fatty acids was negligible in stationary phase cultures. These results were correlated with changes in the specific activity of the Δ12-desaturase. Δ12-Desaturase activity was greatest in microsomal membranes isolated from early- to mid-exponential phase cells, but declined by approximately 50% as cultures progressed towards stationary phase. Membrane fractionation studies revealed that although some differences in fatty acid composition between plasma-membrane, mitochondrial (enriched), and microsomal membrane fractions were evident, the large changes in lipid unsaturation in whole cells of A. castellanii could not be accounted for by differential development of particular subcellular membranes.

Notes: Avila, J. L. & Harris, J. R. (ed.). 1992. Subcellular Biochemistry, Volume 18: Intracellular Parasites. Plenum Press, New York.Fensome, R. A., Taylor, F. J. R., Norris, G., Sarjeant, W. A. S., Wharton, D. I. & Williams, G. L. 1993. A Classification of Living and Fossil Dinoflagellates. Micropaleontology Special Publication No. 7. Micropaleontology Press, American Museum of Natural History, New York.Volberding, P. & Jacobson, M. A. (ed.). 1993. AIDS Clinical Review 1993/1994. Marcel Decker, Inc., New York.Hyde, J. E. (ed.). 1993. Protocols in Molecular Parasitology, Volume 21 in the Methods in Molecular Biology Series. Hurnana Press, Totowa, New Jersey.

Notes: . Four different tubulin genes were identified in the somatic nucleus (macronucleus) of Euplotes focardii, a strictly coldadapted, Antarctic ciliate: one of 1,800 bp for α-tubulin and three of 2,150, 1,900, and 1,600 bp, respectively, for β-tubulin. Preliminarily analysed for restriction fragment length polymorphisms, these genes showed remarkable differences in organisation from tubulin genes of other ciliates which live in temperate areas and were analysed in parallel with E. focardii. The complete coding sequence of the 1,600 bp β-tubulin gene was then determined and shown to contain unique structural features of potential importance for E. focardii microtubule organization and activity. Of eight unique substitutions detected, seven were concentrated in the large amino terminal domain of the molecule that directly interacts with the carboxy terminal region of α-tubulin for heterodimer formation. Sequence analysis of the cloned gene revealed, in addition, a potential new exception in the use of the genetic code by ciliates. A TAG codon was aligned in correspondence with Trp-21 which is strictly conserved in every tubulin sequence so far determined.

Notes: . The mitochondrion appears to be essential for the growth of asexual, intraerythrocytic stages of Plasmodium falciparum and may thus be a suitable chemotherapeutic target. The in vitro activity of almitrine, a mitochondrial ATP synthetase inhibitor used for the treatment of hypoxemia, was compared with other mitochondrial inhibitors against chloroquine-susceptible and chloroquine-resistant P. falciparum using an isotopic semimicro drug susceptibility assay. The 50% inhibitory concentration (IC50) values of almitrine (range: 2.6–19.8 μM) were within similar range of values of other mitochondrial ATP synthetase inhibitors and doxycycline, a mitochondrial protein synthesis inhibitor. Almitrine was equally active against chloroquine-susceptible and chloroquine-resistant parasites. Drug combination studies showed no interaction between chloroquine and almitrine. Our results suggest that almitrine, a clinically safe drug, may represent a lead compound with a specific target against the mitochondrial ATP synthetase which may be useful for antimalarial chemotherapy.

Notes: . Twenty-five in vitro cultures of Giardia duodenalis derived from a Brisbane patient were established to assess the genetic heterogeneity of a population. Each of the established lines carried a predominance of one of two distinct varieties of Giardia. The two varieties were heterogeneous by four unambiguous criteria that were representative of the whole genome. These included restriction enzyme polymorphisms, hybridization with the cloned rDNA repeat and with a gene encoding a cysteine-rich surface protein, electrophoretic karyotyping and DNA fingerprinting. Differences between parasites derived from this patient were greater than have been seen between all other established G. duodenalis in vitro cultures from both human and animals. The culture were heavily selected such that a single Giardia line carried a predominance of one genotype and was not representative of the entire original population.

Notes: . Cross-transmission experiments were performed in order to determine the host specificity in the intermediate and definitive hosts of the four described dihomoxenous Sarcocystis species, S. gallotiae, S. stehlinii, S. simonyi, and S. dugesii from lacertid lizards of the genera Gallotia and Podarcis from the Macaronesian Islands. Sarcocysts of either species from experimentally infected lizards were fed to a variety of laboratory-bred lizard species of the genera Gallotia, Lacerta, and Podarcis. These sarcocysts proved to be infectious to all examined animals, showing no definitive host specificity in the tested genera. Lizards of the genera Chalcides and Tarentola, however, were not susceptible definitive hosts for S. gallotiae. The inoculation of experimentally obtained sporocysts of each of the four Sarcocystis species to various lacertid lizard species revealed varying degrees of intermediate host specificity, generally demonstrating each native host to be the most susceptible.

Notes: . Cells of the ciliate Tetrahymena thermophila failed to establish cultures in lipid-free standard synthetic nutrient medium if the initial population density was 250 cells per ml or less. These cells died within 10 h, but were saved and formed dense cultures if their medium was supplemented with 10 μg per ml of either certain phospholipids, 1,2-di-, 1-monoglycerides, fatty acids, long-chain alcohols, or sterols. Cell multiplication was followed in cultures in which the standard synthetic medium was supplemented with a selection of the compounds listed above. It was observed that the cells in the supplemented cultures in their exponential phases of growth had about the same average doubling times as control cells starting multiplication at 10-fold higher initial cell densities in lipid-free medium. These cells have been grown for decades in lipid-free synthetic nutrient media at short (ca. two-three h) doubling times. Therefore lipids have been considered nutritionally non-essential for growth and multiplication of these cells. We propose that those compounds that rescue the cells at low cell densities act as “proliferation signals,”sensu lato. This effect of lipids and long-chain alcohols has so far remained unnoticed.

Notes: . The expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana was investigated using gelatin polyacrylamide gel electrophoresis. Two prominent bands were detected which distinguished metacyclics from multiplicative promastigotes, lacking detectable cysteine proteinase activity, and amastigotes, with a distinct banding pattern composed of multiple enzymes. A correlation between relative activity of the metacyclic-specific bands and the prevalence of metacyclics was found both during the growth cycle in vitro as metacyclogenesis occurred, and by comparison of stationary phase populations from consecutive subpassages in vitro. Irreversible inhibition of the metacyclic activities using N-benzyloxycarbonyl-phenylalanyl-alanyl diazomethane did not inhibit metacyclic to amastigote transformation in vitro. These activities provide a useful biochemical marker for the metacyclic promastigotes of L. mexicana.

Notes: . Spore suspensions of microsporidian parasites of fish (Microsporidium ovoideum, Glugea stephani, Glugea atherinae and Spraguea lophii) have been analyzed by flow cytometry. Spore nuclei were dyed either by propidium iodide or bis-benzimide (Hoechst 33342). By observation of forward light scatter and fluorescence the four species could be distinguished and the mono- and diplokaryotic populations of S. lophii identified. Staining of DNA by bis-benzimide was better and easier than propidium iodide. Forward light scatter and fluorescence values were characteristic of each species and remained unchanged throughout the year, so flow cytometry can be used for distinction of spores of some microsporidian parasites once their flow cytometric parameters are known. However, special care has to be taken in tool calibration and material preparation for analysis because of the high precision of the technique.

Notes: . The microsporidian species Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis were compared by using sequence data of their rRNA gene segments, which were amplified by polymerized chain reaction and directly sequenced. The forward primer 530f (5′-GTGCCATCCAGCCGCGG-3′) was in the small subunit rRNA (SSU-rRNA) and the reverse primer 580r (5′-GGTCCGTGTTTCAAGACGG-3′) was in the large subunit rRNA (LSU-rRNA). We have utilized these sequence data, the published data on Encephalitozoon cuniculi and Encephalitozoon hellem and our cloned SSU-rRNA genes from E. bieneusi and S. intestinalis to develop a phylogenetic tree for the microsporidia involved in human infection. The higher sequence similarities demonstrated between S. intestinalis and E. cuniculi support the placement of S. intestinalis in the family Encephalitozoonidae. This method of polymerized chain reaction rRNA phylogeny allows the establishment of phylogenetic relationships on limiting material where culture and electron microscopy are difficult or impossible and can be applied to archival material to expand the molecular phylogenetic analysis of the phylum Microspora. In addition, the highly variable region (E. coli numbering 590–650) and intergenic spacer regions in the microsporidia were noted to have structural correspondence, suggesting the possibility that they are coevolving.

Notes: . Ultrastructural studies on Eimeria (syn. Epieimeria) anguillae (Apicomplexa), parasite of the digestive tract of the eel, have shown that the development of this parasite takes place completely within the host cell. Merogony and gamogony are intracellular but in the epicytoplasmic position. Sporogony is also located within the epithelial cells, which agrees with assignment of this coccidian in the family Eimeriidae. However, depending on the intensity of infection and the physiopathological reaction of the host, the gamont may behave in two ways. 1) In massive infections, gamogony stages cause a genuine destruction of intestinal epithelium. Large numbers of gamonts form nodules and parts of the seriously destroyed epithelium peel off and are released into the lumen of the gut and quickly discharged into the outer environment. This discharged epithelium envelops cells containing immature oocysts that then sporulate outside the host. 2) In light infections, the host cells, which are necrotic due to the presence of a zygote, are pushed between the surrounding intact cells towards the base of the epithelium. Closely above its basal lamella, the oocyst then undergoes sporulation. These results show no taxonomically important biological features (e.g. special mode of implantation to the host cell or active movement of the zygote). Because the morphological characteristics of Epieimeria do not differ significantly from Eimeria, we propose to suppress the genus Epieimeria Dyková and Lom, 1981, and relegate its species into the genus Eimeria.

Notes: . The degree of strain and/or species diversity among Pneumocystis carinii isolates is unknown. As a first approach to the study of P. carinii genetic relatedness, we compared the pulsed field gel electrophoretic karyotypes of P. carinii derived from lung homogenates of three immunosuppressed host animals: rats transtracheally inoculated with P. carinii-infected rat lung; mice transtracheally inoculated with P. carinii-infected mouse lung; and ferrets which developed reactivated latent P. carinii pneumonia. Rat P. carinii propagated on HEL299 cells was also examined. Karyotypes of P. carinii DNA from both rat lung homogenate and cell culture were identical (14 bands, 315–680 kb). In contrast, mouse and ferret P. carinii DNA karyotypes were each distinctly different from the rat P. carinii samples (mouse P. carinii 15 bands, 315–610 kb; ferret P. carinii nine bands, 410–760 kb). Three distinct rat P. carinii gene probes reacted with both Southern-transferred rat and mouse P. carinii DNA but not with ferret P. carinii DNA. Thus, P. carinii from rat, mouse, and ferret are genetically diverse. The results are consistent with recently reported antigenic and nucleic acid sequence differences among P. carinii isolates recovered from different hosts.

Notes: . Leishmania donovani promastigotes were collected, washed, resuspended in buffer, and assayed for sucrase activity. No activity was observed in the intact washed cells, but activity was measurable when the cells were permeabilized with Triton X-100. Intracellular sucrase activity was highest in promastigotes grown at pH 7.4, somewhat lower in promastigotes grown at pH 5.5, and significantly lower in “amastigotes” grown at pH 5.5. No trehalase, lactase, or maltase activities were observed. Assay of the medium in which the cells had grown showed that most the sucrase activity was extracellular, i.e. was secreted into the medium during growth.

Notes: . We have studied the action of diphtheria toxin, modeccin and ricin on HeLa cells infected by Trypanosoma cruzi. Parasitized HeLa cells were resistant to diphtheria toxin and modeccin, whereas non-parasitized cells from the same cultures and control cultures showed cytopathological alterations. Protein synthesis, assayed by the incorporation of labelled methionine, diminished in toxin-treated control cultures but remained unaltered in the infected ones, compared to synthesis by untreated infected cells. Ricin, on the other hand, is a toxin that enters the cytoplasm by endocytosis. It has greater cytopathological effects in parasitized cells than in non-parasitized ones from the same cultures or uninfected control cells. Protein synthesis was inhibited in infected cultures treated with ricin.

Notes: . A microsporidian parasite, Nosema muscidifuracis n. sp., has been found in Muscidifurax raptor, a parasitoid of muscoid flies. Stages of the parasite developed in direct contact with the host cell cytoplasm and were detected in midgut epithelium, Malpighian tubules, ovaries (including oocytes) and fat body of larvae and adults. Spores were also detected within eggs deposited on the host. Light and electron microscopy revealed a developmental cycle with diplokaryotic stages dividing by binary fission and disporous sporulation sequences producing diplokaryotic spores of three morphological classes, differing significantly only in length of the polar filament. Two of the classes were found in larvae, pupae and adults. One of these, with about five turns in the coiled polar filament, is presumed to be responsible for transmission from cell to cell within the host (autoinfection) and the other, with about 10 turns, responsible for transmission from host to host. A third class, with about 15 turns in the polar filament, was found in eggs of M. raptor. It is, presumably, either involved in initiation and spread of the infection at eclosion or is responsible for horizontal transmission to a new host individual when eggs are cannibalized.

Notes: . In the ciliate Euplotes crassus a complex series of developmental events lead to formation of a new macronucleus. Millions of telomeres are synthesized during this process. We have shown that transcript levels are tightly regulated throughout Euplotes conjugation and macronuclear development. Thus, expression of gene products needed for macronuclear development and telomere synthesis appears to be controlled at the level of RNA abundance. To learn more about the role played by telomerase and the Euplotes telomere protein during telomere synthesis, we have correlated changes in the abundance of telomerase RNA and telomere protein mRNA transcript with specific developmental events. Telomerase RNA levels increase steadily during the early stages of macronuclear development and reach a peak just after telomere addition. The telomere protein transcript rises and falls twice during conjugation and then rises again at the time of telomere addition. The increases in transcript levels during conjugation parallel micronuclear division suggesting that the telomere protein is synthesized at this time and hence may have a micronuclear function.