Further Studies on in vitro Development of Eimeria bovis and Attempts to Obtain Second-Generation Schizonts*

SYNOPSIS. Eimeria bovis merozoites occurred in tissue culture medium removed from Leighton tube cultures of embryonic bovine tracheal cells beginning 12-14 days after inoculation with 270,000-369,000 sporozoites per tube. The number of merozoites produced in these cultures increased daily until a peak was reached 18-21 days after inoculation. In 3 experiments an average of 2.0–15.6 million merozoites per tube was produced during the 20-day observation period. When such merozoites were frozen in liquid nitrogen and stored 26–42 days, some were motile upon thawing. These merozoites as well as others freshly obtained from cell cultures and from calves were inoculated into 11 different types of cultured mammalian cells including primary, cell line and established cell line cultures. Some merozoites were exposed to substances normally found in the lumen of the gut, before or at the time of inoculation. Altho small numbers of intracellular merozoites were found, no further development was observed. Gametocytes were observed in the cecum of a calf 4 days after merozoites from cell cultures were introduced into a ligated cecum of the calf.     

Ultrastructural Study of Schizogony in Eimeria callospermophili*

SYNOPSIS The development of 1st generation schizonts of Eimeria callospermophili was studied with cell cultures and with experimentally infected host animals, Spermophilus armatus. Sporozoite-shaped schizonts each had 5-10 nuclei and all of the organelles of the sporozoite; each nucleus had a nucleolus and an associated Golgi apparatus. In stages immediately preceding merozoite formation, an intranuclear spindle apparatus with conical polar areas were observed near the outer margin of each nucleus. Two centrioles, each having 9 single peripheral tubules and one central tubule, were observed near each pole in some specimens. Merozoite formation began internally, with anlagen of 2 merozoites developing near each nucleus. The inner membrane of the merozoites first appeared as 2 dense thickenings adjacent to the polar cones and centrioles; subpellicular microtubules appeared simultaneously. Two anterior annuli and the conoid formed between the 2 thickenings. Vesicles, possibly of Golgi origin, were located next to the forming inner membrane. As the forming merozoites underwent elongation, a rhoptries anlage, a Golgi apparatus, refractile bodies, and mitochondria were incorporated into each. Sporozoite-shaped schizonts with merozoite anlagen transformed into spheroid or ovoid schizonts; at this time the conoid, rhoptries, micronemes, and the inner membrane of the pellicle gradually disappeared; several small refractile bodies were formed from the larger one. When development was about 1/3 complete, the immature merozoites began to grow outward from the surface of the schizont. In this phase of development, the single surface membrane of the schizont became the outer membrane of the merozoite's pellicle, and additional organelles, including the nucleus, were incorporated. Finally, the merozoites became pinched off, leaving a residual body. Development in cell cultures and host tissues was similar. This type of schizogony, previously undescribed in Eimeria, is compared with corresponding stages of development in other species of Eimeria and Sporozoa.     

Eimeria scholtysecki n. sp. from Ord's Kangaroo Rat Dipodomys ordii*

SYNOPSIS. Eimeria scholtysecki n. sp. is described from Ord's kangaroo rat Dipodomys ordii. The sporulated oocysts are broadly ovoid to ellipsoid, averaging 24.6 by 19.6 μ. A polar granule is present. A micropyle and oocyst residuum are absent. The ovoid sporocysts average 12.1 by 8.0 μ, and have small, flattened Stieda bodies. The distinctive sporocyst residuum is composed of coarse granules. The mean prepatent period was 8.2 days. Five inoculated rats apparently became reinfected and discharged oocysts for 30 days or more.     

Sensitive detection of mycoplasmalike organisms in field-collected and in vitro propagated plants of Brassica, Hydrangea and Chrysanthemum by polymerase chain reaction

A polymerase chain reaction (PCR) protocol, previously designed for amplification of a DNA fragment from aster yellows mycoplasmalike organism (MLO), was employed to investigate the detection of MLO DNA in field-collected and in vitro micropropagated plants. PCR with template DNA extracted from symptomatic, naturally-infected samples of Brassica, Chrysanthemum and Hydrangea, each yielded a DNA band corresponding to 1.0 Kbp. However, no DNA product was observed when either infected Ranunculus (with phyllody disease) or Gladiolus with (symptoms of ‘germs fins’) was used as source of template nucleic acid for PCR; further experiments indicated absence of target DNA in the case of Ranunculus and the presence of substances in Gladiolus which inhibited the PCR. The MLO-specific DNA was detected by PCR using less than 95 pg of total nucleic acid (equivalent to total nucleic acid from 1.9, ug tissue) in the case of field-collected Hydrangea and less than 11.4 pg of nucleic acid (equivalent to total nucleic acid from 19 ng of tissue) in the case of field-collected Brassica. The findings illustrate highly sensitive detection of MLOs in both field-grown and in vitro micropropagated infected plants.     

Ultrastructure of the Invasion of Eimeria magna Sporozoites into Cultured Cells*†‡

SYNOPSIS. Monolayers of bovine kidney cells were overlaid with Eimeria magna sporozoites and observed with phase-contrast optics until penetration of the cells by the parasites had begun. Cells and penetrating parasites were fixed with glutaraldehyde and OsO₄-containing ruthenium red, dehydrated, and embedded in situ. Cells being penetrated were selected for study in the electron microscope. The lack of intracellular staining with ruthenium red and intact plasmalemmas of cells being penetrated, was accepted as evidence that the sporozoites did not disrupt the plasma membranes. The sporozoite caused invagination of the host cell plasmalemma until the parasite was entirely within the cell, after which the invagination was sealed off by short pseudopodia enclosing the sporozoite within a membrane-lined vacuole inside the cell. Often myelin-forms, apparently of host cell origin, were seen in the space between the sporozoite and the cell.     

Geology,Oil and Gas Potential,Pipelines, and the Geopolitics of the Caspian Sea Region

The Caspian Sea region contains oil and gas reserves that are comparable to those of other of the world's fossil-fuel-producing regions, excluding the Middle East. We review here the economic, environmental, and complicated geopolitical concerns with respect to exploration and recovery of the region's fossil fuels. These include mud volcanoes, gas hydrates, earthquakes, pollution, rapid changes in sea level, desertification, ownership of resources, and the transportation routes of fossil fuels. Significant advances have been made concerning these problems in the short time since the breakup of the Soviet Union, fueling optimism for the future of the region.-     

Mechanism of Mitochondrial Mutation in Yeast

THE yeast Saccharomyces cerevisiae can mutate to the respiratory-incompetent petite colony form. The mutation is probably caused by damage to, or loss of, the yeast's mitochondrial DNA, for petite mutants often lack mitochondrial DNA, possess it in abnormal amounts or with abnormal buoyant density¹. Some of the agents, such as acrifiavine or ethidium bromide, which induce the petite mutation interfere with mitochondrial DNA synthesis^2,3 whereas ethidium bromide also causes or permits degradation of Saccharomyces cerevisiae mitochondrial DNA^2,3. We have observed that nalidixate (50 µg/ml.), an inhibitor of DNA synthesis, can prevent or delay petite mutation induced by ethidium bromide⁴. A similar effect has been observed by Hollenberg and Borst using a higher nalidixate concentration⁵. We have investigated the mechanism of this effect. A diploid prototrophic strain of Saccharomyces cerevisiae (NCYC 239) was used throughout.     

Phylogenetic analysis of the latitude‐niche breadth hypothesis in the butterfly subfamily Nymphalinae

1. One possible explanation for the latitudinal gradient in species richness often demonstrated is a related gradient in niche breadth, which may allow for denser species packing in the more stable environments at low latitudes. 2. The evidence for such a gradient is, however, ambiguous, and the results have varied as much as the methods. Several studies have considered the non‐independence of species, but few have performed explicit phylogenetic analyses. 3. In the present study, we tested for a correlation between diet breadth and latitude of distribution in Nymphalinae butterflies using generalised estimating equations (GEE) and accounting for phylogenetic independence. 4. Using a simple model with only latitude of distribution as a predictor variable revealed a significant positive relationship with diet breadth. Previous studies, however, have shown that diet breadth is also correlated with butterfly range size, and in turn, that range size may be correlated with latitude of distribution. Including geographical range size in the model also turned out to have a profound effect on the results – to the extent that the relationship between latitude of distribution and diet breadth was effectively reversed. 5. We conclude that, at least for this group of butterflies, there is no evidence for a positive correlation between latitude of species distribution and diet breadth when controlling for range size, and that the effect may actually even be reversed.