Plasmodium gallinaceum: ookinete formation and proteolytic enzyme dynamics in highly refractory Aedes aegypti populations

Despite significant progress in the identification of the genetic basis of the refractory phenotype, little is known about the physiological mechanism of refractoriness. This study therefore examined the physiological basis of mosquito refractoriness in the Aedes aegypti/P. gallinaceum system, in which a selected refractory strain does not permit Plasmodium oocyst formation. We examined the kinetics of two major proteolytic enzymes involved in blood meal digestion and the dynamics of ookinete formation for two refractory populations (strains Moyo-R and Formosus) and one susceptible population (strain Red). Healthy ookinetes were observed in both the susceptible and the refractory populations, although the susceptible population generally exhibited higher enzymatic activity for trypsin and aminopeptidase than the refractory populations. Parasite numbers in the susceptible Red population showed a 4- to 7-fold decrease in abundance during the transition from the ookinete stage to the oocyst stage, far less than the refractory populations (30- to 92-fold reduction). Due to its smaller body size, Moyo-R individuals generally ingest a smaller blood meal and thus intake fewer gametocytes than Red individuals. Thus, the possibility that refractoriness in the Moyo-R population results from fewer gametocytes being ingested is examined. We found that the Red population remained highly susceptible and the Moyo-R population stayed refractory when those individuals with similar blood meal size were compared. We conclude that failure of oocyst development in the refractory mosquitoes is not due to ookinete damage by proteolytic enzymes or to fewer gametocytes being ingested, but rather is due to a midgut barrier or to some other mechanism.     

Pseudomonas pellicle in disinfectant testing: electron microscopy, pellicle removal, and effect on test results

Pseudomonas aeruginosa ATCC 15442 is a required organism in the Association of Official Analytical Chemists use-dilution method for disinfectant efficacy testing. When grown in a liquid medium, P. aeruginosa produces a dense mat or pellicle at the broth/air interface. The purpose of this investigation was to examine the pellicle by scanning electron microscopy, to evaluate three pellicle removal methods, and to determine the effect of pellicle fragments on disinfectant efficacy test results. The efficacies of three methods of pellicle removal (decanting, vacuum suction, and filtration) were assessed by quantifying cell numbers on penicylinders. The Association of Official Analytical Chemists use-dilution method was used to determine whether pellicle fragments in the tubes used to inoculate penicylinders affected test results. Scanning electron micrographs showed the pellicle to be a dense mass of intact, interlacing cells at least 10 microns thick. No significant differences in pellicle removal methods were observed, and the presence of pellicle fragments usually increased the number of positive tubes in the use-dilution method significantly.     

Intracellular calcium levels in the Plasmodium berghei ookinete

Ookinetes are the motile and invasive stages of Plasmodium parasites in the mosquito host. Here we explore the role of intracellular Ca2+ in ookinete survival and motility as well as in the formation of oocysts in vitro in the rodent malaria parasite Plasmodium berghei. Treatment with the Ca2+ ionophore A23187 induced death of the parasite, an effect that could be prevented if the ookinetes were co-incubated with insect cells before incubation with the ionophore. Treatment with the intracellular calcium chelator BAPTA/AM resulted in increased formation of oocysts in vitro. Calcium imaging in the ookinete using fluorescent calcium indicators revealed that the purified ookinetes have an intracellular calcium concentration in the range of 100 nm. Intracellular calcium levels decreased substantially when the ookinetes were incubated with insect cells and their motility was concomitantly increased. Our results suggest a pleiotropic role for intracellular calcium in the ookinete.     

Pseudomonas pellicle in disinfectant testing: electron microscopy, pellicle removal, and effect on test results.

Pseudomonas aeruginosa ATCC 15442 is a required organism in the Association of Official Analytical Chemists use-dilution method for disinfectant efficacy testing. When grown in a liquid medium, P. aeruginosa produces a dense mat or pellicle at the broth/air interface. The purpose of this investigation was to examine the pellicle by scanning electron microscopy, to evaluate three pellicle removal methods, and to determine the effect of pellicle fragments on disinfectant efficacy test results. The efficacies of three methods of pellicle removal (decanting, vacuum suction, and filtration) were assessed by quantifying cell numbers on penicylinders. The Association of Official Analytical Chemists use-dilution method was used to determine whether pellicle fragments in the tubes used to inoculate penicylinders affected test results. Scanning electron micrographs showed the pellicle to be a dense mass of intact, interlacing cells at least 10 microns thick. No significant differences in pellicle removal methods were observed, and the presence of pellicle fragments usually increased the number of positive tubes in the use-dilution method significantly.     

Plasmodium gallinaceum: a novel morphology of malaria ookinetes in the midgut of the mosquito vector

Malaria ookinetes invade midgut epithelial cells of the mosquito vector from the bloodmeal in the lumen of the mosquito midgut, but the cellular interactions of ookinetes with the mosquito vector remain poorly described. We describe here a novel morphology of Plasmodium gallinaceum ookinetes in which the central portion of the ookinete is an elongated narrow tube or stalk joining the anterior and posterior portions of the parasite. We propose that the previously undescribed stalkform ookinete may be an adaptation to facilitate parasite locomotion through the cytoplasm of mosquito midgut epithelial cells.     

Observations on the fine structure of the pellicle pores ofEuglena granulata

Summary The structure or granules associated with the pellicle strips ofEuglena granulata (Klebs) Schmitz has been studied using light and electron microscopy. Two types of bodies can be distinguished in this association on the basis of their staining reactions, distribution, ultrastructure, and other characteristics. The first type, called pellicle pores, is distributed with regularity along certain pellicle strips; the pores are very uniform in size (about 300 m in diameter) and in content. They consist of an aperture and a compartment, the latter lined by the plasmalemma; in each compartment a dense, osmiophilic body and a series of tubules occur. The structure of the pellicle strips is modified at the points where the pellicle pores are present; the aperture extends to the cell surface in the groove between two pellicle strips. The second type of body, which undergoes vital staining with neutral red, is called a pellicle vacuole. These are variable in size (ca. 1 to 2 microns in diameter) and contain a variety of components including myelin figures, proliferated membranes, and sheets of electrondense material. The structure of these elements as well as their possible functional significance are discussed in some detail.     

Exoerythrocytic development of Plasmodium gallinaceum in the White Leghorn chicken

Plasmodium gallinaceum typically causes sub-clinical disease with low mortality in its primary host, the Indian jungle fowl Gallus sonnerati. Domestic chickens of European origin, however, are highly susceptible to this avian malaria parasite. Here we describe the development of P. gallinaceum in young White Leghorn chicks with emphasis on the primary exoerythrocytic phase of the infection. Using various regimens for infection, we found that P. gallinaceum induced a transient primary exoerythrocytic infection followed by a fulminant lethal erythrocytic phase. Prerequisite for the appearance of secondary exoerythrocytic stages was the development of a certain level of parasitaemia. Once established, secondary exoerythrocytic stages could be propagated from bird to bird for several generations without causing fatalities. Infected brains contained large secondary exoerythrocytic stages in capillary endothelia, while in the liver primary and secondary erythrocytic stages developed primarily in Kupffer cells and remained smaller. At later stages, livers exhibited focal hepatocyte necrosis, Kupffer cell hyperplasia, stellate cell proliferation, inflammatory cell infiltration and granuloma formation. Because P. gallinaceum selectively infected Kupffer cells in the liver and caused a histopathology strikingly similar to mammalian species, this avian Plasmodium species represents an evolutionarily closely related model for studies on the hepatic phase of mammalian malaria.     

Presentation of cell membranes by freeze-fracture electron microscopy

Freeze-fracturing is especially suitable for the investigation of membrane structures. In contrast to ultrathin sectioning, large areas of the membranes are exposed. The true surface of membranes, however, can be seen only after etching (vacuum sublimation of ice) because during fracturing the frozen membrane is split between the two lipid layers. The representation of the hydrophobic region of the membrane reveals particles representing integral membrane proteins or, exceptionally, micelles of membrane lipids. Special structures on microbial membranes are, e.g., regular particle arrangements, invaginations and lipid domains with a periodic pattern of curvatures. There are still many unsolved questions concerning these structures, but the occurrence or the alteration of such structures as well as the density of “etching holes” on the membrane fracture face can be used as indicators for membrane perturbations.     

Malaria parasite colonisation of the mosquito midgut--placing the Plasmodium ookinete centre stage

Vector-borne diseases constitute an enormous burden on public health across the world. However, despite the importance of interactions between infectious pathogens and their respective vector for disease transmission, the biology of the pathogen in the insect is often less well understood than the forms that cause human infections. Even with the global impact of Plasmodium parasites, the causative agents of malarial disease, no vaccine exists to prevent infection and resistance to all frontline drugs is emerging. Malaria parasite migration through the mosquito host constitutes a major population bottleneck of the lifecycle and therefore represents a powerful, although as yet relatively untapped, target for therapeutic intervention. The understanding of parasite-mosquito interactions has increased in recent years with developments in genome-wide approaches, genomics and proteomics. Each development has shed significant light on the biology of the malaria parasite during the mosquito phase of the lifecycle. Less well understood, however, is the process of midgut colonisation and oocyst formation, the precursor to parasite re-infection from the next mosquito bite. Here, we review the current understanding of cellular and molecular events underlying midgut colonisation centred on the role of the motile ookinete. Further insight into the major interactions between the parasite and the mosquito will help support the broader goal to identify targets for transmission-blocking therapies against malarial disease.     

Scanning electron microscopy of the shell membranes of the hen's egg

Summary Scanning and transmission electron microscopy have been used to study the structure of the hen's shell membranes and their relationship to the shell and to the chorioallantoic membrane.We have confirmed previous observations that the fibres of the outer shell membrane are of larger diameter than those of the inner shell membrane, and that the fibres of the outer shell membrane extend into the mammillary knobs of the shell.The shell membrane fibres are arranged in layers parallel to the surface of the egg and there is no interweaving between the layers. Individual fibres are randomly orientated and may extend for distances of at least 25 m. It is suggested that relative movement between the oviduct and the developing membrane is random in direction and location.Each fibre consists of a core with a covering cortex, but the idea that the core may consist of keratin is criticised. A limiting membrane separating the surface of the albumen from the fibres of the shell membrane is also formed from this cortex. During incubation the chorioallantoic membrane becomes pressed against this inner limiting membrane.No correlation could be found between the positioning of the mammillary knobs and the patterning of the shell membrane fibres. It is suggested that the positioning of the mammillary knobs reflects the pattern of certain secretory cells in the genital tract of the hen.No significant changes in structure of thickness of the shell membrane could be found during incubation. The tips of the mammillary knobs, however, become detached from the shell and remain adherent to the shell membrane.The Cambridge Scientific InstrumentsStereoscan scanning electron microscope was provided by the Science Research Council (UK).We should like to thank Mr.R. F. Moss and Mr.P. S. Reynolds for technical assistance, and Mrs.Jeanne Mills for secretarial assistance.