Ute Mackenstedt

Parasitic apicomplexans harbor a chlorophyll a-D1 complex, the potential target for therapeutic triazines

Ultrastructural evidence is presented for the presence of plastid-like organelles inToxoplasma gondii, Sarcocystis muris, Babesia ovis, andPlasmodium falciparum. In addition, it was shown that merozoites ofT. gondii contain protochlorophyllidaea and traces of chlorophylla bound to the photosynthetic reaction centers I PS I and PS II. ApsbA gene was isolated from merozoites ofS. muris by the polymerase chain reaction (PCR). Partial sequencing of the PCR product revealed that the herbicide-binding region is highly conserved. Therefore, it is likely that the sensitivity of apicomplexans to the herbicide toltrazuril depends on the interaction of the herbicide with the D1 protein of the photosynthetic reaction center of the parasites organelles.

Ultrastructural investigation of matrix-mediated biomineralization in echinoids (Echinodermata, Echinoida)

SummaryThe formation of echinoderm endoskeletons is studied using echinoid teeth as an example. Echinoid teeth grow rapidly. They consist of many calcareous elements each produced by syncytial odontoblasts. The calcification process in echinoderms needs (1) syncytial sclerocytes or odontoblasts, (2) a spacious vacuolar cavity within this syncytium, (3) an organic matrix coat in the cavity. As long as calcite is deposited, the matrix does not touch the interior face of the syncytium. The cooperation between syncytium, interior cavity and matrix coat during the mineralization process is discussed. The proposed hypothesis applies to the formation of larval skeletons, echinoderm ossicles and echinoid teeth.When calcite deposition ceases the syncytium largely splits up into filiform processes, and the skeleton is partly exposed to the extracellular space. However, the syncytium is able to reform a continuous sheath and an equivalent of the cavity and may renew calcite deposition.The tooth odontoblasts come from an apical cluster of proliferative cells, each possessing a cilium. The cilium is lost when the cell becomes a true odontoblast. This suggests that cilia are primitive features of echinoderm cells. The second step in calcification involves the odontoblasts giving rise to calcareous discs which unite the hitherto single tooth elements. During this process the odontoblasts immure themselves. The structures necessary for calcification are maintained until the end of the process.The mineralizing matrix is EDTA-soluble. The applied method preserves the matrix coating the calcite but more is probably incorporated into the mineral phase and dissolved with the calcite.

DNA measurements and ploidy determination of developmental stages in the life cycles ofTheileria annulata andT. parva

The relative DNA levels of different developmental stages ofTheileria annulata andT. parva in the cow and the tick were measured by the cytophotometric DNA technique using the fluorochrome Hoechst 33258 as a staining dye. The results revealed that sporozoites, merozoites, gamonts, and gametes were haploid, whereas multinucleated intralymphocytic schizonts were polyploid. No difference was observed betweenT. parva andT. annulata in these stages. For bothTheileria species, the DNA measurements revealed that fusion of gametes occurred in the gut of the final host, thus providing evidence of sexual reproduction. However, differences were observed between the two parasites in the tick. WhereasT. parva zygotes underwent a two-step meiotic division, a comparable reduction division could not be unequivocally detected inT. annulata. Differences could also be detected in the further development of kinetes, indicating thatTheileria species are not characterized by only one life cycle, which is specific for this genus.

Sexual cycle of Babesia divergens confirmed by DNA measurements.

The DNA content of the developmental stages within the life cycle ofBabesia divergens was measured by means of fluorescence microscopy using the DNA-specific bisbenzimide Hoechst 33258. By comparing the mean relative fluorescence intensities, the assumption of sexual reproduction in the gut of the tick vector (Ixodes ricinus) was confirmed. However, no proof of a pre- or postzygotic meiosis was found.

DNA measurements reveal differences in the life cycles ofBabesia bigemina andB. canis, two typical members of the genusBabesia

The relative DNA levels of different developmental stages ofBabesia bigemina andB. canis were measured by cytophotometry using the fluorochrome Hoechst 33258 as a staining dye. The DNA measurements provided direct proof of sexual reproduction, i.e., fusion of gametes resulting in the formation of zygotes followed by a meiotic division. BothBabesia species are considered to be typical members of the genusBabesia; however, the DNA measurements revealed important differences in the life cycle of these parasites, indicating thatBabesia species are not characterized by a life cycle, which is specific for this genus.

Experimental and comparative morphology of radula renewal in pulmonates (Mollusca, Gastropoda)

SummaryThe continuous renewal of the pulmonate radula and the histology and regeneration of its concomitant epithelia were studied by light and electron microscopy, autoradiography and electron microprobe analysis. The two species investigated show histological differences and the results were compared with those of a preceding study on a prosobranch radula. The radula is an intricate cuticular structure of the foregut. Only the fully grown part, which is active during feeding, lies in the buccal cavity while it is constantly renewed by the coordinated cooperation of specialized cells forming the radular sheath. The end of the sheath is occupied by cells which produce the organic matrix of the radula. In taeniogloss prosobranchs, seven multicellular cushions of small odontoblasts lie at the end of the sheath and produce the seven teeth of each cross-row. In pulmonates, the multidenticular radula is generated by numerous groups of a few voluminuous cells. Despite these histological differences, prosobranchs and pulmonates generate the radula matrix by microvilli, cytoplasmatic protrusions and apocrine secretions. The epithelia of the radular sheath contribute to the transport, tanning and mineralization of the radula. The concomitant epithelia are replaced in limited proliferation zones at the end of the radular sheath and their cells migrate anteriorly to the buccal cavity. The ultrastructure of the sheath cells and the alterations which they undergo in connection with their functions are discussed. The proliferation zone of the superior epithelium is strictly confined and the cells move together with the radula forward. In prosobranchs, the cells of the superior epithelium begin to degenerate in the middle of the radular sheath and the entire epithelium is simply extruded into the buccal cavity. In pulmonates, the opening of the radular sheath is closed by the cuticular collostylar hood which is generated by a distinct epithelium which is proved to be stationary. When leaving the proliferation zone, the superior epithelium differentiates into supporting cells and mineralizing cells; the latter cause the hardening of the radular teeth and already degenerate in the middle of the sheath. The whole superior epithelium degenerates at the border to the collostylar hood-epithelium. In Lymnaea the degeneration zone is strictly confined whereas in Cepaea the collostylar hood and its generating epithelium extend into the radular sheath and the degeneration zone ranges over a distance of 3–5 rows of teeth. The proliferation zone of the inferior epithelium extends over the posterior half of the radular sheath, but the replacement rate is much lower than in the superior epithelium. Although the inferior epithelium carries the radula, it migrates slower than the radula. Obviously the radula has to be transported actively by apical protrusions of the cells, which penetrate into the radular membrane. At the opening of the radular sheath the inferior epithelium generates the adhesive layer and degenerates. During feeding, the adhesive layer has to maintain the firm mechanical connection between radula and distal radular epithelium. Autoradiographic experiments demonstrate that the distal radular epithelium is stationary. Nevertheless, the radula is known to advance to its degeneration zone. Special attention is paid to this problem. We strongly suspect that the transport of the adhesive layer and the radula is based on pseudopodial movements of apical protrusions characteristic for the distal radular epithelium. These protrusions interdigitate with the lower face of the adhesive layer. The mechanical connection has to be maintained and so the respective structures (tonofilaments and hemi-desmosomes) have to be continually renewed. This needs a high amount of energy and obviously results in the conspicuous concentration of mitochondria near the apical surface.

Autoradiographic and morphological studies on the uptake of the triglyceride [3H]-glyceroltrioleate by acanthocephalans

Two eoacanthocephalans, four palaeacanthocephalans, including the infective larvae of one species, and one archiacanthocephalan were exposed in vitro to a labelled triglyceride. The nutrient was almost exclusively incorporated by the tegument of the presoma. The uptake of [3H]-glyceroltriolete started at the anterior half of the presoma, followed by a consecutive labelling of the entire presoma tegument and the lemnisci. In the archiacanthocephalan, however, the general uptake was comparatively low. In the final host of the acanthocephalans, the absorbed nutrient must derive from the hosts intestinal wall, injured by the parasite. In the eoacanthocephalans and the palaeacanthocephalans, the labelled nutrient was most intensively taken up by the tegument of the hooks. Inside the hook tegument, the basal membrane and the outer membrane form a labyrinth of entangling crypts and protuberances. In the surrounding proboscis tegument as well as in the neck tegument, the lipid seemed to be transported along these two membranes. In addition to its absorption by the presoma tegument, the labelled nutrient was intensively incorporated by the apical organ and the paired lateral organ of the eoacanthocephalan presoma and by the terminal part of the uterine endothelium of all female eoacanthocephalans and palaeacanthocephalans. The use of the nutrient by the parasites is discussed.

DNA measurements and ploidy determination of different stages in the life cycle of Sarcocystis muris.

The DNA contents of different stages within the life cycle ofSarcocystis muris were measured cytophotometrically using DNA-specific Feulgen staining. Stages of gamogony were obtained by the transfer of isolated cyst merozoites into cat kidney-cell cultures. For calculation of absolute DNA contents, the amounts of DNA in the parasites were compared with those in chicken erythrocytes, which are known. The measurements revealed that all investigated stages ofS. muris contained haploid DNA except the early zygotes, which were diploid. The further development of the zygotes started with a nuclear division, resulting in two daughter nuclei that again revealed haploid DNA values. The results confirm the existence of zygotic meiosis; thus, a haplo-homophasic life cycle is proposed for the Sarcosporidia.

Effects of pyrantel pamoate on adult and preadultToxocara canis worms: An electron microscope and autoradiography study

Adult as well as preadultToxocara canis isolated from the intestine of a beagle were incubated fro 2, 4, and 14 h in medium containing either different concentrations of pyrantel pamoate (23.6, 236, and 2360 μg/ml medium) or tritiated pyrantel pamoate (2.36 μg/ml medium). These incubations were performed to study the effects of pyrantel pamoate on the morphology of the parasitic nematodes and to obtain information concerning the mode of uptake, the distribution, and the total amount of pyrantel pamoate ingested byT. canis. The results of the ultrastructure studies indicate that the intestine, hypodermis, and muscle cells are the organs that are predominantly affected by the drug. Additionally, it turned out that the duration of the treatment, i.e., the incubation time, was more important in determining the efficacy of pyrantel pamoate againstT. canis than was the concentration itself. Autoradiography studies revealed that the adult worms ingest the drug orally, whereas preadults absorb pyrantel pamoate mainly through the whole body surface. Finally, measurements of the total amount of pyrantel pamoate taken up byT. canis indicated that adult worms can limit or even reduce the ingestion of pyrantel for more than 4 h, but then ingest large amounts of the drug. Preadult worms, however, absorb the drug more or less continuously during the first 14 h through the cuticula, albeit in lower concentrations than the adults. The different experiments elucidate differences in the uptake of pyrantel pamoate as well as in the total amount of drug ingested or absorbed by adult or preadult worms, leading to the assumption that repeated treatment with lower concentrations will be more effective than high concentrations given only once.

Genetic differentiation of pathogenic and nonpathogenic strains of Entamoeba histolytica by random amplified polymorphic DNA polymerase chain reaction.

The random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) method was used to compare pathogenic and nonpathogenic strains ofEntamoeba histolytica. DNA polymorphisms were detected among the different strains and dendrograms were constructed by PHYLIP and PAUP analyses to study the relationship of the strains. Both analyses resulted in identical results, which indicated that pathogenic strains ofE. histolytica are closely related and clearly separated from the nonpathogenic strains. The results of this study agree with classification of the strains based on isoenzyme analyses. This suggests that RAPD-PCR is a valuable method in differentiating between strains of this parasite, and the results are consistent with the concept that pathogenic and nonpathogenicEntamoeba represent two different species.