Our knowledge of ontogenetic development and reproductive biology in lichen-forming fungi is rather poor. Here, we aim to advance our understanding of the reproductive biology of Parmelina carporrhizans and P. quercina for which mycobiont fungi of both species were cultured in aposymbiotic conditions from ascospores. For P. carporrhizans 48 hours were necessary for 98·6% of apothecia to eject spores, while for P. quercina 100% of apothecia ejected spores in the first 24 hours. In P. quercina, large apothecia ejected more spores than smaller ones. In both species the percentage of spores germinating seemed independent of apothecium size. The percentage germination was higher in P. carporrhizans (72·4%) than in P. quercina (14·3%). Moreover, P. carporrhizans was grown more successfully on culture media than P. quercina. These results suggest that these species have different reproductive strategies, given that P. carporrhizans expels larger spores and in greater numbers than P. quercina as well as having different nutritional requirements (since P. carporrhizans grew successfully in the selected media but P. quercina did not). These characteristics may explain the sympatric speciation of these species.

Leishmania virulence and disease development critically depends on the ability of Leishmania promastigotes to infect, differentiate into amastigote forms and replicate inside mammalian host macrophages. Understanding changes associated with amastigote differentiation in axenic culture conditions is a key to identifying virulence factors. Here we compared efficiency of the conventional pH–temperature-dependent shift method to induce amastigote differentiation with the recently identified trigger for differentiation mediated by mitochondrial reactive oxygen species (ROS). Using two different visceral leishmaniasis species, L. infantum and. L. donovani, we show that ROS-generating methods such as iron deprivation or exposure to sub-lethal concentrations of hydrogen peroxide (H2O2) or menadione are significantly more effective in promoting promastigote–amastigote differentiation than the low pH–high temperature shift, leading to higher survival rates, morphological changes and gene expression patterns characteristic of the amastigote stage. Notably, both H2O2 and menadione-mediated differentiation did not require upregulation of the mitochondrial electron transport chain-associated protein p27, suggesting that treatment with oxidants bypasses the necessity to upregulate mitochondrial activity, a precondition for mROS generation. Our findings confirm that ROS-induced differentiation occurs in multiple Leishmania species, including the medically important visceralizing species, and provide mechanistic rationale for earlier reports demonstrating markedly increased virulence of L. infantum promastigotes pre-treated with oxidative reagents.

Giardia infections were detected in 14 out of 32 weaned, 1 to 2-month-old pigs, used in experiments with coccidia. Giardia trophozoites were present in the small intestine from the duodenum to the ileum with maximum numbers in the cranial part of the upper jejunum. They were localized mainly on the surface of intestinal crypts. No histological changes accompanied the infection and the infected animals were asymptomatic. Giardia trophozoites obtained from the intestines of necropsied pigs were isolated in axenic culture in a bile-supplemented TYI-S-33 medium. Morphological features of the trophozoites revealed by both light and scanning electron microscopy proved that the organism belongs to the Giardia intestinalis group.

A complete developmental sequence of Leishmania mexicana has been produced in axenic culture for the first time. This was achieved by manipulation of media, pH and temperature conditions over a period of 16 days. All experiments were initiated with lesion amastigotes that were transformed to multiplicative promastigotes by culture in HOMEM, 10% foetal calf serum, pH 7·5, at 25 °C. Metacyclogenesis was induced by subpassage in Schneider's Drosophila medium, 20% foetal calf serum, pH 5·5, and the resulting forms transformed to axenically growing amastigotes by subpassage in the same medium and raising the temperature to 32 °C. Parasites from each day were characterized with respect to their general morphology using light microscopy of Giemsa-stained smears, and biochemically by analysis of total protein content, proteinases, nucleases and secretory acid phosphatase. The results demonstrated that the three main stages identified - amastigotes, multiplicative promastigotes and metacyclic promastigotes - each exhibited the expected suite of biochemical properties. Further, the changes in morphology observed as the developmental sequence proceeded from stage to stage were accompanied by appropriate changes in biochemical properties. These results provide both useful biochemical markers and a culture system in which to examine the regulation of differentiation and transformation during the Leishmania life-cycle.

A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigotelike forms in Schneider's Drosophila medium supplemented with 2% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32–33 °C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5·4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.

The study aimed to optimize culture conditions and nutrient requirements for theproduction of secondary metabolites by the cultured mycobiont Lobaria spathulata. This species proved to be an excellent model system for such studies, as the complete chemosyndromefound in the natural lichen thallus was repeatedly formed in the cultured mycobiont with differentiated, aerial mycelia. Nutrient media containing the disaccharide, sucrose, were found to favour both rapid growth and the production of typical lichen substances. Higher proportions of the secondary compounds were detected in the developing mycobiont than in mature lichen thalli.

The excretion of extracellular polymeric substances (EPS) by an axenic culture of the benthic diatom Cylindrotheca closterium was investigated. Two sequential extraction steps proved to be sufficient to remove the bulk of the EPS present. Soluble EPS was recovered by a simple centrifugation step and represented a fraction that was not or was only loosely associated with diatom cells. For the extraction of bound EPS, different procedures were compared. The best results were obtained using distilled water as extraction solvent (1 h, 30 °C). The sugars that were recovered using this procedure were typically associated with aggregates of diatoms. In addition to the distinct differences in localization of the different types of EPS, their temporal dynamics differed in relation to the light–dark cycle. Soluble EPS were continuously released into the medium at a rate of 1·6 pg cell−1 day−1. In contrast, the production of bound EPS was highly light-dependent. In the dark, this bound EPS rapidly disappeared, probably as the result of its utilization by the diatoms.