The amoebae (and many other protists) have traditionally been considered as asexual organisms, but suspicion has been growing that these organisms are cryptically sexual or are at least related to sexual lineages. This contention is mainly based on genome studies in which the presence of ‘meiotic genes’ has been discovered. Using RNA-seq (next-generation shotgun sequencing, identifying and quantifying the RNA species in a sample), we have found that the entire repertoire of meiotic genes is expressed in exponentially growing Acanthamoeba and we argue that these so-called meiotic genes are involved in the related process of homologous recombination in this amoeba. We contend that they are only involved in meiosis in other organisms that indulge in sexual reproduction and that homologous recombination is important in asexual protists as a guard against the accumulation of mutations. We also suggest that asexual reproduction is the ancestral state.

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause foetal damage and abortion in both animals and humans. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localisation and analyses of the Toxoplasma ceramide synthases Tg CerS1 and Tg CerS2 and, using a conditional gene regulation approach, establish their roles in pathogenicity and parasite fitness. Interestingly, we observed that whilst Tg CerS1 was a fully functional orthologue of the yeast Lag1p capable of catalysing the conversion of sphinganine to ceramide, in contrast Tg CerS2 was catalytically inactive. Furthermore, genomic deletion of Tg CerS1 using CRISPR/Cas-9 led to viable but slow growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of Tg CerS2 was only accessible utilising the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this ‘pseudo’ ceramide synthase, Tg CerS2, has an even greater role in parasite fitness than its catalytically active orthologue (Tg CerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa arose through gene duplication. However, in the Apicomplexa the duplicated copy subsequently evolved into a non-functional ‘pseudo’ ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites, a feature that could potentially be exploited in the development of new antiprotozoals.

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii, instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 μm in diameter, are the smallest so far described in genus Vannella. Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others.

Toxoplasma gondii is a widespread single-celled intracellular eukaryotic apicomplexan protozoan parasite primarily associated with mammalian foetal impairment and miscarriage, including in humans. Is estimated that approximately one third of the human population worldwide is infected by this parasite. Here we used cutting-edge, label-free 3D quantitative optical diffraction holotomography to capture and evaluate the Toxoplasma lytic cycle (invasion, proliferation and egress) in real-time based on the refractive index distribution. In addition, we used this technology to analyse an engineered CRISPR-Cas9 Toxoplasma mutant to reveal differences in cellular physical properties when compared to the parental line. Collectively, these data support the use of holotomography as a powerful tool for the study of protozoan parasites and their interactions with their host cells.

Acanthamoeba is a widely distributed opportunistic parasite which causes a vision-threatening keratitis and a life-threatening encephalitis. The cyst stage of this amoeba is especially resistant to currently used therapeutics and so alternative agents are urgently required. Growing evidence supports the existence of a programmed cell death system (PCD) in Acanthamoeba and while some features are shared by higher eukaryote cells, others differ. It is hoped that by understanding these differences we can exploit them as targets for novel drug intervention to activate PCD pathways in the amoebae but not the invaded human tissue. Here, we use the aminoglycoside G418 to activate PCD in Acanthamoeba. This drug caused a shape change in the treated amoebae. Cells rounded up and contracted, and after 6 h fragments of cells resembling the ‘apoptotic bodies’ of vertebrate cells were observed. G418 causes an increase in intracellular calcium from a resting level of 24 nM to 60 nM after 6 h of treatment. Mitochondrial function as assayed by the ΔΨm reporting dye JC-1 and CTC a redox dye becomes inhibited during treatment and we have found that cytochrome c is released from the mitochondria. Cells stained with Hoechst showed first an alteration in chromatin structure and then a vesiculation of the nucleus with G418 treatment, although we found no obvious breakdown in genomic DNA in the early stages of PCD.Electronic supplementary materialThe online version of this article (10.1007/s00436-018-6192-0) contains supplementary material, which is available to authorized users.