Morphological convergence in distantly related myxozoans

Myxozoa sind wenige Mikrometer große Parasiten, welche zu den Cnidaria gehören. Zurzeit sind ungefähr 2200 Arten innerhalb der Myxozoa beschrieben. Sie leben meistens als Endoparasiten innerhalb aquatischer Habitate und besitzen einen komplizierten Wirtswechsel zwischen Evertebraten und Wirbeltieren (am häufigsten Fischen). In den letzten Jahren zeichnete sich immer deutlicher ab, dass es einen Zusammenhang zwischen Form, Oberflächenstruktur (Rillen, Höhlen…) und Anhängen (Schwänze, Flügel, Lappen, Haare…) der Sporstadien und dem ökologischen Habitat, dem Wirtsgewebe und andern Faktoren geben muss. Somit müsste es auch möglich sein, Sporen anhand der beeinflussenden Faktoren als Ökotypen zu beschreiben. Diese Studie zeigt, dass das Infektionshabitat eine bedeutende Rolle in der Sporentwicklung einnimmt. Sporstadien aus dem Süßwasser zeigen immer eine Form von Oberflächenstrukturen (meistens Rillen), da sie ihre Oberfläche und so auch ihr Volumen vergrößern, um die im Wasser vorhandenen Strömungs- und Auftriebskräfte zu nutzen. Marine Arten sind in den meisten Fällen glatt, da im Salzwasser der Auftrieb aufgrund der höheren Dichte des Wassers größer ist. Ausnahmen lassen sich häufig durch die Verwandtschaftsbeziehungen erklären, da einige Arten ihr Habitat während ihrer Evolution wechselten. Weitere Unterschiede bei der Oberflächenstruktur und den Anhängen konnten nur in langsam fließenden Gewässern festgestellt werden, obwohl die Fließgeschwindigkeit am ehesten die Form der Sporen verändert. Die Form verändert sich jedoch wesentlicher in Abhängigkeit vom Organsystem innerhalb des Fisches, während die Oberflächenstruktur geringere Veränderungen aufweist. Zusammenfassend, konnte diese Studie jedoch bestätigen, dass sowohl Organsysteme als auch Faktoren des externen Habitats für die meisten morphologischen Veränderungen verantwortlich sind. Somit konnte bestätigt werden, dass die Sporen von Myxozoen tatsächlich Ökotypen darstellen.Myxozoans are miniaturized endoparasites belonging to the Cnidaria, with roughly 2200 species currently described. They are characterized by a two-host live cycle, including an invertebrate and a vertebrate host (mainly fish). Spores are formed as durable transmission stages between hosts and they represent pluricellular stages in a wide variety of shapes, sometimes with ornamentation and appendages. Myxozoan taxonomy has been based predominantly on these morphological features, however the incongruence of spore morphology and phylogenetic clustering of myxozoans is obvious. Different spore morphotypes, i.e. genera, can be extremely closely related while other morphotypes appear to have emerged more than once during the myxozoan evolution. The aim of the present study was to determine whether a relationship exists between the spore features and their habitats, both within the host and the environment. Spores of 10 species were collected, their SSU rDNA was sequenced and their surface structure characteristics studied by SEM. Furthermore, they were included in a large database of 258 taxa to perform statistical analyses in R. The most prominent correlation was found between spore characteristics and external habitat. Spores in freshwater habitats are usually characterized by some form of surface structure enlarging their surface area and increasing their buoyancy, whereas marine spores are predominantly smooth. Most exceptions can be explained by the habitat of the ancestor (as determined by SSU rDNA phylogeny). Despite significant differences between projections and ornamentation in slow flowing water, the water current is more likely to influence the shape of the spore than its ornamentation. Characteristic spore shapes exist also for each host organ system, while ornamentation is more variable. In summary, the present study shows that intrapiscine and external environment account for most of the variation in morphological spore features, demonstrating that spores are in fact ecotypes

Characterization of Cystoisospora suis sexual stages

Dissertation - University of Veterinary Medicine Vienna - 2022Cystoisospora suis, an apicomplexan enteral parasite of pigs, causes severe economic losses in the livestock industry worldwide. It is characterized by a complex life cycle, during which asexual multiplication, with sporogony and merogony, is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Previous life cycle studies give an overview of the development in vivo; however, the detailed development of the sexual stages, i.e. fusion of macro- and microgametes and proteins involved in this is not yet known. However, this crucial step in the life cycle of C. suis may constitute a promising intervention target to interrupt parasite development and to prevent further formation of transmissible stages. This thesis aimed to provide a detailed morphological characterisation of all stages of the sexual development of C. suis, including fertilization, and to identify and characterise stage-specific proteins linked to sexual development by various cell cultivation, molecular and imaging techniques. First and foremost, we could demonstrate that the detailed life cycle of C. suis can be produced in vitro and all life stages can be found at distinct timeslots. Early sexual stages of C. suis are first observed on day 8 of cultivation (doc) and look like sunny-side-up eggs, are immobile and have a size of 11.6×15.6 μm. Late sexual stages comprise of two types of gamonts, micro- and macrogamonts, which are similar in size and morphology. Mature micro- and macrogametes are mainly found at 9-11 doc and are often observed in close proximity to each other to support fusion. Mature microgametes consist of an uninucleate body (3-5 μm), with two flagella of 10.8-12.3 μm length on opposite sides, which makes them actively motile. Macrogametes are immobile, spherical with a smooth surface and have a diameter of 11.5-13.0 μm. After fusion a zygote is formed which is the stage preceding the infectious, transmissible sporozoites. From the proteins of the macrogamete an oocyst wall forms, and the environmentally resistant oocysts can be found in vitro between 10-13 doc. The present work also identified genes linked to the developmental cycle of C. suis by RNASeq analysis and showed the expression changes of selected genes by quantitative real time PCR during the course of development. Genes related to the sexual stages (micro- or macrogametes) showed an upregulation from 6 doc onwards with a peak on the days when gamonts were also present in vitro (10-13 doc). For microgamonts and macrogametes an increased expression of DLC1 and HAP2, proteins related to fertilization, was shown. The occurrence of macrogamonts and macrogametes was correlated with elevated OWP1 and TyRP expression, proteins necessary for oocyst wall formation, both of which increased steadily during cultivation. Moreover, a new host cell free culture system was developed during this project. For this, merozoites collected from in vitro IPEC monolayer cultures infected with C. suis were transferred to a host cell-free environment on the 6th doc developed further into sexual stages and thus continued their life cycle in the same time slots as in the cell culture system. Also, the gene expression levels of sexual stage specific genes were comparable in the host cell-free culture system to the already established cell in vitro system. This novel system provides a new tool for detailed research on the development of C. suis and possibly other Coccidia and will also be useful for evaluation of novel drug or vaccine targets in these parasites. Furthermore, the gene transcribing for the TyRP in C. suis could be identified, and the native protein was bound by a recombinant anti-TyRP-antibody in host cell-free culture. Inhibition of TyRP stunned the formation of the oocyst wall, rendering the parasite non-infectious. Hence, during the course of this project, a possible candidate for the interruption of the C. suis life cycle could be described. Overall, this work sheds light on the sexual development of an important intestinal parasite of pigs, and paves the ways to more applied research for the development of tools and methods to effectively interrupt the parasite’s development as the basis of novel control options.Dissertation - Veterinärmedizinische Universität Wien - 202

Sexual Development in Non-Human Parasitic Apicomplexa: Just Biology or Targets for Control?

The phylum Apicomplexa is a major group of protozoan parasites including gregarines, coccidia, haemogregarines, haemosporidia and piroplasms, with more than 6000 named species. Three of these subgroups, the coccidia, hemosporidia, and piroplasms, contain parasites that cause important diseases of humans and animals worldwide. All of them have complex life cycles involving a switch between asexual and sexual reproduction, which is key to their development. Fertilization (i.e., fusion of female and male cells) results in the formation of a zygote that undergoes meiosis, forming a new generation of asexual stages. In eukaryotes, sexual reproduction is the predominant mode of recombination and segregation of DNA. Sex is well documented in many protist groups, and together with meiosis, is frequently linked with transmission to new hosts. Apicomplexan sexual stages constitute a bottleneck in the life cycle of these parasites, as they are obligatory for the development of new transmissible stages. Consequently, the sexual stages represent attractive targets for vaccination. Detailed understanding of apicomplexan sexual biology will pave the way for the design and implementation of effective transmission-blocking strategies for parasite control. This article reviews the current knowledge on the sexual development of Apicomplexa and the progress in transmission-blocking vaccines for their control, their advantages and limitations and outstanding questions for the future