Summary:

Surprisingly, in many field studies of natural host-parasite systems genetic changes have been investigated for one player only – the host. This is astonishing as coevolution requires changes in the frequencies of both players. The previous restrictions were mainly caused by limited access to molecular markers for unculturable microparasites. Recently, we have established a NGS protocol to shed light on the genetic changes within populations of Caullerya mesnili (Ichthyosporea), the parasite of the waterflea Daphnia. This parasite is one of the most common pathogens of European Daphnia species that inhabit large, permanent lakes. We show that the genetic structure of parasite populations varies both over space and, most interestingly, over time. Given its high virulence (up to 95% fecundity reduction), strong genetic specificity for infection, large prevalence in natural populations (up to 40%), and currently established molecular tools - Caullerya is a strong candidate to become a model parasite for future coevolutionary studies of natural host-parasite systems. Our preliminary studies show that the genetic structure of parasite populations varies both over space and, most interestingly, over time. In the near future, the new molecular and bioinformatical techniques will allow to track changes in genetic population structure on a large scale for the often unstudied member of a coevolutionary pair – the parasite.

Summary:

The role of hybridization in evolution has been underestimated for a long time. For example, hybrids were often considered less fit then their parental species, due to genetic incompatibility. However, hybridization occurs frequently in plants and in animals. Our recently collected field data showed that a Daphnia community of a small quarry lake in Munich, normally consisting of parental species and hybrids belonging to the D. longispina complex, has become dominated by a single hybrid clone - the “super clone”. By creating artificial communities consisting of the “super clone” and other clones of the D. longispina complex, we proved the competitive strength of the “super clone”. After ~6 generations the “super clone” had increased from 8% to 100% in some of the artificial communities. Additionally, we studied fitness parameters of these clones, kept under two different temperatures. Here, there was no special performance of the “super clone”. Therefore we are currently comparing the carrying capacity of “super clone” with the other clones, by measuring the change in numbers of Daphnia over time, in a certain volume of water. Additionally, we will study fitness parameters of the clones under crowding (and control) conditions. The crowded media will be obtained from crowded stock cultures. We expect the “super clone” to have higher carrying capacity as well as to be less affected by crowded media than the other clones, thereby preventing the other clones from reproducing and establishing within the population. Finding the pattern that makes the “super clone” so special will contribute to a general knowledge about hybrid-specific traits and their contribution to evolutionary processes.

Summary:

In order to track temporal-evolutionary changes in the plakton communities, as an important issue remains the proper identification of species and their hybrids. For the analyses of Daphnia longispina complex, microsatellie genotyping is a commonly used method. However, we found these length-based markers unsuitable when analyzing poor quality samples. Therefore the historical, formaldehyde preserved samples remain inaccessible. To overcome this problem, we propose SNP based genotyping, due to possibility of shorter fragment amplification. Furthermore, this method allows not only high-throughput genotyping, but the calibration among laboratories is also relatively precise. Therefore, we aim to develop a reliable method to identify species of the D. longispina complex and their hybrids by SNP genotyping. By comparing the transcriptome of D. galeata with D. pulex genome (wfleabase.org) we are identifying genes and their chromosomal location in order to obtain multi-loci markers, and corresponding primers are then being designed. After the sequencing and alignment of these genes for each species in the complex (D. cucullata, D. galeata and D. longispina), candidate SNPs are being identified. For the small scale confirmation of the diagnostic value of these candidate SNPs, we are sequencing a set of genetically well-defined clones from species and hybrids originating from diverse locations across Europe. For the large-scale screenings we are optimizing multiplex PCR reaction of short amplicons and SNP detection via SnaPshot Multiplex kit. To validate the results, we are applying the developed assay for the samples, which were previously analyzed with microsatellite markers. By multi-locus SNP genotyping we will be able to assess the population structures in long-term formaldehyde preserved samples of a hybridizing species complex.

Summary:

Caullerya mesnili (Opisthokonta, Ichthyosporea) is an endoparasite infecting Daphnia (Crustacea, Cladocera) gut. This protozoan has high virulence and a strong genetic specificity for the infection, thus it seems to be a good model to study host-parasite coevolution dynamics. However, little is known about population structure of this microparasite. Previous work based on cloned sequences has shown that variation of the ITS region (internal transcribed spacer of ribosomal DNA) can be used to analyse spatial and temporal variation in C. mesnili. However, high-throughput next generation sequencing (NGS) allows much larger scale analyses. In this work we will present a bioinformatic pipeline analysing 392 bp long ITS amplicons from Caullerya obtained from 454 pyrosequencing. With this approach, it is possible to study in detail aspects such as the spatio-temporal distribution of Caullerya in different host populations or clones. We will also demonstrate the comparison of patterns obtained by cloning with the NGS approach.