The development of transgene Daphnia in a molecular reconstruction of a host-parasite coevolution.

Antagonistic interactions between hosts and parasites are a key structuring force in natural host populations leading to coevolution. The omnipresence of parasites in combination with their short generation time and their virulence effects, enables parasites to reduce the density and to influence the genetic structure of their host populations. In the arms race with their fast evolving parasites, there will be selection in the host against defence mechanisms that are abundant in the momentary interaction, as parasites adapt to the most abundant host genotypes. This results in dynamic interactions over time. It is, however, notoriously difficult to study coevolutionary dynamics and the under-laying genetic mechanisms in nature, because time series over many generations are needed. Consequently, empirical evidence for the process of host-parasite coevolution in natural systems is lacking. An elegant way to overcome the time aspect problem is by using aquatic organisms that produce dormant stages, such as the crustacean Daphnia. These dormant stages settle down in layered pond sediments producing dormant stage banks and can be reactivated even after many years of settlement and dormancy (resurrection ecology). As such an archive of gene pools and evolutionary changes in the past is provided. We earlier performed a historical reconstruction of a host-parasite coevolution in a natural setting, based on the dormant stage bank of both the crustacean Daphnia host and its endo-bacterial parasite, Pasteuria ramosa. In this study, coevolution was documented as changes in the host and the parasite fitness in cross-infection experiments. Daphnia clones were exposed to parasites of different depths of a sediment core, isolated from a pond (Abdij van t Park, Heverlee) in which Daphnia and Pasteuria co-occurred approximately fifty years. The different sediment depths reflect different time frames of the Daphnia-Pasteuria coevolution. We here want to reproduce a more direct approach to obtain insight into the mechanism of host-parasite coevolution by direct registration of changes in the Daphnia genes involved in the interaction with Pasteuria (Pasteuria target genes). An elegant and effective way to do this would be to exchange allelic variants of these Pasteuria target genes between Daphnia clones of different depths of a pond sediment core (corresponding to different time intervals) and to investigate Pasteuria adaptation to these transgenic Daphnia clones. The water flea Daphnia is an ecologically very important organism, playing a central role in freshwater ecosystems as it is a primary grazer of algae and serves as preferred food for fish. Further, Daphnia is an ideal model organism for evolutionary and eco(toxico)logical functional genomics, because a lot of ecological and evolutionary background information is available, they have a high evolutionary potential due to their relative short generation time, and one can work with clonal lineages. A rapidly increasing amount of genomic data is becoming available, including the full genome sequence of Daphnia pulex, through the concerted effort of the Daphnia Genomics Consortium (see http://daphnia.cgb.indiana.edu). Daphnia has an important advantage compared to classic model organisms, because a lot of information is available on the natural environment and selection factors to which animals are exposed, inclusive host-parasite interactions. Further, being a crustacean, Daphnia is reasonably allied to insects, with important model organisms such as the fruit fly Drosophila and the mosquito Anopheles. The crustacean and insect classes both belong to the same phylum of Arthropoda within the invertebrates and can thus be expected to share a lot of genes. Yet, these two classes of animals have evolved in two different habitats: crustaceans are predominantly adapted to aquatic habitats, whereas insects are predominantly adapted to terrestrial habitats. Daphnia have a specialized reproduction mode, called cyclical parthenogenesis, which involves a switch between sexual and asexual reproduction depending on the environmental conditions. Sexual reproduction is associated with the production of dormant eggs, whereas subitaneously developing eggs are parthenogenetic. Cyclical parthenogenesis involves a sex determination mechanism and is derived from obligate sexual reproduction, which suggests that it has evolved by modifications in the arthropod reproductive genes. Genomic studies with Daphnia as a model organism are in full development. The Daphnia gene annotation project has been set-up to decipher the function of the full list of genes with the advent of the Daphnia genome. At the moment, however, research aiming at creating genetic tools such as transgenic Daphnia is limited and the Daphnia community needs efficient techniques to test gene functions. Innovative and Creative aspects A key aim of this study is the creation of in vivo transgenic Daphnia via stable genetic transfection. Our final aim is to create genetic transformation in vivo in Daphnia, in which the gene expression of different allelic variants of genes that are involved in the Pasteuria interaction will be exchanged. In vitro transfection of Daphnia has been realized via viral vectors in embryonic primary cell cultures, but in vivo genetic transfection and transformation was not performed so far. Moreover, it is difficult to immortalize viable primary Daphnia cells from parthenogenetic Daphnia embryos and to establish continuous Daphnia cell lines. Our new technique would thus confer a revolutionary break-through in this domain (but also in others) with an impact that will transcend the application for which we will develop the technique. Although, there is no guarantee that the exchange of genetic material in vivo in Daphnia will work, RNA-interference and gene transfer techniques have proven their success already in a lot of other organisms, including the crayfish Pacifastacus, a crustacean aquatic invertebrate that is very closely allied to Daphnia. A second creative aspect of this study is the application of transgenic organisms in evolutionary ecological research based on the use of dormant stages. Here, we will use historical populations of Daphnia and her micro-parasites, which provide a unique opportunity to reconstruct molecular coevolution. In Decaestecker et al. (2007, Nature), we were able to unravel the infectivity and virulence aspects of the coevolutionary dynamics, but we did not elucidate the genetic background of the process. By creating and using transgenic organisms, we will be able to characterize which molecular mechanisms and associated immune processes in Daphnia are important against natural bacterial infections and by such structure Daphnia-parasite co-evolution. More specifically, we will create genetic transformation in vivo in Daphnia, in which ten gene expression of different allelic variants of genes that are involved in the Pasteuria interaction will be exchanged. Specific Aims We aim to (i) create stable genetic transfection in vivo in Daphinia via gene transfer and to (ii) unravel the genes, involved in the Daphnia-Pasteuria interaction ('Pasteuria target genes') based on gene silencing in vivo in Daphnia. Further, we aim to (iii) quantify gene expression and allele frequency changes of the 'Pasteuria target genes' over time via a historical reconstruction based on Daphnia dormant stage banks. Molecular historical reconstructions haven been performed earlier, but so far only based on neutral molecular markers. Finally (iv), we aim a historical molecular reconstruction of a coevolutionary process by using transgenic Daphnia in which we exchange allelic variants of the 'Pasteuria target genes' and test Pasteuria adaptation over time. This would be the first molecular reconstruction of a coevolutionary process that elucidates the genetic background and molecular processes of an organism's defences against one of its antagonists.

Keywords:Daphnia, Host-parasite coevolution, Transgenetics, Reconstruction, Resting stages, Pasteuria, Immunity genes

Disciplines:Microbiology, Animal biology, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering