Evolution of Insect Germline Specification

The germ cells are a unique group cells that can give rise to all cell types and regenerate themselves, thereby passing genetic information from generation to generation in sexually reproducing animals. Germline establishment is a process through which germ cells are specified from zygote. Among animals, there are two major strategies that establish the germline during embryogenesis. The prevalent mode is zygotic induction, which is also the ancestral mode. The germ cells are induced by inductive signals after the zygotic genome has been activated, and usually depends on the interactions of other already specified tissue types. The other mode is considered as the derived mode of zygotic induction, which is maternal provision mode. Unlike zygotic induction mode, the germ cells are the first group of cells specified during embryogenesis by inheritance of the germ plasm, which contains germline determinants synthesized by nurse cell and deposited in the oocyte during oogenesis. Some of the species in Holometabola use maternal provision mode, while none of insect species in Hemimetabola use maternal provision mode instead use zygotic induction, which suggests that the origin of maternal provision mode in Insecta is occurred early in the evolution of the Holometabola. For example, the honeybee Apis, the silkworm Bombyx, the red flour beetle Tribolium, etc. use zygotic induction mode. Interestingly, over the repetitive transitions between the two modes, there was no other mode involved. This suggests that the mechanisms for germline specification are labile over the course of evolution. Therefore, identification of the core regulatory network of the germline specification that must be maintained, identification of the parts that are novel or variable, are important pieces of information to understand the evolution of the germline specification. The process of germline specification is very diverse, even within the same mechanism. Take maternal provision as an example, the detailed process of germline development in the fly Drosophila is different from the wasp Nasonia. The Drosophila germ plasm is in a form of polar granules in the posterior pole of the embryo. They are small and static. However, the Nasonia germ plasm is named the oosome and is a large, tightly integrated, spherical structure that can move around in the posterior region of the embryos. At the beginning of pole cell formation in Drosophila, each of the nuclei associates with several polar granules and buds out of the embryo separately, resulting in several pole cells, whereas the Nasonia embryo makes a single bud and the oosome associated with several nuclei enter in this bud to pinch off the embryo and divided into several pole cells. Since Drosophila is a well-documented example for germline specification by maternal provision mode, identification of the genes that are involved in Nasonia germline development and comparison of them with Drosophila would help us understand what genes are conserved and what genes contribute to their unique features. In chapter 2, we used next-generation sequencing to identify the RNA components of the Nasonia oosome. Besides the conserved genes, we found dozens of novel genes that either do not exist or do not play roles in germline development in Drosophila. We characterized some of the novel genes' functions by embryonic RNA interference and found that they all play roles in Nasonia germline development. Although the preliminary functional studies did not give us details on these genes functions, they showed that the strategies that we used worked well for our purpose. Future deep functional studies are required to understand how those genes play their roles in Nasonia germline development. To gain a fuller understanding of maternal provision mode, we also chose the bean beetle Callosobruchus to dissect the germ plasm assembly during oogenesis. Because Callosobruchus uses maternal provision mode and its oogenesis is telotrophic, which is different from the polytrophic oogenesis in Nasonia and Drosophila. In addition, its close related insect Tribolium uses maternal provision mode. Through our studies on germ plasm assembly, we will gain understanding of what genes are required for germ plasm assembly and what are lineage-specific. So far, we found that oskar, vasa and tudor are need for germ plasm assembly. Since we did not have proper germline markers, we were not able to observe the knockdown effects of these genes on germ plasm assembly. We also found that Callosobruchus bruno is localized at the anterior pole of the oocyte, which is a novelty. The specific localization might suggest an anterior role of bruno in Callosobruchus. As I mentioned above, the germline specification mechanisms are labile over the course of evolution. Since Callosobruchus and Tribolium use different modes of germline specification, we tried to knock in the Callosobruchus oskar gene into the Tribolium genome to test how liable the two mechanisms are. I tried to knock in genes by CRISPR/Cas9, but I was not able to gain transgenic lines. Overall my thesis was aimed at revealing the evolutionary relationships between the two modes of germline specification. We want to find out what the core regulatory network is and what genes contribute to lineage-specific features. Beside the conserved genes, we found dozens of novel genes in Nasonia, which could contribute to the unique process of Nasonia germline specification. Preliminary studies in Callosobruchus already showed unique features of the germ plasm assembly. More results would be expected if we are able to find suitable germline marker.