In this amended proposal Dr. Karr proposes to continue his studies to understand the cellular and molecular basis for cytoplasmic incompatibility (CI) in insects. In CI, crosses of strains of the same species fail to yield many progeny.In such crosses the sperm is able to enter the egg, but it is unable to contribute its genetic material to the developing embryo. The syndrome is associated with a rickettsial endosymbiont (a Wolbachia species). CI is inherited maternally, but acts by affecting the sperm of males carrying the incompatibility factor. Crosses between CI males and uninfected females are unsuccessful, while all other combinations are successful. In preliminary studies Dr. Karr has made a number of valuable findings. He has shown a bidirectional incompatibility system in Drosophila simulans, he has developed a technique for monitoring the endosymbiont via DNA staining in early embryos, he has isolated Wolbachia specific monoclonal antibodies, he has shown an association between Wolbachia and the centrosome of the host in the early embryo, and he has developed a partial purification scheme for the endosymbiont. Perhaps his most important preliminary result is the successful transfer of the endosymbiont from Drosophila simulans to tetracycline-cured D. simulans and to Drosophila melanogaster. Furthermore, via a selection scheme he has achieved a high level of CI in D. melanogaster. The proposed research is aimed to a large extent toward placing many of the assumptions about CI on a more rigorous experimental basis, and on developing tools that will be essential for the long term success of the research program. In the first set of proposed experiments, Dr. Karr will test the hypothesis that the presence of Wolbachia in pole cells is sufficient for vertical transmission of CI via pole cell transplantation experiments. He also proposes to obtain definitive evidence that Wolbachia is the cause of CI by microinjection of purified bacteria, and by showing that infected lines can be produced which display CI. He will also quantify CI and Wolbachia levels during the reestablishment of the syndrome after antibiotic treatment. In further experiments he will examine the intracellular localization of Wolbachia during gametogenesis. In another line of research Dr. Karr will attempt to determine the basis for the bidirectional incompatibility found in D. simulans. In these experiments bacteria from one type of strain will be introduced by transplantation into cured strains of the other crossing type. He will also introduce each type of bacteria into D. melanogaster and then determine if bidirectional CI is displayed by the resulting stocks. In preliminary results, Dr. Karr has identified a protein found in sperm from CI carrying males, but not uninfected males. He will purify enough of this protein for microsequencing. He will then use the sequence information to synthesize oligonucleotides that will allow him to clone the gene that encodes the protein. He will then raise antibodies to the protein and determine its cytological location and behavior during CI.
