Insect Molecular Biology (1998) 7(2), 101±105 Analysis of Wolbachia protein synthesis in Drosophila in vivo T. Sasaki,* H. R. Braig and S. L. O'Neill Section of Vector Biology, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut, USA Abstract Intracellular Wolbachia infections are extremely common in arthropods and exert profound control over the reproductive biology of the host. However, very little is known about the underlying molecular mechanisms which mediate these interactions with the host. We examined protein synthesis by Wolbachia in a Drosophila host in vivo by selective metabolic labelling of prokaryotic proteins and subsequent analysis by 1D and 2D gel electrophoresis. Using this method we could identify the major proteins synthesized by Wol- bachia in ovaries and testes of ¯ies. Of these proteins the most abundant was of low molecular weight and showed size variation between Wolbachia strains which correlated with the reproductive phenotype they generated in ¯ies. Using the gel systems we employed it was not possible to identify any proteins of Wolbachia origin in the mature sperm cells of infected ¯ies. Keywords: Drosophila, Wolbachia, cytoplasmic incompatibility, protein synthesis. Introduction Wolbachia are intracellular inherited bacteria of arthropods, best known for the various reproductive phenotypes they cause in dierent hosts, including cytoplasmic incompatibility, parthenogenesis and the feminization of genetic males (Werren, 1997). Esti- mates based on ®eld surveys indicate that as many as 15±20% of all insect species may be naturally infected with this agent (Werren et al., 1995). Despite this wide- spread distribution and the obvious applied potential for utilizing Wolbachia as a means of manipulating insect populations (Sinkins et al., 1997), virtually nothing is known about the molecular mechanism underlying the interactions between Wolbachia and the host. One major factor contributing to this lack of knowledge is the inherent diculty of working with Wolbachia. Until recently there has been no in vitro system for culturing Wolbachia and the only described culture system to date depends on growing Wolbachia within insect cells (O'Neill et al., 1997). The fastidious nature of Wolbachia, together with the diculty in purifying large quantities of the microorganism from host tissue, has severely impeded progress in the study of the mechanistic basis of the reproductive phenomena caused by Wolbachia. Of the dierent reproductive phenotypes associated with Wolbachia infection, cytoplasmic incompatibility is the most commonly described. This phenotype results in the death of the young insect embryo due to a disruption of the normal kinetics of sperm condensa- tion/decondensation reactions during fertilization (Callaini et al., 1997; Lassy & Karr, 1996). Cytoplasmic incompatibility is most commonly expressed when sperm from an infected male fertilizes the egg of an uninfected female. Wolbachia is not present in mature sperm, being excluded during sperm maturation. As such, Wolbachia must exert its eect during spermatid development and this eect must be carried by the mature spermatozoan in the absence of Wolbachia. Crosses between insects in which both sexes carry the same Wolbachia strain are fully compatible, indicating that the presence of Wolbachia in the young egg can rescue a Wolbachia modi®ed sperm cell to normal function. In addition, crosses between insects carrying dierent strains of Wolbachia may be bidirectionally incompatible, suggesting that the egg rescue process is Wolbachia strain speci®c. To begin to understand the basis for the interaction of Wolbachia and a model host, Drosophila simulans, we examined protein synthesis of Wolbachia in vivo *Present address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113, Japan. Received 10 June 1997; accepted 25 August 1997. Correspondence: Dr S. L. O'Neill, Section of Vector Biology, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, New Haven, CT 06510, USA. e-mail: scott.oneill@yale.edu. # 1998 Blackwell Science Ltd 101