Wolbachia is an intracellular endosymbiont present in most arthropod and filarial nematode species. Transmission between hosts is primarily vertical, taking place exclusively through the female germ line, although horizontal transmission has also been documented. The results of several studies indicate that Wolbachia spp. can undergo transfer between somatic and germ line cells during nematode development and in adult flies. However, the mechanisms underlying horizontal cellto-cell transfer remain largely unexplored. Here, we establish a tractable system for probing horizontal transfer of Wolbachia cells between Drosophila melanogaster cells in culture using fluorescence in situ hybridization (FISH). First, we show that horizontal transfer is independent of cell-to-cell contact and can efficiently take place through the culture medium within hours. Further, we demonstrate that efficient transfer utilizes host cell phagocytic and clathrin/dynamin-dependent endocytic machinery. Lastly, we provide evidence that this process is conserved between species, showing that horizontal transfer from mosquito to Drosophila cells takes place in a similar fashion. Altogether, our results indicate that Wolbachia utilizes host internalization machinery during infection, and this mechanism is conserved across insect species. IMPORTANCE Our work has broad implications for the control and treatment of tropical diseases. Wolbachia can confer resistance against a variety of human pathogens in mosquito vectors. Elucidating the mechanisms of horizontal transfer will be useful for efforts to more efficiently infect nonnatural insect hosts with Wolbachia as a biological control agent. Further, as Wolbachia is essential for the survival of filarial nematodes, understanding horizontal transfer might provide new approaches to treating human infections by targeting Wolbachia. Finally, this work provides a key first step toward the genetic manipulation of Wolbachia.KEYWORDS Drosophila, Wolbachia, endocytosis, entry, horizontal, invasion, phagocytosis, transfer, transmission W olbachia spp. are intracellular bacteria that are transmitted through the female germ lines of arthropods and filarial nematodes (1, 2). In arthropods, Wolbachia spp. function as either a mutualist or a parasite, while in filarial nematodes, Wolbachia spp. are essential for host survival. Efficient maternal transmission of Wolbachia cells in Drosophila melanogaster requires their localization to the posterior cortex of the developing embryo, as this is the future site of the germ line (3). In filarial nematodes, Wolbachia cells undergo a precise pattern of migration during host development that involves not only asymmetric mitotic segregation but also the invasion of germ line precursors from somatic cells (4). Thus, the ability of Wolbachia spp. to undergo cell-to-cell transfer plays an important role in maintaining vertical transmission (5).
