SummaryThe maternally inherited bacterium Wolbachia infects the germline of most arthropod species. Using Drosophila simulans and D. melanogaster, we demonstrate that localization of Wolbachia to the fat bodies and adult brain is likely also a conserved feature of Wolbachia infection. Examination of three Wolbachia strains (WMel, WRiv, WPop) revealed that the bacteria preferentially concentrate in the central brain with low titres in the optic lobes. Distribution within regions of the central brain is largely determined by the Wolbachia strain, while the titre is influenced by both, the host species and the bacteria strain. In neurons of the central brain and ventral nerve cord, Wolbachia preferentially localizes to the neuronal cell bodies but not to axons. All examined Wolbachia strains are present intracellularly or in extracellular clusters, with the pathogenic W Pop strain exhibiting the largest and most abundant clusters. We also discovered that 16 of 40 lines from the Drosophila Genetic Reference Panel are Wolbachia infected. Direct comparison of Wolbachia infected and cured lines from this panel reveals that differences in physiological traits (chill coma recovery, starvation, longevity) are partially due to host line influences. In addition, a tetracycline-induced increase in Drosophila longevity was detected many generations after treatment.

Wolbachia is a globally distributed bacterial endosymbiont present in arthropods and nematodes. The advent of sensitive PCR-based approaches has greatly facilitated the identification of Wolbachia-infected individuals and analysis of population infection levels. Here, a complementary visual fluorescence-based Wolbachia screening approach is described. Through the use of the fluorescent dye Syto-11, Wolbachia can be efficiently detected in various Drosophila tissues, including ovaries. Syto-11 also stains Wolbachia in other insects. Because Wolbachia is inherited through the maternal germ line, bacteria reside in the ovaries of flies in infected populations. An advantage of this staining approach is that it informs about Wolbachia titer as well as its tissue and cellular distribution. Using this method, the infection status of insect populations in two central California locations was determined, and variants with unusually low or high Wolbachia titers were isolated. In addition, a variant with ovarioles containing both infected and uninfected egg chambers was identified. Syto-11 staining of Cardinium-and Spiroplasma-infected insects was also analyzed.Wolbachia species are obligate intracellular, bacterial endosymbionts present in over 60% of all insect species (10). Manipulation of host reproduction and efficient maternal transmission have facilitated the global spread of Wolbachia in arthropods. Under optimal laboratory conditions, Wolbachia transmission in Drosophila melanogaster (11) and Drosophila simulans (10a) is 100%, but infection rates in the field can be highly variable with location, season, and host species (10a, 11). In addition, different Wolbachia strains that have various effects on host reproduction and fecundity seem to exist in single collection sites (24). Determining the Wolbachia status quickly and reliably is important for ecological work, as well as for biochemical and genetics studies of host-pathogen interaction. It has been estimated that 30% of the strains in the Bloomington Stock Center are Wolbachia infected (4). Because Wolbachia is present in a number of somatic tissues (6), including the adult brain (1, 17), an infection may influence fly behavior and metabolism (12,14,18), thus having implications for the larger Drosophila research community.The infection status of fly stocks is commonly determined by PCR (2, 11a, 18), which involves a 3-step process to first prepare the DNA, followed by the actual PCR step and gel electrophoreses (19,25). The PCR method is efficient, especially when many samples are analyzed. However, the infection status of some species has been underestimated with conventional PCR and was revealed only after "long PCR" involving 2 polymerase enzymes (13). On the other hand, too-sensitive PCR conditions may produce false positives. Standard PCR methods do not reveal the degree of Wolbachia infection, tissue of origin, and variations in Wolbachia localization or titers in the host. To complement the PCR detection, a quick method to visualize Wolbachia in live i...