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Background  Little is known about how bacterial endosymbionts colonize host tissues. Because many insect endosymbionts are maternally transmitted, egg colonization is critical for endosymbiont success.  Wolbachia  bacteria, carried by approximately half of all insect species, provide an excellent model for characterizing endosymbiont infection dynamics. To date, technical limitations have precluded stepwise analysis of germline colonization by  Wolbachia.  It is not clear to what extent titer-altering effects are primarily mediated by growth rates of  Wolbachia  within cell lineages or migration of  Wolbachia  between cells.    Results  The objective of this work is to inform mechanisms of germline colonization through use of optimized methodology. The approaches are framed in terms of nutritional impacts on  Wolbachia . Yeast-rich diets in particular have been shown to suppress  Wolbachia  titer in the  Drosophila melanogaster  germline. To determine the extent of  Wolbachia  sensitivity to diet, we optimized 3-dimensional, multi-stage quantification of  Wolbachia  titer in maternal germline cells. Technical and statistical validation confirmed the identity of  Wolbachia  in vivo ,  the reproducibility of  Wolbachia  quantification and the statistical power to detect these effects. The data from adult feeding experiments demonstrated that germline  Wolbachia  titer is distinctly sensitive to yeast-rich host diets in late oogenesis. To investigate the physiological basis for these nutritional impacts, we optimized methodology for absolute  Wolbachia  quantification by real-time qPCR. We found that yeast-rich diets exerted no significant effect on bodywide  Wolbachia  titer, although ovarian titers were significantly reduced. This suggests that host diets affects  Wolbachia  distribution between the soma and late stage germline cells. Notably, relative qPCR methods distorted apparent  wsp  abundance, due to altered host DNA copy number in yeast-rich conditions. This highlights the importance of absolute quantification data for testing mechanistic hypotheses.    Conclusions  We demonstrate that absolute quantification of  Wolbachia,  using well-controlled cytological and qPCR-based methods, creates new opportunities to determine how bacterial abundance within the germline relates to bacterial distribution within the body. This methodology can be applied to further test germline infection dynamics in response to chemical treatments, genetic conditions, new host/endosymbiont combinations, or potentially adapted to analyze other cell and tissue types.    Electronic supplementary material  The online version of this article (10.1186/s12866-019-1579-3) contains supplementary material, which is available to authorized users.

Quantitative methods for assessing local and bodywide contributions to Wolbachia titer in maternal germline cells of Drosophila