Quantitative methods for assessing local and bodywide contributions to Wolbachia titer in maternal germline cells of Drosophila
Quantitative methods for assessing local and bodywide contributions to Wolbachia titer in maternal germline cells of Drosophila
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DOI：
10.1186/s12866-019-1579-3
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发表时间：
2019-09-03
期刊：
影响因子：
4.2
通讯作者：
Serbus, Laura R.
中科院分区：
文献类型：
--
作者：
Christensen, Steen;Camacho, Moises;Serbus, Laura R.
背景细菌内共生体是如何在宿主组织中定殖的还知之甚少。由于许多昆虫内共生体是母系传播的，卵定殖是内共生体成功的关键。沃尔巴克氏体细菌，携带的所有昆虫物种的大约一半，提供了一个很好的模型，表征内共生感染动力学。到目前为止，技术上的限制已经排除了逐步分析生殖系定植沃尔巴克氏体。目前尚不清楚在何种程度上滴度改变效应主要由细胞谱系内的沃尔巴克氏体的生长速率或细胞之间的沃尔巴克氏体的迁移介导。结果本研究的目的是通过优化的方法学研究生殖系定植的机制。这些方法的框架在营养方面的影响沃尔巴克氏体。特别是富含酵母的饮食已经显示出抑制黑腹果蝇种系中的沃尔巴克氏体滴度。为了确定沃尔巴克氏体对饮食的敏感程度，我们优化了母体生殖细胞中沃尔巴克氏体滴度的三维多阶段定量。技术和统计验证证实了体内沃尔巴克氏体的身份，沃尔巴克氏体定量的重现性和检测这些影响的统计能力。成虫饲养实验数据表明，生殖系沃尔巴克氏体滴度在卵子发生后期对富含酵母的宿主饲料明显敏感。为了研究这些营养影响的生理基础，我们优化了通过实时qPCR进行绝对沃尔巴克氏体定量的方法。我们发现，富含酵母的饮食对全身沃尔巴克氏体效价没有显着影响，尽管卵巢效价显着降低。这表明宿主饮食影响沃尔巴克氏体在索马和晚期生殖细胞之间的分布。值得注意的是，由于在富含酵母的条件下改变了宿主DNA拷贝数，相对qPCR方法扭曲了表观wsp丰度。这突出了绝对定量数据对检验机制假设的重要性。结论我们证明，沃尔巴克氏体的绝对定量，使用良好控制的细胞学和qPCR为基础的方法，创造了新的机会，以确定如何细菌丰度的生殖系内涉及到体内的细菌分布。这种方法可以应用于进一步测试生殖系感染动态响应化学处理，遗传条件，新的主机/内共生体组合，或可能适用于分析其他细胞和组织类型。
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.