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16s rRNA sequencing of Wolbachia-infected and uninfected Drosophila male gut


Dou, Weihao (2023), 16s rRNA sequencing of Wolbachia-infected and uninfected Drosophila male gut , Dryad, Dataset,


Wolbachia are the most widely distributed intracellular bacteria, and their most common effect on host phenotype is cytoplasmic incompatibility (CI). A variety of models have been proposed to decipher the molecular mechanism of CI, in which the HM (host modification) model predicts that the effectors of Wolbachia play an important role in sperm modification. However, due to the complexity of spermatogenesis and cell-type heterogeneity in the testis, we still do not know whether Wolbachia have different effects on cells at different stages of spermatogenesis, nor whether these effects are linked with CI. Therefore, we used single-cell RNA sequencing to analyze gene expression profiles in the adult male Drosophila testes with or without Wolbachia infection. We found that Wolbachia significantly affected the proportion of different types of germ cells and affected multiple metabolic pathways in germ cells. Most importantly, Wolbachia had the greatest impact on germline stem cells (GSCs), resulting in the dysregulation expression of genes related to nucleosome assembly and CI, Wolbachia infection also influenced the histone-to-protamine transition in the late stage of sperm development. These results suggest that future studies of Wolbachia-mediated sperm modification should focus more on cells in the early stages of spermatogenesis.


One-day-old Wolbachia-infected (named as Wmale samples) and -uninfected male flies (named as Tmale samples) were used for intestinal genome extraction and 16S rRNA gene amplicon sequencing (see Supplement Text for details). For Wolbachia-infected and uninfected sample collection, flies are anesthetized on ice, washed with 75% ethanol and then rewashed two times with sterile water. The whole gut was dissected in sterile PBS, 10 individuals were collected as a sample and ten biological replicates were obtained for each line. Genomic DNA was extracted using Qiagen DNeasy blood and tissue kit (Qiagen, Germany). The V4 hypervariable regions of 16S rRNA gene were amplified by specific primers (forward: 5-GTGCCAGCMGCCGCGGTAA-3, reverse: 5-GGACTACHVGGGTWTCTAAT-3). All purified DNA products were prepared as a paired-end (2 x 250 bp) DNA library and sequenced on the Illumina HiSeq 2500 platform (Novogene, Tianjin, china). On average, 100,000 sequences were obtained from each sample.

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National Natural Science Foundation of China, Award: 31830084

National Natural Science Foundation of China, Award: 31970440

National Natural Science Foundation of China, Award: 32170497