The total DNA of each sample was extracted from whole bodies of individual flies using the DNeasy tissue kit (QIAGEN Inc., Valencia, CA) following the manufacturer’s instructions. The DNA extracted was eluted in 200 μl of the elution buffer. Then, 30 μl of the extracted genomic DNA from individual samples were pooled (six females in one pool and two males in another pool), and the DNA concentrations in the pooled DNA were determined using a spectrophotometer (Synergy H1 Multi-Mode Reader, BioTek, Instruments, Inc., United States). The pooled DNAs were diluted to obtain equal final DNA concentrations (4 ng/μl) and 5 μl of the diluted DNA was analyzed by qPCR on a CFX96 real time PCR detection system (Bio-Rad, Hercules, CA) as described previously (Abd-Alla et al., 2009a). The tsetse housekeeping β-tubulin gene was used to normalize the qPCR reactions (Boucias et al., 2013) to give the virus density. The primers and the qPCR conditions are given in Supplementary Table 1.

The aliquots of the DNA extracted at the time-points outlined in section “In vivo Virus Replication in Intra-Hemocoelic Injected Adults” were used to quantify the densities of bacterial symbionts normalized to the housekeeping β-tubulin gene as previously described (Boucias et al., 2013). Sodalis, Wigglesworthia and Wolbachia densities were quantified in both males and females and at different dpi by qPCR using primers that target fliC (Weiss et al., 2012), thiC (Yamada et al., 2007) and Wolbachia 16R rRNA genes, respectively (Demirbas-Uzel et al., 2018a). The primers and the PCR and qPCR conditions are given in Supplementary Table 1.

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