Impact of thermal environment on Spiroplasma titre in D. hydei

Results of qPCR analysis of relative titre of Spiroplasma in D. hydei. Brief methods: D. hydei carrying Spiroplasma were reared at 18 C or 25 C. These were allowed to breed, and progeny reared at the focal temperature before being culled within 24h of entry to pupation. DNA from Individual pupae was purified using the phenol-chloroform methods. Real-time qPCR reactions were then carried out for the dnaA gene (Spiroplasma) and the rp49 gene (D. hydei reference), using the LightCycler 480 (Roche). Column A: Replicate (A or B)Column B: Temperature treatment (18 or 25 C)Column C: Biological replicateColumn D: dnaA/rp49 ratio across three technical replicatesColumn E: Median dnaA/rp49 ratio for 18 C block A, used as standardColumn F:dnaA/rp49 ratio standardised against median dnaA/rp49 for 18C block AColumn G: Median ratio for each treatment/blockColumn H: Median fold change in Spiroplasma titre for 25 vs 18 for each block.