(A, B, and C) Fifty 3–6-d-old males, per sample, were injected with DCV, and their survival was followed daily. (A) Flies w¹¹¹⁸ iso, VF-0058–3, and VF-0058–3 raised on tetracycline for one generation were injected with 500 TCID₅₀ DCV. (B) Flies w¹¹¹⁸ iso and w¹¹¹⁸ iso raised on tetracycline were injected with 50 TCID₅₀ DCV. (C) Flies w¹¹¹⁸ iso, VF-0058–3, VF-0058–3t, VF-0097–3, and VF-0097–3t were injected with 500 TCID₅₀ DCV. Each assay was repeated once with males and twice with females with similar results.
(D) Fifty 3–6-d-old males, per sample, of VF-0058–3 and VF-0058–3t lines were injected with 50 mM Tris-HCl, pH 7.5, kept at 18°C and their survival was followed daily.
(E) Extracts of VF-0058–3 and VF-0058–3t flies 3 and 6 d after injection with 500 TCID₅₀ DCV or not injected were probed in a Western blot with anti-DCV. Anti-tubulin was used as a loading control.
(F) Titration, in cell culture, of DCV levels per fly of VF-0058–3 and VF-0058–3t flies 3 and 6 d after injection with 500 TCID₅₀ DCV. Squares are replicates (four per sample), lines are geometric means of replicates. Virus titres in VF-0058–3 and VF-0058–3t are significantly different on both days post-infection (Mann-Whitney test, p = 0.0287 for both comparisons).

(A) Two independent sets of isofemales lines were established from VF-0058–3 flies raised on a sub-optimal dose of tetracycline (lines 1–10 and 11–33). The presence of Wolbachia in these lines was tested by PCR amplification using wsp primers on DNA extracts of adult flies, PCR with mt 12S rRNA primers was done as a control. Three–six-d-old males of each line were injected with 500 TCID₅₀ DCV, collected 6 d later, and DCV levels analysed by Western blot with anti-DCV. Anti-tubulin was used as a loading control.
(B) Six wild-type lines infected with Wolbachia (lines 1–6) and six wild-type lines not infected (lines 7–12) were identified by PCR amplification with wsp primers (more information on wild-type lines identity can be found in Materials and Methods). Each stock was treated with tetracycline for two generations and then transferred to tetracycline-free food for at least two generations. Treated and non-treated stocks were re-tested for presence of Wolbachia by PCR amplification with wsp primers. Seven–twelve PCR amplifications were all negative, not shown. Three–six-d-old males of each stock (lines 1–12, tetracycline treated and non-treated) were injected with 500 TCID₅₀ DCV, collected 6 d later, and DCV levels analysed by Western blot with anti-DCV. Anti-tubulin was used as a loading control.

(A) Extracts of flies 6 d after injection with 500 TCID₅₀ DCV were probed in a Western blot with anti-DCV. Anti-tubulin was used as a loading control. Flies used were: w¹¹¹⁸ iso ; w¹¹¹⁸ iso raised with VF-0058–3; VF-0058–3; and progeny of VF-0058–3 raised with w¹¹¹⁸ iso.
(B and C) Fifty 3–6-d-old males, per sample, were injected with 500 TCID₅₀ DCV, and their survival followed daily. (B) Males and females from resistant (VF-0058–3) and sensitive (VF-0058–3t) stocks were crossed in the four possible combinations, and their progeny were tested for DCV resistance. The assay was repeated with females with similar results. (C) VF-0058–3 embryos were surface sterilized with bleach, raised to adults, and their resistance to DCV compared with non-treated VF-0058–3 and w¹¹¹⁸ iso flies. The assay was repeated with females with similar results.
(D) DNA staining, with propidium iodide, of 0–2 h embryos of VF-0058–3, VF-0058–3t, w¹¹¹⁸ iso, VF-0097–3, and VF-0097–3t. Extranuclear DNA staining corresponds to bacteria. Scale bar, 10μm.
(E) PCR amplification with wsp and wspB primers on DNA extracts of VF-0058–3t, VF-0058–3, VF-0097–3t, and VF-0097–3 embryos. PCR amplification with mt 12S rRNA primers was done as a DNA extraction control.
(F) PCR amplification with primers specific for Spiroplasma 16S rRNA gene on DNA extracts of RED-67, VF-0058–3, wt 1, wt 2, wt 3, wt 4, wt 5, and wt 6 adults. PCR amplification with mt 12S rRNA primers was done as a DNA extraction control.

(A) RT-PCR was done with Nora virus primers on RNA of VF-0058–3, VF-0058–3t, and Oregon R flies (left). PCR with RpL32 was done as control. Three–six-d-old males of VF-0058–3 and VF-0058–3t lines were injected with a virus extract of Oregon R flies and collected 3 d later. RNA was extracted and RT-PCR done with primers for Nora virus and RpL32. The same number of PCR cycles was done for both samples. The assay was repeated three more times, from infection of flies with virus extract, with similar results.
(B) Fifty 3–6-d-old males, per sample, of VF-0058–3 and VF-0058–3t lines were injected with 50 TCID₅₀ FHV, and their survival was followed daily. The assay was repeated twice with males and once with females with similar results.
(C) Extracts of VF-0058–3 and VF-0058–3t flies 3, 6, and 9 d after injection with 50 TCID₅₀ FHV or not injected were probed in a Western blot with anti-FHV. Anti-tubulin was used as a loading control.
(D) Titration, in cell culture, of FHV levels per fly of VF-0058–3 and VF-0058–3t flies 6 d after injection with 50 TCID₅₀ FHV. Squares are replicates (10 per sample), lines are geometric means of replicates. Virus titres in VF-0058–3 and VF-0058–3t are not significantly different (Mann-Whitney test, p = 0.05764).
(E) Fifty 3–6-d-old males, per sample, of VF-0058–3 and VF-0058–3t lines were injected with 1,000 TCID₅₀ IIV-6 or buffer, and their survival followed. The assay was repeated once with males and the IIV-6 injected flies survival curves were also repeated with females, with similar results.
(F) Iridescent-infected male 20 d after injection with 1000 TCID₅₀ IIV-6 (right) and not infected same age male (left) are shown.
(G) Titration, in cell culture, of IIV-6 levels per fly of VF-0058–3 and VF-0058–3t flies 10 days after injection with 1000 TCID₅₀ IIV-6. Squares are replicates (10 per sample), lines are geometric means of replicates. Virus titres in VF-0058–3 and VF-0058–3t are not significantly different (Mann-Whitney test, p = 0.08118).