Fig 1.
Histology of Wolbachia infection in the Aedes aegypti wAlbB2-F4 strain.
Wolbachia infection was observed across mosquito organs/tissue types by immunofluorescence analysis (IFA) using a rabbit polyclonal antibody against the Wolbachia surface protein (WSP) as the primary antibody and Alexa Fluor 488-conjugated donkey anti-rabbit antibody as the secondary antibody. DNA was stained using DAPI. (A) Example of whole body section showing IFA staining. (B-E) High resolution images of Wolbachia staining in oocytes, midgut, salivary gland and heads, respectively. (F) Quantification of Wolbachia staining density. Staining areas were quantified by image analysis and expressed as a ratio of Wolbachia staining over DAPI staining for each organ/tissue. The median staining densities differed significantly between groups (Kruskal-Wallis statistic = 95.37, N = 124). P values are reported for comparisons where medians differed significantly by Dunn’s multiple comparison test (α = 0.05, 21 comparisons). Green, Wolbachia,. Blue DNA. h, head. f.m., flight muscles. m, midgut. o, ovary. ooc, oocyte. p, proboscis. s.g., salivary glands. t.g., thoracic ganglia. Scale bars: A: 1.00 mm, B, D: 0.10 mm. C, E: 0.25 mm.
Fig 2.
Wolbachia inhibit dengue 2 infections in Ae. aegypti wAlbB2-F4 mosquitoes.
(A) DENV-2 virus prevalence in the bodies, legs and wings, and saliva of Ae. aegypti wAlbB2-F4 and wild type mosquitoes 14 d after feeding on a blood meal containing 1 × 106.6 CCID50/ml (in C6/36 cells) of DENV-2 virus. P values are included for comparisons with significant differences in mosquito infection rates between wAlbB2-F4 and wild type mosquitoes (Fisher’s Exact test). (B) DENV-2 infection intensity in mosquitoes in A. Virus copy numbers were determined from bodies, legs and wings and saliva samples using quantitative reverse-transcriptase PCR (qRT-PCR). P values are shown for comparisons with significant different median virus copy numbers between wAlbB2-F4 and wild type mosquitoes (Mann-Whitney test). (C) Example whole body midsagittal section from a wild type mosquito dual stained for Wolbachia (green) and DENV-2 (red). (D) Whole body section of a wAlbB2-F4 female showing restriction of virus to the midgut. (E-F) High resolution images of midguts from wild type and wAlbB2-F4 mosquitoes showing lower DENV-2 staining density in the wAlbB2-F4 midgut. (G-H) High resolution images of salivary glands from wild type and wAlbB2-F4 mosquitoes showing dense Wolbachia infection and absence of DENV-2 infection in the latter. a, abdomen. h, head. LOD, limit of detection. m, midgut. s.g., salivary glands. t, thorax. Scale bars: C-D, 1 mm. E-F, 0.25 mm, G-H: 0.10 mm.
Fig 3.
Wolbachia inhibit ZIKV infections in Ae. aegypti wAlbB2-F4 mosquitoes.
(A) ZIKV virus infection prevalence in Ae. aegypti wild type and wAlbB2-F4 mosquitoes 14 d after feeding on a blood meal containing 1 × 108.5 CCID50/ml (in C6/36 cells) of Zika virus. P values are included for comparisons with significant differences in mosquito infection rates between wAlbB2-F4 and wild type mosquitoes (Fisher’s Exact test). (B). ZIKV infection densities in mosquitoes from A. Virus copy numbers were determined from bodies, legs and wings and saliva samples using qRT-PCR. P values are shown for comparisons with significant different median virus copy numbers between wAlbB2-F4 and wild type mosquitoes (Mann-Whitney test). (C-D) Example whole body midsagittal sections of wild type and wAlbB2-F4 mosquitoes dual stained for Wolbachia (green) and ZIKV (red). (E-F) High resolution images of midguts from wild type and wAlbB2-F4 mosquitoes, respectively, showing relatively lower staining density for wAlbB2-F4 mosquitoes. (G-H) High resolution images of midguts from wild type and wAlbB2-F4 mosquitoes, respectively. Infection was limited and spatially restricted in wAlbB2-F4 mosquitoes. a, abdomen. h, head. LOD, limit of detection. m, midgut. s.g. salivary gland. t, thorax. Scale bars: C-D, 1 mm. E-F, 0.25 mm, G-H: 0.10 mm.
Fig 4.
Analysis of genetic equivalency of wAlbB2-F4 strain to Australian Aedes aegypti.
(A) All mosquitoes from the wAlbB2-F4 strain have >98% ancestry (Q-values) from the Australian wild-type strain (and not the WB2 strain), indicating a successful backcrossing procedure. (B) The Wolbachia infected Aedes aegypti wAlbB2-F4 strain has equivalent insecticide susceptibility to the parental Queensland wild type mosquitoes and Ae. aegypti wAlbB2 strains. Survival rates of mosquitoes were recorded following exposure to diagnostic doses of alpha-cypermethrin, cypermethrin and lambda-cyhalothrin using CDC bottle bioassays. Survival rates following exposure to a diagnostic dose of bifenthrin were determined using WHO filter paper assays.