Fig 1.
Constant and fluctuating temperature regimes.
Temperature set points for the maintenance of mosquitoes in the constant and fluctuating temperature regimes. Relative humidity was set at 75% and a 12:12 h day:night light cycle was used.
Table 1.
Infection, dissemination and transmission percentages of ZIKVBR in Ae. aegypti and Ae. albopictus maintained at a 28°C constant or fluctuating temperature conditions.
Fig 2.
ZIKV RNA copies in Ae. aegypti and Ae. albopictus bodies.
Comparison of ZIKV RNA copies in the bodies of Ae. aegypti and Ae. albopictus maintained at 28°C constant or fluctuating temperature conditions. The amount of ZIKV RNA copies in mosquito bodies were quantified by qRT-PCR at 3, 7 and 14 dpi. Each point on the plot represents an individual mosquito. All plots show the median value ± interquartile range (IQR).
Fig 3.
ZIKV RNA copies in Ae. aegypti and Ae. albopictus wings and legs.
Comparison of ZIKV RNA copies in the wings and legs of Ae. aegypti and Ae. albopictus maintained at 28°C constant or fluctuating temperature conditions. The amount of ZIKV RNA copies in mosquito wings and legs were quantified by qRT-PCR at 3, 7 and 14 dpi. Each point on the plot represents an individual mosquito. All plots show the median value ± interquartile range (IQR).
Fig 4.
In vivo distribution of ZIKV infection in Ae. aegypti mosquitoes using immunofluorescence assay (IFA) with whole mosquito microscopy.
Mosquitoes were examined by IFA for ZIKV by staining with an anti-Flavivirus NS1 protein monoclonal antibody (green) and DAPI staining for DNA (blue). (A) An example of a whole mosquito body section showing ZIKV infection in the midgut (m), head (h) and salivary glands (s). (B) Quantification of anti-ZIKV staining density. Staining areas were quantified by image analysis and were expressed as the area of ZIKV staining divided by the area of DAPI staining for each organ/tissue. Data are presented for midguts, bodies (whole mosquitoes minus the area covered by midgut tissue), heads and salivary glands for mosquitoes that had infection in at least one tissue/organ. Post hoc comparisons of the main effect of days post infection on ZIKV staining density was carried out for each organ by Sidak’s method. Lines join comparisons where significant increases in ZIKV staining density had occurred for the main effect of days post infection. (C-E) High resolution images of ZIKV in infected mosquito midgut, head and salivary glands, respectively.
Fig 5.
Immunofluorescence of whole Ae. aegypti mosquito sections.
Whole Ae. aegypti mosquito sections showing infection and dissemination of ZIKV throughout mosquito tissues over a 14 day incubation period. Representative sections from different mosquitoes were selected at various time points. (A) 3 dpi (B) 7 dpi (C) 10 dpi and (D) 14 dpi. Immunofluorescence staining was performed as described for Fig 4. Green, ZIKV infection. Blue, DNA.
Fig 6.
ZIKV infection in Ae. aegypti ovaries.
(A) Quantification of ZIKV staining density relative to DNA over time, calculated as described for Fig 4. (B) High resolution image of ZIKV staining in the follicular epithelium of mature oocytes within Ae. aegypti ovaries. Green, ZIKV infection. Blue, DNA. Post hoc comparisons of the main effect of days post infection on ZIKV staining density was carried out for each organ by Sidak’s method. Lines join comparisons where significant increases in ZIKV staining density had occurred for the main effect of days post infection.