Distinct Roles of Hemocytes at Different Stages of Infection by Dengue and Zika Viruses in Aedes aegypti Mosquitoes

Aedes aegypti mosquitoes are vectors for arboviruses of medical importance such as dengue (DENV) and Zika (ZIKV) viruses. Different innate immune pathways contribute to the control of arboviruses in the mosquito vector including RNA interference, Toll and Jak-STAT pathways. However, the role of cellular responses mediated by circulating macrophage-like cells known as hemocytes remains unclear. Here we show that hemocytes are recruited to the midgut of Ae. aegypti mosquitoes in response to DENV or ZIKV. Blockade of the phagocytic function of hemocytes using latex beads induced increased accumulation of hemocytes in the midgut and a reduction in virus infection levels in this organ. In contrast, inhibition of phagocytosis by hemocytes led to increased systemic dissemination and replication of DENV and ZIKV. Hence, our work reveals a dual role for hemocytes in Ae. aegypti mosquitoes, whereby phagocytosis is not required to control viral infection in the midgut but is essential to restrict systemic dissemination. Further understanding of the mechanism behind this duality could help the design of vector-based strategies to prevent transmission of arboviruses.


Supplementary Figure Legends
Supplementary Figure 1 -Experimental design for the quantification of the midgut infection area. Infection area computing was performed using ImageJ following 9 steps as detailed in the figure: Step 1, color-deconvolution was used to isolate red, green and blue spectra and select the image corresponding to virus infection staining.
Step 2, since a series of z-stack confocal images were acquired, a projection final image was generated using all z-series, "Image > Stacks > Z-project function ". Step 3, the projection image was processed into 8 bits image type.
Step 4, the midgut outline was delimited.
Step 5, the area outside of midgut delimitation was erased by using the "clear outside" function. Steps 6 and 7, optical density was assessed by setting a "threshold" using the "threshold tool", and a maximum threshold was set. Steps 8 and 9, the function "Measure" in the 'Analyze' tool menu was used to calculate the optical density and compute the midgut infection area.
Supplementary Figure 2 -Latex beads do not significantly affect numbers of hemocytes but efficiently block phagocytosis. (A) Mosquitoes injected with latex beads were blood fed two days later. Mosquitoes kept fed on sugar during the whole period were used as controls. Hemocytes were counted after perfusion of mosquitoes at 4 and 8 days post feeding. Total number of mosquitoes is indicated above each box plot. Each dot represents an individual mosquito. Upper, middle and lower bars in the boxplot represent the 75th percentile, the median and the 25th percentile, respectively. Statistical analyses were performed using the Mann-Whitney-Wilcoxon test. (B) Mosquitoes were injected with latex beads or buffer and two days later again injected with fluorescent beads. Hemocytes were counted after perfusion of mosquitoes and the number of cells with and without fluorescent beads was determined at 4 and 8 days post injection. The percentage of cells with red fluorescent beads was plotted as the phagocytic index. The number of mosquitoes analyzed is indicated above each boxplot. Upper, middle and lower bars in the boxplot represent the 75th percentile, the median and the 25th percentile, respectively. Statistical analyses were performed using a general linear model with the ratios of phagocytic activity as response variable and explanatory factors of buffer vs beads, days post feeding, experiment replicate and all second-order interactions. The model used simplified by progressively removing terms. Terms were only removed if it did not cause a significant drop in the proportion of the total deviance explained. In the final models residuals were checked for normality and for the presence of outliers with high leverage. Latex beads were injected into mosquitoes that were kept on sugar (A) or blood fed 2 days after injection (B). Mosquitoes kept on sugar feeding were used as controls. The number of midgut associated hemocytes was determined in mosquitoes at 4 and 8 days post feeding. The number of midguts analyzed in indicated above each boxplot. Each dot represents an individual midgut. Upper, middle and lower bars in the boxplot represent the 75th percentile, the median and the 25th percentile, respectively. Statistical analyses were performed using the Mann-Whitney-Wilcoxon test.     (""%