Effect of repeat human blood feeding on Wolbachia density and dengue virus infection in Aedes aegypti

Abstract

Background: The introduction of the endosymbiotic bacterium, Wolbachia into Aedes aegypti populations is a novel approach to reduce disease transmission. The presence of Wolbachia limits the ability of the mosquito to transmit dengue virus (DENV) and the strength of this effect appears to correlate with Wolbachia densities in the mosquito. There is also some evidence that Wolbachia densities may increase following the consumption of a bloodmeal. Here we have examined whether multiple blood feeds lead to increases in density or associated changes in Wolbachia-mediated blocking of DENV.

Methods: The Wolbachia infected Aedes aegypti mosquito line was used for the study. There were three treatment groups; a non-blood fed control, a second group fed once and a third group fed twice on human blood. All groups were orally infected with DENV-2 and then their midguts and salivary glands were dissected 10-11 days post infection. RNA/DNA was simultaneously extracted from each tissue and subsequently used for DENV RNA copies and Wolbachia density quantification, respectively.

Results: We found variation between replicate vector competence experiments and no clear evidence that Wolbachia numbers increased in either the salivary glands or remainder of the body with feeding and hence saw no corresponding improvements in DENV blocking.

Conclusions: Aedes aegypti are "sip" feeders returning often to obtain bloodmeals and hence it is important to assess whether repeat blood feeding improved the efficacy of Wolbachia-based DENV blocking. Our work suggests in the laboratory context when Wolbachia densities are high that repeat feeding does not improve blocking and hence this ability should likely be stable with respect to feeding cycle in the field.

Figure 1

Wolbachia density in salivary gland, midgut and body of wMel.F mosquitoes. Significantly more Wolbachia was found in the remainder of the mosquito body compared to the salivary glands and midguts (P < 0.0001). Salivary glands had significantly more Wolbachia than the midguts (P = 0.0009). Y-axis shows ratio of wsp/Rps17. Letters denote distinct statistical groups and error bars are standard error of the mean of 16–17 biological replicates.

Figure 2

DENV-2 RNA copies in salivary glands of wMel.F and Wildtype mosquitoes. In replicate A, wMel.F mosquitoes fed twice on human bloodmeal (Fed 2x) prior to being infected had complete viral blocking 10–11 dpi. This was significantly lower (P = 0.02) than the wMel.F mosquitoes which were not fed on human bloodmeal (Unfed) prior to being infected. The Wildtype mosquitoes which had two repeat human bloodmeals (Fed 2x) had significantly lower copies of DENV-2 RNA compared to those which did not have human bloodmeal (Unfed) (P = 0.005) and those which had only one human bloodmeal (Fed 1x) (P = 0.005). In replicate B, there was complete DENV-2 blocking in the wMel.F salivary glands in all three treatment groups and in the Wildtype mosquitoes, the effect of repeat blood feeding was not significant. Comparisons were made within mosquito lines and across treatment groups. Letters represent distinct statistical groups. Bars denote medians and each point represents individual salivary gland.

Figure 3

Wolbachia density in wMel.F mosquito salivary glands. wMel.F mosquitoes fed once and twice on human bloodmeals (Fed 1x and Fed 2x) prior to being challenged with DENV-2 did not have a significant change in Wolbachia density in both replicates A and B compared to the Unfed controls which were not blood fed. Y-axis shows ratio of wsp/Rps17. Letters represent distinct statistical group. Error bars are standard error of the mean of 11–19 salivary glands.

Figure 4

DENV-2 RNA copies in midgut of wMel.F and Wildtype mosquitoes. DENV-2 RNA copies in midguts of wMel.F and Wildtype mosquitoes fed once and twice on human bloodmeal (Fed 1x and Fed 2x) did not change significantly compared to those which were not blood fed (Unfed) in both replicates A and B. Comparisons were made within mosquito lines and within treatment groups. Letters represent distinct statistical group. Bars denote medians and each point represents an individual midgut.

Figure 5

Wolbachia density in wMel.F mosquito midguts. In replicate A, midgut Wolbachia density in wMel.F mosquitoes decreased in mosquitoes fed twice on human bloodmeal (Fed 2x) before being challenged with DENV-2 compared to those which were not blood fed (Unfed) (P = 0.002) and those fed only once on human blood (Fed 1x) (P = 0.03). However in replicate B, there was no significant change in Wolbachia density across treatment groups. Y-axis shows ratio of wsp/Rps17. Letters represent distinct statistical groups. Error bars are standard error of the mean of 14-21individual midguts.

Figure 6

DENV-2 RNA copies in the body of wMel.F and Wildtype mosquitoes. In replicate A, wMel.F mosquitoes that fed twice on human bloodmeal (Fed 2x) prior to being infected had complete viral blocking in the remainder of the mosquito body 10–11 dpi. This was significantly lower (P = 0.02) than the wMel.F mosquitoes which were not fed human bloodmeal (Unfed) prior to being infected. Repeat blood feeding did not have a significant effect on DENV-2 RNA copies in the remains of the Wildtype mosquito body. In replicate B, there was no significant change in DENV-2 RNA copies between treatment groups in both wMel.F and Wildtype mosquitoes. Letters represent distinct statistical groups. Bars denote medians and each point represents an individual carcass/remainder of the mosquito body.

Figure 7

Wolbachia density in the remainder/carcass of the mosquito body. There was no significant change in Wolbachia density in wMel.F remainder/carcass of the mosquito body across treatment groups in both the replicates A and B. Y-axis shows ratio of wsp/Rps17. Letters represent distinct statistical groups. Error bars are standard error of the mean of 17–22 individual remainder/carcass of the mosquito body.