Implications of successive blood feeding on Wolbachia-mediated dengue virus inhibition in Aedes aegypti mosquitoes

Abstract

Wolbachia is a promising strategy to inhibit dengue virus (DENV) transmission by Ae. aegypti mosquitoes. Laboratory studies assessing DENV inhibition by Wolbachia typically have not considered natural frequent mosquito blood feeding behavior. Here, we determine the impact of successive feeding on DENV-2 transmission by Ae. aegypti in the presence or absence of Wolbachia (wAlbB and wMelM strains). We show that successive feeding shortens the extrinsic incubation period (EIP) in wildtype (WT; without Wolbachia) and wAlbB mosquitoes through enhanced dissemination. Feeding empirical data into models showed that successive feeding increases the probability of WT and wAlbB mosquitoes surviving beyond the EIP. Importantly, the more epidemiologically relevant comparison of the odds of wAlbB mosquitoes surviving beyond the EIP relative to WT, reveals a larger impact of successive feeding on WT than wAlbB. This indicates a strong inhibitory effect of Wolbachia even in the context of natural frequent mosquito blood feeding behavior.

Fig. 1. Transinfection with wAlbB or wMelM Wolbachia reduces DENV-2 infection in Ae. aegypti and successive feeding leads to higher rates of dissemination in wildtype (WT) and wAlbB mosquitoes at 7 dpi.

a Experimental design for initial infection and dissemination studies of single- and double-fed wildtype mosquitoes lacking Wolbachia (WT), wAlbB, and wMelM mosquitoes. Created in BioRender. Brackney, D. (2025) https://BioRender.com/bg8atjz. b Proportion of infected single- and double-fed WT, wAlbB, and wMelM mosquitoes 7 dpi. Numbers indicate infected mosquitoes over total fed mosquitoes. wMelM single-fed vs double-fed p = 0.0448. c Proportion of single- and double-fed WT, wAlbB, and wMelM mosquitoes with disseminated infection 7 dpi. Numbers indicate mosquitoes with disseminated infection (measured using legs + wings) over infected mosquitoes. WT single-fed vs double-fed p = 0.0361 and wAlbB single-fed vs double-fed p = 0.0015. Comparisons were made using two-sided Fisher’s exact tests. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001. Blue = single-fed, red = double-fed. Lines indicate mean ± standard error of the mean of the total sample proportions. Data was collected across 4 replicates for WT and wAlbB groups and 5 replicates for wMelM groups. Source data for (b, c) are provided as a Source Data file.

Fig. 2. Comparison of DENV-2 titers and relative Wolbachia densities at 7 dpi by feeding and dissemination status.

a Body DENV-2 titers in single- and double-fed wildtype mosquitoes lacking Wolbachia (WT), wAlbB, and wMelM mosquitoes. WT SF n = 34, WT DF n = 42, wAlbB SF n = 46, wAlbB DF n = 35, wMelM SF n = 11, wMelM DF n = 27. WT single-fed vs double-fed p = 0.0001 and wAlbB single-fed vs double-fed p < 0.0001. b Relative body Wolbachia density in single- and double-fed wAlbB and wMelM mosquitoes. Numbers tested are as follows: wAlbB SF n = 74, wAlbB DF n = 61, wMelM SF n = 119, and wMelM DF n = 148. wAlbB single-fed vs double-fed p = 0.0027. c Body DENV-2 titers in wAlbB mosquitoes by dissemination status and feeding status. Total Dis n = 46 and Not Dis n = 35. Dis SF n = 18, Dis DF n = 28, Not Dis SF n = 28, and Not Dis DF n = 7. Dis vs Not Dis p < 0.0001. d Relative body Wolbachia densities in wAlbB mosquitoes by infection, dissemination, and feeding status. Total Dis n = 46, Not Dis n = 35, and Not Inf n = 54. Dis SF n = 18, Dis DF n = 28, Not Dis SF n = 28, Not Dis DF n = 7, Not Inf SF n = 28, and Not Inf DF n = 26. Comparisons were made using two-tailed Mann-Whitney U tests (a–c) or a Kruskal-Wallis test with Dunn’s multiple comparisons (d). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001. Blue = single-fed, red = double-fed. GE = genome equivalents, Dis = mosquitoes with a disseminated infection (i.e., dengue infected bodies and legs), Not Dis = mosquitoes with a non-disseminated infection (i.e., dengue infected bodies, but not legs), Not Inf = mosquitoes that were exposed but are not infected with DENV-2. Lines indicate median with 95% confidence interval. Data was collected across 4 replicates for WT and wAlbB groups and 5 replicates for wMelM groups. Source data are provided as a Source Data file.

Fig. 3. Successive feeding accelerates DENV-2 dissemination and shortens the extrinsic incubation period in both WT and wAlbB Wolbachia-transinfected Ae. aegypti.

a Experimental design for infection and dissemination time course studies of single- and double-fed wildtype mosquitoes lacking Wolbachia. Created in BioRender. Brackney, D. (2025) https://BioRender.com/53u2wpv (WT) and wAlbB mosquitoes. b Percentage of infected single- and double-fed WT mosquitoes 5–10 dpi. Numbers indicate infected mosquitoes over total fed mosquitoes. c Percentage of single- and double-fed WT mosquitoes with disseminated infection 5–10 dpi. Numbers indicate mosquitoes with disseminated infection (measured using legs) over infected mosquitoes. WT single-fed vs double-fed day 5 p = 0.0001. WT single-fed vs double-fed day 6 p = 0.0294. d Percentage of infected single- and double-fed wAlbB mosquitoes 5–10 dpi. Numbers indicate infected mosquitoes over total fed mosquitoes. e Percentage of single- and double-fed wAlbB mosquitoes with disseminated infection 5–10 dpi. wAlbB single-fed vs double-fed day 6 p = 0.0378. wAlbB single-fed vs double-fed day 7 p = 0.0494. Numbers indicate mosquitoes with disseminated infection (measured using legs) over infected mosquitoes. Comparisons were made using two-sided Fisher’s exact tests. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001. Blue = single-fed, red = double-fed. Lines indicate mean ± standard error of the mean of the total sample proportions. Data for time course experiments were collected over 2 replicates. Source data for (b–e) are provided as a Source Data file.

Fig. 4. Modeling of timing of dissemination in single and double-fed WT and wAlbB Wolbachia mosquitoes.

Modeling of timing of dissemination using empirical data shown in Fig. 3 for a single-fed WT mosquitoes (wildtype mosquitoes without Wolbachia), b double-fed WT mosquitoes, c single-fed wAlbB mosquitoes, and d double-fed wAlbB mosquitoes. Experimental data are shown as black dots, with black lines indicating the raw uncertainty in the proportions disseminated. Dark blue or red lines show different draws from the posterior distribution of parameters and indicate the model’s uncertainty. Bright blue and red lines represent the model’s maximum a posteriori prediction. Black lines with arrows mark the timing of 50% dissemination (EIP₅₀) with 95% credible intervals marked by flanking gray lines with arrows. For (c) 50% dissemination = 8.38 days post-infection (95% credible interval [CrI]: 7.72–9.01. For (d) 50% dissemination = 6.86 days post-infection (95% credible interval [CrI]: 6.03–7.62. Blue = single-fed, red = double-fed. SF = single-fed, DF = double-fed. Data used to model the dissemination time course was collected over 2 replicates.

Fig. 5. Impact of feeding behavior on the probability of a mosquito surviving beyond the extrinsic incubation period (EIP).

a Probability of single- and double-fed wildtype mosquitoes without Wolbachia (WT) and wAlbB mosquitoes surviving past the EIP assuming an average mosquito lifespan of 4, 7, or 10 days. b Odds ratio of surviving past the EIP (wAlbB:WT) given a mosquito lifespan of 4, 7, or 10 days. Center lines indicate median posterior predictions. Boxes and whiskers indicate 50% and 95% posterior prediction intervals, respectively. Circles indicate posterior samples falling beyond the 95% posterior prediction intervals. Blue = single-fed, red = double-fed. SF = single-fed, DF = double-fed.