Fig 1.
Partial blood meals result in intermediate levels of midgut expansion, collagen IV damage, and basal lamina disorder.
(A) Whole mosquitoes and dissected midguts 0 hpbm when given a full blood meal (Full), partial blood meal (Partial), or no blood meal (Unfed). (B) Midgut collagen IV damage in unfed, partially fed, and fully fed mosquitoes 24 hpbm as assessed through CHP binding to pools of 3 midguts (n = 15 pools/treatment of 3 midguts each). Differences were measured using a one-way ANOVA with a Tukey’s multiple comparisons post-test. Center lines represent means and bars represent SEM. (C) TEM images of midgut basal lamina (arrows) sections from unfed, partially fed, and fully fed mosquitoes 24 hpbm. Scale bars are 1μm and 500 nm. (D) Basal lamina thickness measurements from two mosquitoes per treatment with 5 TEM images per mosquito and 5 measurements per image that were averaged for 10 measurements per treatment. Differences between treatments were evaluated using a Kruskal-Wallis test with a Dunn’s multiple comparisons test comparing mean ranks between groups. Center lines represent median values and error bars represent a 95% confidence interval. On graphs, n.s. = not significant, (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001. Exact p values can be found in text.
Fig 2.
Secondary partial blood meals are sufficient to induce midgut basal lamina damage and a higher prevalence of DENV-2 dissemination.
(A) Experimental design schematic for double fed mosquitoes. (B) Midgut collagen IV damage 24 hours after the time of the second blood meal in mosquitoes given a single full blood meal (Single-full), a full blood meal and then a partial blood meal (Full-partial), or two full blood meals (Double-full). CHP binding was assessed using pools of 3 midguts (n = 15 pools/treatment of 3 midguts each) and differences were measured using a one-way ANOVA with a Tukey’s multiple comparisons post-test. Center lines represent means and bars represent SEM. (C) Percent of mosquitoes infected with DENV-2 at 7 dpi as assessed through testing mosquito bodies. Differences were evaluated using a two-tailed Fisher’s exact test. (D) Percent of mosquitoes with disseminated DENV-2 infection 7 dpi as assessed through testing mosquito legs. Data were analyzed using a two-tailed Fisher’s exact test. For C and D, center values represent the proportion and bars represent the binomial SE of sample proportions. On graphs, n.s. = not significant, (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001. Exact p values can be found in text.
Fig 3.
Midgut basal lamina damage is comparable in naïve and minimally fed midguts, yet minimal blood meals still lead to infection and dissemination.
(A) Mosquito midguts 0 hpbm when given either no blood meal (Unfed) or a minimal 15 sec blood meal (Minimal). (B) Midgut basal lamina collagen IV damage 0 hpbm. CHP binding was assessed using 3 pools/treatment of 4 midguts each (n = 3) and an unpaired t-test. Center lines represent means and bars represent SEM. (C) Percent of minimally fed mosquitoes with a DENV-2 infection (I) or disseminated infection (DI) 10 dpi as assessed through testing mosquito bodies and legs, respectively. (D) Percent of fully fed mosquitoes with a DENV-2 infection (I) or disseminated infection (DI) 10 dpi. For C and D, center values represent the proportion positive for DENV-2 and bars represent the binomial SE of sample proportions. On graphs, n.s. = not significant, (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001. Exact p values can be found in text.
Fig 4.
Basal lamina damage 24 hpbm increases with blood meal number but repairs by 96 hpbm.
(A) Experimental design schematic for mosquitoes given multiple successive blood meals. (B) Midgut collagen IV damage 24 hours after 1 (BM1), 2 (BM2), 3 (BM3), or no blood meal (Unfed). CHP binding assays used pools of 3 midguts with 12–14 pools/treatment (Unfed n = 14, BM1 n = 12, BM2 n = 12, BM3 n = 14). Data was analyzed using a Kruskal-Wallis test with a Dunn’s multiple comparisons test comparing mean ranks between groups. Center lines represent median values and error bars represent a 95% confidence interval. (C) Midgut collagen IV damage 96 hours after 1, 2, 3, or no blood meal using pools of 3 midguts with 11–13 pools per treatment (Unfed n = 13, BM1 n = 11, BM2 n = 12, BM3 n = 13). Pools were tested for CHP binding and evaluated using a one-way ANOVA with a Tukey’s multiple comparisons post-test. Center lines represent means and bars represent SEM. On graphs, n.s. = not significant, (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001. Exact p values can be found in text.