Article Figures & Data
Figures
- FIG 1
Additional feeding differentially impacts rodent and human malaria parasite survival. Experimental overview of additional feeding experiments in A. gambiae (A). Following an initial infection (day 0) with Plasmodium berghei or Plasmodium falciparum (dark red circle), blood fed mosquitoes were maintained on sugar or were challenged with an additional blood (pink circle) or protein (blue circle) meal at either 4 or 8 days postinfection. The effects of each experimental condition on oocyst numbers were then evaluated at 8 or 10 days postinfection, respectively. The influence of an additional feeding on P. berghei oocyst numbers was examined on day 8 for mosquitoes receiving an additional blood (B) or protein meal (C) 4 days postinfection. Similar experiments were also performed with P. falciparum, where oocyst numbers were evaluated on day 8 in mosquitoes that received an additional blood or protein meal 4 days postinfection (D). Potential temporal effects of the timing of feeding on oocyst survival were also examined, where mosquitoes infected with P. berghei or P. falciparum were maintained on sugar or received an additional uninfected blood meal 8 days postinfection (E and F). Oocyst numbers were evaluated at 10 days postinfection for P. berghei (E) and P. falciparum (F). For all experiments, each dot represents the number of parasites on an individual midgut, with the median value denoted by a horizontal red line. Mosquito infection data were pooled from three or more independent experiments (B to D) or from two independent experiments (E and F). Statistical analysis was performed using nonparametric tests for individual comparisons (Mann-Whitney U) or multiple comparisons (Kruskal-Wallis with a Dunn’s multiple-comparison test) using GraphPad Prism 7 software. Asterisks denote significance (**, P < 0.01; ****, P < 0.0001). n, number of mosquitoes examined per group; ns, not significant.
- FIG 2
Additional protein or blood feeding similarly influence mosquito physiology. To examine the physiological impacts of additional feeding, P. berghei-infected mosquitoes (dark red circle) were challenged 4 days postinfection with an additional protein (blue circle) or blood meal (pink circle). Approximately 24 h after an additional feeding, samples were collected to examine the effects on the microbiome using bacterial 16S rRNA expression (A), vitellogenesis using vitellogenin (Vg) expression (B), and TEP1 expression as a proxy for the immune system (C). Gene expression was examined by reverse transcription-quantitative PCR (qRT-PCR) using either dissected midgut samples (A) or whole-mosquito samples (B and C) using three or more independent biological replicates. Statistical analysis was performed using Mann-Whitney analysis with GraphPad Prism 7 software. Asterisks denote significance (*, P < 0.05). ns, not significant.
- FIG 3
Mosquito feeding promotes the degradation of the midgut basal lamina. Using a fluorescein-labeled collagen hybridizing peptide (CHP) to detect degraded collagen, midgut basal lamina integrity was examined temporally at 3, 6, 18, 24, and 48 h following blood or protein feeding (A). Heat-treated midguts (70°C for 10 min in 1× phosphate-buffered saline [PBS]) were used as a positive (+) control sample. The CHP fluorescence signal was quantified with ImageJ for each sample, and used to determine the relative fluorescence at each time point following blood feeding (B) or protein feeding (C). CHP binding analysis was performed in three independent experiments under blood-fed conditions and in two independent experiments with protein feeding. For each time point, three or more midgut samples were examined by fluorescence microscopy with images analyzed using ImageJ. Relative fluorescence was calculated using the 0-h time point as the baseline measurement, then examined across multiple time points using a one-way analysis of variance (ANOVA) with a Holm-Sidak multiple-comparison test using GraphPad Prism 7 software. Asterisks denote significance (***, P < 0.001; ****, P < 0.0001).
- FIG 4
Additional feeding enables the recognition and killing of P. berghei oocysts by mosquito complement. Immunofluorescence assays were performed to examine TEP1 localization on developing oocysts when maintained on sugar or following an additional blood meal. P. berghei oocysts were identified by circumsporozoite protein (CSP) staining and residual signal from the mCherry-parasite background, enabling determination of the percentage of TEP1+ oocysts from both experimental conditions (A). Similar experiments were performed following P. falciparum infection (B). Additional feeding experiments were performed on either wild-type (WT) or mutant TEP1 (ΔTEP1) lines to confirm the involvement of mosquito complement in P. berghei oocyst recognition and killing (C). Oocyst numbers were evaluated 8 days postinfection. (D) Model for the role of mosquito complement via TEP1 recognition and killing of P. berghei oocysts. The percentage of TEP1+ oocysts for P. berghei and P. falciparum studies are displayed as the mean (+ standard error of the mean [SEM]) and analyzed by Mann-Whitney U test for direct comparison (**, P < 0.01; ****, P < 0.0001). Bar, 10 μm. n, number of mosquitoes examined; ns, not significant. For each figure, the feeding status is designated by a dark red circle demonstrating an initial P. berghei or P. falciparum parasite infection. Additional blood (pink circle) or protein (blue circle) meals at either 4 days postinfection are designated by their respective colors.
- FIG 5
Human malaria parasites utilize host resources provided by an additional feeding to enhance their growth. P. falciparum (A) or P. berghei (B) oocysts were examined at 8 days postinfection. The size of individual oocysts from mosquitoes maintained on sugar or that received an additional blood or protein meal were measured using ImageJ and compared by relative size across conditions. Representative images are shown on the right. Based on growth differences and supporting literature, we propose a model in which human malaria parasites are able to utilize host resources to increase growth and increase the chances of transmission (C). Oocyst measurements were performed in ImageJ using infected midgut micrographs with oocyst measurements in sugar-fed samples from two independent experiments serving as the baseline for relative comparisons using Kruskal-Wallis and Dunn’s multiple-comparison tests in GraphPad Prism 7 software. Asterisks denote significance (****, P < 0.0001). n, number of oocysts examined; ns, not significant. For each figure, the feeding status is designated by a dark red circle demonstrating an initial P. berghei or P. falciparum parasite infection. Additional blood (pink circle) or protein (blue circle) meals at either 4 days postinfection are designated by their respective colors.
Supplemental Material
FIG S1
Experimental overview of additional feeding studies. Experimental overview of feeding experiments where Anopheles gambiae mosquitoes were initially challenged with Plasmodium berghei (A) or Plasmodium falciparum (B). They were then either maintained on sugar or received an additional uninfected blood or protein meal 4 days postinfection. For both panels A and B, oocyst numbers were examined at 8 days postinfection. Similar experiments were performed for both P. berghei or P. falciparum mosquitoes (C) in which an additional blood meal was provided at 8 days postinfection. Oocyst numbers were evaluated at 10 days postinfection. Download FIG S1, TIF file, 2.9 MB.
Copyright © 2021 Kwon et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S1
Raw data for additional blood- and protein-feeding experiments. Download Table S1, XLSX file, 0.02 MB.
Copyright © 2021 Kwon et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Low-melt agarose causes mortality in P. berghei-infected mosquitoes. P. berghei-infected mosquitoes were challenged with an additional meal of low-melt agarose at various concentrations (0.05, 0.1, and 0.2%) 4 days postinfection. After feeding, mosquitoes were maintained at 19°C, the standard temperature for P. berghei infection. Mosquito mortality was assessed at 24 h for each concentration of low-melt agarose. Each condition resulted in complete mortality of all of the mosquitoes that fed on the low-melt agarose. n, number of mosquitoes examined per treatment. Download FIG S2, TIF file, 0.08 MB.
Copyright © 2021 Kwon et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
Raw data for P. berghei infection experiments with wild-type (WT) and TEP1 mutant lines. Download Table S2, XLSX file, 0.01 MB.
Copyright © 2021 Kwon et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S3
Primers used for reverse transcription-quantitative PCR (qRT-PCR) analysis. Download Table S3, DOCX file, 0.01 MB.
Copyright © 2021 Kwon et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
