Sensory Discrimination of Blood and Floral Nectar by Aedes aegypti Mosquitoes

Abstract

Blood-feeding mosquitoes survive by feeding on nectar for metabolic energy but require a blood meal to develop eggs. Aedes aegypti females must accurately discriminate blood and nectar because each meal promotes mutually exclusive feeding programs with distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. We investigated the syringe-like blood-feeding appendage, the stylet, and discovered that sexually dimorphic stylet neurons taste blood. Using pan-neuronal calcium imaging, we found that blood is detected by four functionally distinct stylet neuron classes, each tuned to specific blood components associated with diverse taste qualities. Stylet neurons are insensitive to nectar-specific sugars and respond to glucose only in the presence of additional blood components. The distinction between blood and nectar is therefore encoded in specialized neurons at the very first level of sensory detection in mosquitoes. This innate ability to recognize blood is the basis of vector-borne disease transmission to millions of people worldwide.

Keywords: Aedes aegypti; GCaMP calcium imaging; blood-feeding behavior; chemogenetics; gustatory receptors; ionotropic receptors; mosquito; nectar-feeding behavior; stylet; taste.

Figure 1.. Sensory Detection Prior to Blood and Nectar Feeding

(A,C) An Ae. aegypti female feeding on human skin (A, Photo: Benjamin Matthews) or flower nectar (C, Photo: Eric Eaton). (B,D) Transmitted light image of the female stylet (B) or labium (D). Scale bars: 25 μm (E,F) Volume of meal consumed after presenting blood (E) or sugar (F). Unfed controls were not given the option to feed and therefore represent the baseline for the assay. Each data point represents 1 female (mean ± SD, N=37-46; * p < 0.05 Mann-Whitney test). (G) Ae. aegypti female with a blood meal in the midgut (red) and a 10% sucrose meal in the crop (green). Green food dye added to 10% sucrose to visualize meal location. (H) Schematic of blood- (top) and nectar-feeding (bottom) behavior assay. (I) Confocal image of dTomato expression in Gr4>dTomato-T2A-TRPV1 labium with transmitted light overlay. Scale bar: 50 μm. (J) Volume of meal consumed by the indicated genotypes. Each data point represents 1 female: 10% sucrose N=30-40 females/genotype; water N=41-60 females/genotype; water + 50 μM capsaicin (red chili pepper): Gr4 N=61, TRPV1 N=62, Gr4>TRPV1 N=124 females. (K) Female mosquitoes following 15 min exposure to different meals. Scale bar, 1 cm. (L) Sampled weight measurements from data for engorged females offered blood or unfed controls not offered any meal; N=10-19 weight measurements/meal (mean ± SEM; * p < 0.05 unpaired t-test). (M) Female engorgement on the indicated meal delivered via Glytube. Each data point denotes 1 trial with 15-20 females/trial: N=5-11 trials/meal. In (J, M) data labeled with different letters are significantly different from each other (mean ± SD; Kruskal-Wallis test with Dunn’s multiple comparison, p < 0.05). See Figure S1 for chemogenetic and blood-feeding behavioral experiments.

Figure 2.. The Stylet is Poised to Evaluate Meal Quality Prior to Blood Feeding

(A) Still video frames of female in biteOscope assay when stylet contacted meal for the first (left panel) or last (middle panel) time during the trial. Inset at right is from middle panel. (B) biteOscope ethogram of landing events (gray boxes), stylet piercing events (pink boxes), and engorgement events (black boxes) for individual females provided water (N=8 females), saline (N=7 females), or 1mM ATP in saline (N=10 females) over 700 sec trial. Each row is an ethogram from 1 female. (C-E) Summary statistics from individual female ethograms in (A) for cumulative piercing duration during trial (C), # of landings (D), and # of piercings (E) for indicated meal. Each dot denotes 1 female, filled dot represents an engorged female. In C,E, data labeled with different letters are significantly different from each other (mean ± SD; Kruskal-Wallis test with Dunn’s multiple comparison, p < 0.05). In D, data labeled with different letters are significantly different from each other (mean ± SD; one-way ANOVA with Tukey’s multiple comparisons test). See Video 1 representative biteOscope movies.

Figure 3.. Sensory Neurons in the Female Stylet are Sexually Dimorphic and Project to a Unique Subesophageal Zone Region

(A,B) Confocal image with transmitted light overlay of TO-PRO-3 nuclear staining (cyan) in wild-type female (A, left) and male (A, right) stylets, and dTomato expression (gray) in Brp>dTomato-T2A-GCaMP6s female (B, left) and male (B, right) stylets. (C,D) Average # of TO-PRO-3 nuclei/stylet for most distal 300 μm (C, N=7 females, N=6 males), and dTomato neurons/stylet (D, N=10 females, N=16 males). Each dot denotes 1 animal (mean ± SD, * p < 0.05 Mann-Whitney test). (E) Confocal image of transmitted light (top) and dTomato (gray, bottom) in Brp>dTomato-T2A-GCaMP6s female (left) and male (right) stylet tip. (F) Confocal image with transmitted light overlay of phalloidin-594 (red) staining in wild-type female (left) and male (right) stylets. (G, J) Schematic of stylet (G) and double (J) dye-fill experiment set-up performed in (I) and (L), respectively. (H, K) Schematic of mosquito brain region captured in (I), and subesophageal zone optical sections captured in (L). (I) Stylet neuron projection pattern (magenta) revealed by dextran-595 dye-fill. Neuropil stained with anti-Drosophila Brp (gray). (L) Optical subesophageal zone sections from most anterior (top row) to most posterior (bottom row) of stylet (left, magenta) and labium (middle, green) projection pattern revealed by dual dextran-494 and dextran-595 dye-fill. Scale bar: 50 μm (I), 25 μm (A,B,L), 10 μm (E,F). See Figure S2 for additional analysis of stylet sexual dimorphism and Video 2 for confocal Z-stacks of dual dye-fills.

Figure 4.. Sexually Dimorphic Stylet Neurons Directly Sense Blood

(A) Schematic of ex vivo stylet imaging preparation. (B) Wide-field image of dTomato (top) and baseline GCaMP6s (bottom, scale: arbitrary units) for a representative stylet, oriented proximal to distal. (C) Representative image of GCaMP6s fluorescence increase to bulk neuronal depolarization with 500 mM KCl (bottom) compared to baseline (top). (D) Representative bright-field image before (top) and during (bottom) delivery of sheep blood to the stylet tip via the BioPen. (E,F) Representative image of GCaMP6s fluorescence increase to indicated blood presentation (bottom, E) or water control (bottom, F), compared to baseline (top). (G) Heat maps of peak ΔF/F₀ response to the indicated ligand. Each square is the average of the peak ΔF/F₀ measured in 3 separate trials. Each column represents 1 neuron and each row represents the response to indicated ligand for all neurons from 1 individual female, with neurons ordered from proximal to distal. N=6 individual females. In (B-F) scale bar: 25 μm. 0.0002% fluorescein was added to blood and water stimuli to visualize ligand delivery zone. See Video 3, 4 for representative movies of BioPen stimulus delivery and stylet responses to blood or water, and Figure S3 for details on calcium imaging analysis.

Figure 5.. Stylet Neurons Integrate Across Taste Modalities to Detect Blood

A) Representative engorged Ae. aegypti female following 15-min exposure to blood (top) or Mix+ATP (bottom) via Glytube assay. (B) Female engorgement on blood (N=5 trials) and Mix+ATP (N=6 trials) delivered via Glytube (lines denote mean ± SD, 15–20 females/trial, p = 0.0714, Mann-Whitney test). (C) Representative image of GCaMP6s fluorescence increase (scale: arbitrary units) to blood (bottom, left) or Mix+ATP (bottom, right), compared to baseline (top). Scale bar: 25 μm. (D) Heat maps of peak ΔF/F₀ response to the indicated ligand. Each square is the average of 3 ligand exposures and each column represents one neuron. Each row represents the response to indicated ligand for all neurons from 1 individual female, with neurons ordered from proximal to distal. N=6 individual females. (E) Summary of % neurons with ≥ 0.25 peak ΔF/F₀ to the indicated ligand from (D), each column represents 1 female. (F) Scatter plot comparing peak ΔF/F₀ in response to Mix+ATP (y-axis) and blood (x-axis) summarized across N=6 females from (D,E). Each dot represents 1 neuron, dots that fall on the dashed line have the same peak ΔF/F₀ in response to blood and Mix+ATP. Dots that fall above the line respond more to Mix+ATP than to blood and dots that fall below the line respond more to blood than to Mix+ATP. (G-I) 5 clusters of blood-sensitive neurons identified by unsupervised hierarchical clustering of peak ΔF/F responses to the ligands indicated in (G). Clustering removes proximal-distal ordering and female identity. N=5 females (* p < 0.05, one-sample Wilcoxon signed-rank test). In (A-I) and all subsequent experiments “Mix” is 4.5 mM glucose, 25 mM NaHCO₃, 115 mM NaCl and “Mix+ATP” is Mix supplemented with 1 mM ATP. To visualize ligand delivery zone, 0.0002% and 0.00002% fluorescein was added to blood and Mix+ATP, respectively, in BioPen experiments. See Video 5 for representative movies of stylets responding to blood and Mix+ATP, Data File 1 for raw imaging data and p values for Figure 5I, Figure S4 for responses of individual females to blood components, and Figure S5 for details of the hierarchical clustering method.

Figure 6.. Ir7a and Ir7f Mark the NaHCO₃ and Integrator Neurons

(A,B) Confocal image with transmitted light overlay of dTomato expression (gray) in the female stylet (left panel), male stylet (middle panel), and female labium (right panel) of Ir7a>dTomato-T2A-GCaMP6s (A) and Ir7f>dTomato-T2A-GCaMP6s (B) animals. Ir7a expression: 10/13 females = 2 neurons, 2/13 females = 1 neuron, 1/13 females = 0 neurons. Ir7f expression: 6/11 females = 4 neurons, 5/11 females = 3 neurons. (C-F) mCD8:GFP expression (magenta, white arrow) of Ir7a>mCD8:GFP (C,E) and Ir7f>mCD8:GFP (D,F) in female (left) and male (right) brain (top) and subesophageal zone (bottom). Neuropil in C and D is labeled with anti-Drosophila Brp (gray). The brain and subesophageal zone images in C-F were acquired from different individuals. (G,H) Heat maps of peak ΔF/F₀ response to the indicated ligand in Ir7a>dTomato-T2A-GCaMP6s (G) and Ir7f>dTomato-T2A-GCaMP6s (H) neurons across N=5 females. Each square is the average of 3 ligand exposures and each column represents one neuron. Columns are sorted by largest to smallest peak ΔF/F₀ in response to blood. (I,K) Raw F₀ traces from individual neurons in response to indicated ligand. (J,L) For blood-sensitive neurons, peak ΔF/F₀ to indicated ligand. Each data point denotes the response from 1 neuron and responses from the same neuron are connected by a line (* p < 0.05, one-sample Wilcoxon signed-rank test). In (A-F) scale bar: 25 μm. 0.0002% fluorescein was added to blood and 140 mM NaCl, and 0.00002% was added to Mix and 25 mM NaHCO₃ in the BioPen to visualize ligand delivery zone. See Video 6 for confocal Z-stack movies of Ir7a- and Ir7f- labeled neurons, Figure S6 for RNA-seq data and behavioral analysis of Ir7a and Ir7a mutants and chemogenetic manipulation, and Data File 1 for p values for Figure 6J,L.

Figure 7.. The Stylet is Specialized to Detect Blood over Nectar

(A) Venn diagram schematizing the similarity and differences between nectar (left circle) and blood (right circle) components. (B,C) Volume of indicated meal consumed in the nectar-feeding (B) and blood-feeding (C) assay. Each data point represents 1 female: water N=36-40; sucrose N=53–60; fructose N=40-74; glucose N=55-59. Blood in (C) is a positive control for blood-feeding assay, N=76 females. (D) Sweet taste receptor expression from RNA-seq analysis of the indicated tissues. N=4 replicates/tissue. Median indicated by black line, bounds of box represent first and third quartile, whiskers are 1.5 times the inter-quartile range, and dots represent TPM value from each biological replicate. The outlier is denoted by a dot without whisker. (E) Confocal image with transmitted light overlay of dTomato expression (gray) in the female stylet (left panel), male stylet (middle panel), and female labium (right panel) of Gr4>dTomato-T2A-GCaMP6s animals. Scale bar: 25 μm. (F) Representative image of GCaMP6s fluorescence increase to indicated 298 mM sugar presentation (bottom) compared to baseline (top). Flower/blood symbol (3^rd from left) indicates that sugar is found in nectar and blood. (G) Quantification of % neurons with ≥ 0.25 peak ΔF/F₀ to the indicated ligand, each data point denotes the response from 1 female, responses from the same female are connected by a line, N=6 females. (H,I) For Integrator neurons, peak ΔF/F₀ to 298 mM glucose (H, N=8 neurons) and 4.5 mM glucose (I, N=5 neurons). Each dot represents 1 neuron (mean ± SD, * p < 0.05 Mann-Whitney test). (J) For Integrator neurons, peak ΔF/F₀ to indicated ligand(s). Each data point denotes the response from 1 neuron, N=8 neurons. Data labeled with different letters are significantly different from each other (one-way repeated measures ANOVA, with the Geisser-Greenhouse correction and Tukey’s multiple comparisons test, p < 0.05). In (B,C,G) data labeled with different letters are significantly different from each other (mean ± SD; Kruskal-Wallis test with Dunn’s multiple comparison, p < 0.05) and in (H, I, J) responses from the same neuron are connected by a line. See Figure S7 for behavioral and imaging data with nectar sugars.