Evans Blue as a Simple Method to Discriminate Mosquitoes’ Feeding Choice on Small Laboratory Animals

<div><p>Background</p><p>Temperature, humidity, vision, and particularly odor, are external cues that play essential roles to mosquito blood feeding and oviposition. Entomological and behavioral studies employ well-established methods to evaluate mosquito attraction or repellency and to identify the source of the blood meal. Despite the efficacy of such methods, the costs involved in the production or acquisition of all parts, components and the chemical reagents involved are unaffordable for most researchers from poor countries. Thus, a simple and relatively low-cost method capable of evaluating mosquito preferences and the blood volume ingested is desirable.</p><p>Principal Findings</p><p>By using Evans blue (EB) vital dye and few standard laboratory supplies, we developed and validated a system capable of evaluating mosquito’s choice between two different host sources of blood. EB-injected and PBS-injected mice submitted to a number of situations were placed side by side on the top of a rounded recipient covered with tulle fabric and containing <i>Aedes aegypti</i> mosquitoes. Homogenates from engorged mosquitoes clearly revealed the blood source (EB- or PBS-injected host), either visually or spectrometrically. This method was able to estimate the number of engorded mosquitoes, the volume of blood ingested, the efficacy of a commercial repellent and the attractant effects of black color and human sweat.</p><p>Significance</p><p>Despite the obvious limitations due to its simplicity and to the dependence of a live source of blood, the present method can be used to assess a number of host variables (diet, aging, immunity, etc) and optimized for several aspects of mosquito blood feeding and vector-host interactions. Thus, it is proposed as an alternative to field studies, and it could be used for initial screenings of chemical compound candidates for repellents or attractants, since it replicates natural conditions of exposure to mosquitoes in a laboratory environment.</p></div

Presence of EB is detected either visually or spectrophotometrically in blood samples or in homogenates of engorged mosquitoes.

<p><i>A.</i> Visual comparison of blood dilutions from PBS- and EB-injected mice (20 and 200 mg/kg). <i>B.</i> Absorbance comparison of blood from PBS- and EB-injected mice at 540 and 620 nm. <i>C.</i> Linear regression of blood volume (X axis) <i>versus</i> absorbance (Y axis) at 540 nm and 620 nm from PBS- and EB-injected mice. <i>D.</i> Experimental scheme and typical visual profile of homogenates from <i>A. aegypti</i> mosquitoes fed on PBS-injected (red) or EB-injected (blue) mice. <i>E.</i> Absorbance profiles of homogenates from individual <i>A. aegypti</i> mosquitoes fed on PBS- or EB-injected mice at 540 nm and 620 nm.</p

Effect of coat color and fresh human sweat on attraction of <i>A. aegypti</i> to mice.

<p><i>A</i>. Proportion of mosquitoes that chose the C57BL/6 mouse (black coat). <i>B.</i> Proportion of mosquitoes that chose sweat-applied mice (EB-injected or PBS-injected). The bars represent the 95% confidence interval.</p

Basic laboratory reagents and supplies needed to evaluate/discriminate the blood source from engorged mosquitoes.

<p>Materials are: <i>1.</i> Tulle fabric; <i>2.</i> Rounded container; <i>3.</i> Glass beaker containing distilled water; <i>4.</i> 96-well plate, flat bottom; <i>5.</i> 1 mL syringe with needle; <i>6.</i> plastic pestle; <i>7.</i> 1.7 mL microcentrifuge tube; <i>8.</i> 20–200 µL universal tip; <i>9.</i> 15 mL tube; <i>10.</i> EB solution; <i>11.</i> 20–200 µL pipette.</p

UV-Visible absorption spectra of blood and EB diluted in water.

<p><i>A.</i> Five microliters of a BALB/c mouse blood were drawn and diluted to 250 µL (1∶50 dilution) with distilled water, centrifuged and the supernatant collected for analysis. Indicated peaks are: 1) protein amide backbone and nucleic acids (220 nm); 2) proteins with chromophoric amino acids and other small chromophoric molecules (280 nm); 3) globin-heme interaction (340 nm); 4) soret band (420 nm); 5) oxyhemoglobin <i>β</i>-band (∼540 nm); and 6) oxyhemoglobin α-band (∼575 nm). <i>B.</i> Evans blue (EB) solution was prepared in distilled water at 200 µg/mL. Indicated peaks are: 1′) unkown (∼320 nm); 2′) EB (∼620 nm). <i>C.</i> Mixture of A and B solutions v/v. Arrows show the common wavelength to measure hemoglobin (540 nm) and EB (620 nm).</p

EB model is useful to evaluate the activity of a commercial repellent against <i>A. aegypti</i> mosquitoes.

<p><i>A</i> and <i>D</i>. Experimental scheme and typical visual profile of homogenate from <i>A. aegypti</i> mosquitoes fed on PBS-injected (red) and EB-injected (blue) mice that received a commercial repellent before exposure to mosquitoes. <i>B</i> and <i>E</i>. Proportion of mosquitoes that chose the PBS and EB mice without repellent, respectively. The bars represent the 95% confidence interval. <i>C</i> and <i>F</i>. Boxplots of the blood volumes from engorged mosquitoes fed on PBS- or EB-injected mice (2.5–97.5 percentile). Dark dots represent the outliers.</p

EB presence in blood circulation does not affect <i>A. aegypti</i> attractiveness to mice.

<p><i>A</i>. Experimental scheme and typical visual profile of homogenates from <i>A. aegypti</i> mosquitoes fed on PBS-injected (red) and EB-injected (blue) mice. <i>B.</i> Proportion of mosquitoes that chose the EB-injected mouse. The bars represent the 95% confidence interval. <i>C.</i> Boxplots of the blood volumes from engorged mosquitoes fed on PBS- or EB-injected mice (2.5–97.5 percentile). Dark dots represent the outliers.</p

B and T cells are increased in the BAL of mice sensitized by mosquito bites followed by SGE challenge.

<p>BAL collection from control and sensitized groups was performed 24 h after the last challenge with PBS or SGE. Differential analyses of lymphocytes were performed by flow cytometry in cells stained with florescence-conjugated antibodies for cell surface markers. <b>(A)</b> Sensitization protocol; <b>(B)</b> total number of CD19⁺ cells, <b>(C)</b> total number of CD4⁺ cells; <b>(D)</b> total number of CD8⁺ cells. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 when compared with PBS group; ^<i>#</i><i>p</i> < 0.05 when compared with the 1x group.</p

Mice sensitized by mosquito bites develop eosinophilic airway inflammation after intranasal SGE challenge.

<p>BAL collection from control and sensitized groups was performed 24 h after the last challenge with PBS or SGE. Total cell number and differential cell counts were analyzed by microscopic evaluation of cytocentrifuged slides. <b>(A)</b> Sensitization protocol; <b>(B)</b> total cells; <b>(C)</b> eosinophils; <b>(D)</b> neutrophils; <b>(E)</b> macrophages; <b>(F)</b> lymphocytes. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 when compared with PBS group; ^<i>#</i><i>p</i> < 0.05 when compared with the 1x group.</p

Th2 cytokine levels are upregulated in BAL of sensitized mice in response to SGE challenge.

<p>BAL of control and sensitized mice was collected 24 h after the last challenge with PBS or SGE, and the cytokine levels in the free-cell supernatant were determined by ELISA. <b>(A)</b> Sensitization protocol; <b>(B)</b> IL-4; <b>(C)</b> IL-5; <b>(D)</b> IL-13; <b>(E)</b> IFN-γ; <b>(F)</b> IL-17. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 compared with PBS group; ^<i>#</i><i>p</i> < 0.05 compared with SGE group.</p