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12 juillet 19151915/07/12 (A44).Appartient à l’ensemble documentaire : PoitouCh

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

Brazil study site sampling locations.

<p>A) The study site, Itaberaba, a suburb of the city of Juazeiro, Bahia State, Brazil. B) MRR release points (numbered squares) and sampling locations (green circles) within households distributed across the sampling grid at the study site.</p

Dispersal kernels.

<p>Examples of different kernel interpretations for the negative exponential (A, B and C) and exponential power (D, E and F) kernels. The distance pdf is shown in panels A and D. The density with respect to distance is shown in panels B and E and the density pdf is illustrated in panels C and F (after Cousens <i>et al</i>. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004156#pntd.0004156.ref025" target="_blank">25</a>]). Kernels in A, D, C and F integrate to unity (in 1 dimension for the distance pdfs and 2 dimensions for the density pdfs).</p

Brazil model coefficient estimates.

<p>GLM coefficient estimates and associated standard errors, z-value and p-values from the optimal model for the Brazil analysis. Distance was transformed using the exponential power kernel.</p><p>*Overall significance level p<0.0001 (χ² = 46.50, 3df).</p><p>Brazil model coefficient estimates.</p

Dispersal kernel comparison.

<p>A comparison of the A) distance pdf and B) density with respect to distance for estimates using MRR data from Brazil (solid blue line) and Malaysia (dashed pink line). The comparison highlights the similarity in estimated kernels for experiments conducted on different continents, in different habitats.</p

Summary of a literature review of male <i>Ae</i>. <i>aegypti</i> dispersal estimates.

<p>Summary of a literature review of male <i>Ae</i>. <i>aegypti</i> dispersal estimates.</p

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

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