Assessing the Effects of <i>Aedes aegypti kdr</i> Mutations on Pyrethroid Resistance and Its Fitness Cost

<div><p>Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (Na_V), inducing the knockdown effect. In <i>Aedes aegypti</i>, the main dengue vector, the AaNa_V substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (<i>kdr</i>) mutations. We evaluated the fitness cost of these <i>kdr</i> mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both <i>kdr</i> mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the <i>kdr</i> mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the <i>Ae. aegypti kdr</i> mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the <i>kdr</i> mutation is found before new adaptive alleles can be selected for diminishing the <i>kdr</i> deleterious effects.</p> </div

Comparison of larval development time between Rockefeller and Rock-kdr <i>Ae. aegypti</i> strains.

<p>Numbers represent the cumulative daily proportion of Rock and Rock-kdr pupae formation after larvae eclosion under standard laboratory conditions. SEM is indicated. Gray dotted line indicates equal proportion (rate = 1) between strains.</p

Activity of enzymes related to insecticide metabolic resistance in <i>Aedes aegypti</i> strains.

<p>The cut-offs are (dashed lines) determined by the Rockefeller 99 percentile value of each enzyme (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060878#pone.0060878-Montella1" target="_blank">[11]</a>). Rockefeller is a reference strain of insecticide susceptibility and vigor. Distributions with less than 15% of individuals beyond the cut-off are considered unaltered. Between 15 and 50% are altered and above 50% are highly altered. CIT-32 is the original <i>kdr</i> strain, derived from a pyrethroid resistant Brazilian <i>Aedes aegypti</i> population. Rock-kdr is the <i>kdr</i> strain, backcrossed for eight generations with Rockefeller in order to reduce the contribution of detoxification enzymes to pyrethroid resistance.</p

Oviposition performance of Rockefeller and Rock-kdr <i>Ae. aegypti</i> females.

<p>Oviposition performance of Rockefeller and Rock-kdr <i>Ae. aegypti</i> females.</p

Developmental timing of <i>Ae. aegypti</i> Rock and Rock-kdr male adult emergence competing under a stringent condition.

<p>A – Cumulative rate of male emergence up to the 8th day after the beginning of adult emergence when the controls Rock and Rock-kdr were reared separately (‘intra-strain’ conditions). B – Cumulative proportion of Rock or Rock-kdr male emergence from the inter-strain competition. Male strain was daily determined by randomly genotyping 30% of emerging individuals.</p

Locomotor activity Rock and Rock-kdr <i>Ae. aegypti</i> strains.

<p>Locomotor activity of susceptible (Rockefeller strain – blue line) and pyrethroid resistant (Rock-kdr – red line) <i>Aedes aegypti</i> females exposed two days under LD 12∶12, at 25°C. Dotted lines represent standard errors.</p

Number of eggs laid by females from Rock and Rock-kdr <i>Ae. aegypti</i> strains.

<p>Each dot represents a single female. Only females that laid at least one egg were included. Median value with interquartile range is shown for each distribution. Dotted line points 50 eggs/female, which was herein empirically considered as discriminative of successful insemination. ***Difference between strains was highly significant by <i>t</i> test.</p