Assessing the effects of Aedes aegypti kdr mutations on pyrethroid resistance and its fitness cost

Abstract

Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr 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 kdr 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 kdr 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 Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects.

Figure 1. Activity of enzymes related to insecticide metabolic resistance in Aedes aegypti strains.

The cut-offs are (dashed lines) determined by the Rockefeller 99 percentile value of each enzyme (see [11]). 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 kdr strain, derived from a pyrethroid resistant Brazilian Aedes aegypti population. Rock-kdr is the kdr strain, backcrossed for eight generations with Rockefeller in order to reduce the contribution of detoxification enzymes to pyrethroid resistance.

Figure 2. Linear regression curves of Aedes aegypti mortality after exposure to deltamethrin impregnated papers.

Strains evaluated correspond to the susceptibility control (Rock), the 1016 Ile/Ile selected strains with genetic background from a natural population (CIT-32) or from Rock (Rock-kdr), and the F1 offspring between Rock-kdr and Rock (Hib-F₁).

Figure 3. Comparison of larval development time between Rockefeller and Rock-kdr Ae. aegypti strains.

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.

Figure 4. Locomotor activity Rock and Rock-kdr Ae. aegypti strains.

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

Figure 5. Number of eggs laid by females from Rock and Rock-kdr Ae. aegypti strains.

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 t test.

Figure 6. Developmental timing of Ae. aegypti Rock and Rock-kdr male adult emergence competing under a stringent condition.

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.

Figure 7. Population cage assays with pyrethroid resistant Aedes aegypti (Rock-kdr) and Rockefeller strains.

The frequency of AaNa_V alleles in the 1016 site was followed in independent cages kept under the same conditions, without insecticide exposure for 15 generations. The initial frequency of the 1016Ile kdr allele in cages 1–3 (A) was 0.50 and in cages 3–6 (B) was 0.75. Lines represent the linear regression analysis taken by the means of the mutant allele frequencies of the respective three cages in A (r² = 0.5273, p = 0,0006) and B (r² = 0,5690, p = 0,0003).