High Frequencies of kdr Mutation and Chikungunya Infection in Aedes aegypti Population from Minas Gerais, Brazil.

The Chikungunya virus (CHIKV) presents global health challenges, with Brazil experiencing outbreaks since its introduction in 2014. In 2023, following a CHIKV outbreak in Minas Gerais (MG), social media was used to optimize an entomological survey aimed at identifying vectors and viral lineages and assessing insecticide resistance. Following Instagram posts, residents with suspected CHIKV infection were able to schedule mosquito aspirations. In total, 421 mosquitoes (165 Aedes aegypti and 256 Culex quinquefasciatus) were captured from 40 households in Salinas city (MG) and tested for the Dengue, Zika, and Chikungunya viruses through RT-qPCR. Twelve of 57 pools (10 Ae. aegypti and two Cx. quinquefasciatus) tested positive for CHIKV RNA. Viral RNA was also detected in the heads of nine Ae. aegypti, indicating viral dissemination but not in Cx. quinquefasciatus. Genome sequencing yielded the first near-complete genome from the 2023 outbreak, unveiling that the CHIKV strain belonged to the East/Central/South African (ECSA) genotype. Additionally, genetic analyses revealed high frequencies of kdr alleles, including in CHIKV-infected mosquitoes, suggesting resistance to pyrethroid insecticides in this Ae. aegypti population. Social media was important for guiding mosquito-capture efforts in CHIKV transmission hotspots, thus optimizing the opportunity for viral detection. These findings emphasize the urgent need for innovative vector studies and control strategies, as well as interdisciplinary approaches in public health interventions.

Insecticide resistance and underlying targets-site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan.

Insecticide resistant Aedes populations have recently been reported in Pakistan, imposing a threat to their control. We aimed to evaluate the susceptibility of Aedes aegypti and Aedes albopictus populations from Lahore to WHO-recommended insecticides and to investigate metabolic and target-site resistance mechanisms. For this purpose, we first carried out bioassays with the larvicides temephos and pyriproxyfen, and the adulticides malathion, permethrin, deltamethrin, alpha-cypermethrin, and etofenprox. We looked for Knockdown resistance mutations (kdr) by qPCR, High-Resolution Melt (HRM), and sequencing. In order to explore the role of detoxifying enzymes in resistance, we carried out synergist bioassay with both species and then checked the expression of CYP9M6, CYP9J10, CYP9J28, CYP6BB2, CCAe3a, and SAP2 genes in Ae. aegypti. Both species were susceptible to organophosphates and the insect growth regulator, however resistant to all pyrethroids. We are reporting the kdr haplotypes 1520Ile + 1534Cys and T1520 + 1534Cys in high frequencies in Ae. aegypti while Ae. albopictus only exhibited the alteration L882M. PBO increased the sensitivity to permethrin in Ae. aegypti, suggesting the participation of P450 genes in conferring resistance, and indeed, CYP928 was highly expressed. We presume that dengue vectors in Lahore city are resistant to pyrethroids, probably due to multiple mechanisms, such as kdr mutations and P450 overexpression.

Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018.

Insecticide resistance is currently a threat to the control of Aedes agypti, the main vector of arboviruses in urban centers. Mutations in the voltage gated sodium channel (NaV), known as kdr (knockdown resistance), constitute an important selection mechanism for resistance against pyrethroids. In the present study, we investigated the kdr distribution for the Val1016Ile and Phe1534Cys alterations in Ae. aegypti from 123 Brazilian municipalities, based on SNP genotyping assays in over 5,500 mosquitoes. The alleles NaVS (1016Val+ + 1534Phe+), NaVR1 (1016Val+ + 1534Cyskdr) and NaVR2 (1016Ilekdr + 1534Cyskdr) were consistently observed, whereas kdr alleles have rapidly spread and increased in frequency. NaVS was the less frequent allele, mostly found in Northeastern populations. The highest allelic frequencies were observed for NaVR1, especially in the North, which was fixed in one Amazonian population. The double kdr NaVR2 was more prevalent in the Central-west and South-eastern populations. We introduce the 'kdr index', which revealed significant spatial patterns highlighting two to three distinct Brazilian regions. The 410L kdr mutation was additionally evaluated in 25 localities, evidencing that it generally occurs in the NaVR2 allele. This nationwide screening of a genetic mechanism for insecticide resistance is an important indication on how pyrethroid resistance in Ae. aegypti is evolving in Brazil.

Levels of Resistance to Pyrethroid among Distinct kdr Alleles in Aedes aegypti Laboratory Lines and Frequency of kdr Alleles in 27 Natural Populations from Rio de Janeiro, Brazil.

BACKGROUND: Several mutations in voltage gated sodium channel (NaV) have been identified in Aedes aegypti populations worldwide. However, only few are related to knockdown resistance to pyrethroids, most of which with variations in the 1016 and 1534 NaV sites. In Brazil, at least two NaV alleles are known: NaVR1, with a substitution in the 1534 (1016 Val+ + 1534 Ile kdr ) and NaVR2, with substitutions in both 1016 and sites (1016Ilekdr + 1534Cys kdr ). There is also the duplication in the NaV gene, with one copy carrying the substitution Ile1011Met, although its effects on pyrethroid resistance remain to be clarified. Our goals in this study were (1) to determine the role of each kdr NaV allele and the duplication on pyrethroid resistance and (2) to screen the frequency of the kdr alleles in 27 several natural Ae. aegypti populations from the metropolitan region of Rio de Janeiro. METHODS: Pyrethroid resistance was evaluated by a knockdown time (KdT) assay, an adaptation of the WHO test tubes with paper impregnated with deltamethrin. We used laboratory-selected Ae. aegypti lineages: R1R1 and R2R2 (homozygous for the kdr NaVR1 and NaVR2 alleles, respectively), Dup (with duplication in the NaV gene), Rockefeller (the susceptibility reference control), and F1 hybrids among them. Genotyping of both 1016 and 1534 NaV sites was performed in 811 Ae. aegypti sampled from 27 localities from Rio de Janeiro (17), Niterói (6) and Nova Iguaçu (4) cities, Rio de Janeiro State, Brazil, with a TaqMan real time PCR approach. RESULTS: The laboratory lineages R1R1, R2R2, and R1R2 were the only ones that needed more than 60 minutes to knock down all the insects exposed to the pyrethroid, being the KdT R2R2 > R1R2 > R1R1, corroborating the recessive nature of the kdr mutations. Frequency of kdr alleles NaVR1 and NaVR2 in field-caught mosquitoes varied from 0 to 52% and 43 to 86%, respectively, evidencing high levels of "resistant genotypes" (R1R1, R1R2, and R2R2), which together summed 60 to 100% in Ae. aegypti populations from Rio de Janeiro. CONCLUSIONS: The NaVR1 and NaVR2 kdr alleles confer resistance to the pyrethroid deltamethrin in homozygotes and R1R2 heterozygotes, being the R2R2 most resistant genotype. The allele containing duplication in the NaV gene, with a mutation in the 1011 site, did not confer resistance under the tested conditions. The frequencies of the "resistant genotypes" are elevated in Ae. aegypti natural populations from Rio de Janeiro.

Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations.

BACKGROUND: The chemical control of the mosquito Aedes aegypti, the major vector of dengue, is being seriously threatened due to the development of pyrethroid resistance. Substitutions in the 1016 and 1534 sites of the voltage gated sodium channel (AaNaV), commonly known as kdr mutations, confer the mosquito with knockdown resistance. Our aim was to evaluate the allelic composition of natural populations of Brazilian Ae. aegypti at both kdr sites. METHODS: The AaNaV IIIS6 region was cloned and sequenced from three Brazilian populations. Additionally, individual mosquitoes from 30 populations throughout the country were genotyped for 1016 and 1534 sites, based in allele-specific PCR. For individual genotypes both sites were considered as a single locus. RESULTS: The 350 bp sequence spanning the IIIS6 region of the AaNaV gene revealed the occurrence of the kdr mutation Phe1534Cys in Brazil. Concerning the individual genotyping, beyond the susceptible wild-type (NaVS), two kdr alleles were identified: substitutions restricted to the 1534 position (NaVR1) or simultaneous substitutions in both 1016 and 1534 sites (NaVR2). A clear regional distribution pattern of these alleles was observed. The NaVR1kdr allele occurred in all localities, while NaVR2 was more frequent in the Central and Southeastern localities. Locations that were sampled multiple times in the course of a decade revealed an increase in frequency of the kdr mutations, mainly the double mutant allele NaVR2. Recent samples also indicate that NaVR2 is spreading towards the Northern region. CONCLUSIONS: We have found that in addition to the previously reported Val1016Ile kdr mutation, the Phe1534Cys mutation also occurs in Brazil. Allelic composition at both sites was important to elucidate the actual distribution of kdr mutations throughout the country. Studies to determine gene flow and the fitness costs of these kdr alleles are underway and will be important to better understand the dynamics of Ae. aegypti pyrethroid resistance.

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

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.

Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti.

BACKGROUND AND OBJECTIVES: Mutations in the voltage-gated sodium channel gene (NaV), known as kdr mutations, are associated with pyrethroid and DDT insecticide resistance in a number of species. In the mosquito dengue vector Aedes aegypti, besides kdr, other polymorphisms allowed grouping AaNaV sequences as type 'A' or 'B'. Here, we point a series of evidences that these polymorphisms are actually involved in a gene duplication event. METHODOLOGY: Four series of methods were employed: (i) genotypying, with allele-specific PCR (AS-PCR), of two AaNaV sites that can harbor kdr mutations (Ile1011Met and Val1016Ile), (ii) cloning and sequencing of part of the AaNaV gene, (iii) crosses with specific lineages and analysis of the offspring genotypes and (iv) copy number variation assays, with TaqMan quantitative real-time PCR. RESULTS: kdr mutations in 1011 and 1016 sites were present only in type 'A' sequences, but never in the same haplotype. In addition, although the 1011Met-mutant allele is widely disseminated, no homozygous (1011Met/Met) was detected. Sequencing revealed three distinct haplotypes in some individuals, raising the hypothesis of gene duplication, which was supported by the genotype frequencies in the offspring of specific crosses. Furthermore, it was estimated that a laboratory strain selected for insecticide resistance had 5-fold more copies of the sodium channel gene compared with a susceptible reference strain. CONCLUSIONS AND IMPLICATIONS: The AaNaV duplication here found might be a recent adaptive response to the intense use of insecticides, maintaining together wild-type and mutant alleles in the same organism, conferring resistance and reducing some of its deleterious effects.