Reversing insecticide resistance with allelic-drive in Drosophila melanogaster

A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.

Analysis of the Knockdown Resistance Locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. for Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia

The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

Insecticide susceptibility status and knockdown resistance (kdr) mutation in Aedes albopictus in China

Background Aedes (Stegomyia) albopictus (Skuse, 1894) is the main vector of dengue virus in China. The resistance to insecticides is a huge obstacle for the control of this species, and determining its resistance status and mechanisms in China is essential for the implementation of vector management strategies. Methods We have investigated the larval and adult resistance status of Ae. albopictus to deltamethrin in eight field populations in China. Mutations at the voltage-gated sodium channel gene, related to the knockdown resistance (kdr) effect, were detected by sequencing of PCR products. The eight field populations were examined for pyrethroid resistance using the World Health Organization standard bioassays, and the association between the mutations and phenotypic resistance was tested. Results The eight field populations of larvae of Ae. albopictus in China exhibited high resistance to deltamethrin; the RR50 values ranged from 12 (ZJ) to 44 (GZ). Adult bioassay revealed that Ae. albopictus populations were resistant to deltamethrin (mortality rate < 90%), except ZJ population (probably resistant, mortality rate = 93.5%). Long knockdown time in the field populations was consistent with low mortality rates in adult bioassay. F1534S mutation showed increased protection against deltamethrin in all populations except BJ and SJZ populations, whereas I1532T mutation showed increased protection against deltamethrin in only BJ population. Conclusion There were different degrees of resistance to deltamethrin in field Ae. albopictus populations in China. The longest knockdown time and lowest mortality rate observed in Ae. albopictus population in Guangzhou indicate the severity of high resistance to deltamethrin. The patchy distribution of deltamethrin resistance and kdr mutations in Ae. albopictus mosquitoes suggests the necessity for resistance management and developing counter measures to mitigate the spread of resistance. Graphical Abstract

Putative pleiotropic effects of the knockdown resistance (L1014F) allele on the life-history traits of Anopheles gambiae

Background Existing mechanisms of insecticide resistance are known to help the survival of mosquitoes following contact with chemical compounds, even though they could negatively affect the life-history traits of resistant malaria vectors. In West Africa, the knockdown resistance mechanism kdrR (L1014F) is the most common. However, little knowledge is available on its effects on mosquito life-history traits. The fitness effects associated with this knockdown resistance allele in Anopheles gambiae sensu stricto (s.s.) were investigated in an insecticide-free laboratory environment. Methods The life-history traits of Kisumu (susceptible) and KisKdr (kdr resistant) strains of An. gambiae s.s. were compared. Larval survivorship and pupation rate were assessed as well as fecundity and fertility of adult females. Female mosquitoes of both strains were directly blood fed through artificial membrane assays and then the blood-feeding success, blood volume and adult survivorship post-blood meal were assessed. Results The An. gambiae mosquitoes carrying the kdrR allele (KisKdr) laid a reduced number of eggs. The mean number of larvae in the susceptible strain Kisumu was three-fold overall higher than that seen in the KisKdr strain with a significant difference in hatching rates (81.89% in Kisumu vs 72.89% in KisKdr). The KisKdr larvae had a significant higher survivorship than that of Kisumu. The blood-feeding success was significantly higher in the resistant mosquitoes (84%) compared to the susceptible ones (34.75%). However, the mean blood volume was 1.36 µL/mg, 1.45 µL/mg and 1.68 µL/mg in Kisumu, homozygote and heterozygote KisKdr mosquitoes, respectively. After blood-feeding, the heterozygote KisKdr mosquitoes displayed highest survivorship when compared to that of Kisumu. Conclusions The presence of the knockdown resistance allele appears to impact the life-history traits, such as fecundity, fertility, larval survivorship, and blood-feeding behaviour in An. gambiae. These data could help to guide the implementation of more reliable strategies for the control of malaria vectors.

Detection of Knockdown-Resistance Mutations (V1016G and F1534C) in Dengue Vector from Urban Park, Surabaya, Indonesia

An urban park is potentially a source of vector-borne disease transmission due to it being a natural and artificial mosquito breeding habitats combined with people's continuous presence. Thus, this study aims to screen the occurrence of knockdown-resistance (kdr) mutant alleles (V1016G and F1534C) in mosquito populations collected from urban parks in Surabaya, Indonesia. Cross sectional study was conducted in July 2019. A total of 28 ovitraps were installed in seven urban parks, having four ovitraps installed in each park. In total, 1,662 eggs were collected, and only 187 emerged into adult mosquitoes, consisting of 97 Aedes (Stegomyia) aegypti and 90 Aedes (Stegomyia) albopictus. All-female adult mosquitoes (n=55) were tested using allele-specific polymerase chain reaction assay (AS-PCR) to detect voltage gated sodium channel (VGSC) gene mutations. This study found no mutations in Valine to Glysine mutation in point 1016 (V1016G) and Phenylalanine to Cysteine in point 1534 (F1534C) alleles in both two species. All of mosquito samples have wild type genotype of kdr alleles (V1016V and F1534F). Data were analysed using R Studio 1.4 Version by Genetics package. Results showed that the frequency of resistant alleles (G1016 and C1534) was zero, and the frequency of susceptible allele was 1 (V1016 and F1534). Insecticide bioassay could not be established due to the limited number of adult mosquitoes, so insecticide resistance status could not be determined. However, this study can be used as preliminary monitoring for the vector control program.

Levels of cross-resistance to pyrethroids conferred by the Vssc knockdown resistance allele 410L+1016I+1534C in Aedes aegypti

Aedes aegypti is a primary vector of viral pathogens and is responsible for millions of human infections annually that represent critical public health and economic costs. Pyrethroids are one of the most commonly used classes of insecticides to control adult A. aegypti. The insecticidal activity of pyrethroids depends on their ability to bind and disrupt the voltage-sensitive sodium channel (VSSC). In mosquitoes, a common mechanism of resistance to pyrethroids is due to mutations in Vssc (hereafter referred as knockdown resistance, kdr). In this study, we found that a kdr (410L+V1016I+1534C) allele was the main mechanism of resistance in a pyrethroid-resistant strain of A. aegypti collected in Colombia. To characterize the level of resistance these mutations confer, we isolated a pyrethroid resistant strain (LMRKDR:RK, LKR) that was congenic to the susceptible Rockefeller (ROCK) strain. The full-length cDNA of Vssc was cloned from LKR and no additional resistance mutations were present. The levels of resistance to different pyrethroids varied from 3.9- to 56-fold. We compared the levels of resistance to pyrethroids, DCJW and DDT between LKR and what was previously reported in two other congenic strains that share the same pyrethroid-susceptible background (the ROCK strain), but carry different kdr alleles (F1534C or S989P + V1016G). The resistance conferred by kdr alleles can vary depending on the stereochemistry of the pyrethroid. The 410L+1016I+1534C kdr allele does not confer higher levels of resistance to six of ten pyrethroids, relative to the 1534C allele. The importance of these results to understand the evolution of insecticide resistance and mosquito control are discussed.

Knockdown Resistance (kdr) Mutations I1532T and F1534S Were Identified in Aedes albopictus Field Populations in Zhejiang Province, Central China

Aedes albopictus is the only vector that can transmit the dengue virus in Zhejiang Province, central China, and it can develop insecticide resistance due to long-term exposure to pyrethroids. The presence of knockdown resistance (kdr) mutations is one of the mechanisms responsible for pyrethroid resistance, and has been reported in some Ae. albopictus populations in southern China. However, little is known about the DNA diversity of the voltage-gated sodium channel (VGSC) gene in Ae. albopictus populations in central China. Four Ae. albopictus field populations were collected, in Yiwu (YW), Quzhou (QZ), Wenzhou (WZ), and Jiaxing (JX) from Zhejiang Province, central China. The susceptibility of Ae. albopictus adults to three pyrethroids (beta-cypermethrin, deltamethrin, and permethrin) was tested using the WHO tube assay, and Kdr mutations were identified via PCR and sequencing. The relationship between kdr mutations and pyrethroid phenotypes was also analyzed. Of the four populations, none was sensitive to any pyrethroid tested, and the YW population showed the strongest pyrethroid resistance. Non-synonymous kdr mutations were detected in codons 1532 and 1534, domain III. At codon 1534, one mutant allele, TCC(S), was detected in the four populations with a frequency of 42.08%, while at codon 1532, one mutant allele, ACC(T), was detected in the JX and QZ populations, with frequencies of 4.22 and 3.03%, respectively. The F1534S mutant allele was positively correlated with both beta-cypermethrin and deltamethrin resistance phenotypes (OR &gt; 1, P &lt; 0.05), whereas the I1532T mutant allele was possibly negatively correlated with beta-cypermethrin, deltamethrin, and permethrin resistance phenotypes (OR &lt; 1, P &gt; 0.05). In conclusion, resistance and resistance mutations regarding to three pyrethroids are already present in the Ae. Albopictus populations from Zhejiang, central China, which prompts the need to use non-insecticide-based methods of insect control.

Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance, recovery, and death

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site—the voltage-gated sodium channel (VGSC)—and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.