Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae

How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S) in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr) to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs.     

Frequency of kdr gene in house fly field populations: correlation of pyrethroid resistance and kdr frequency

The frequency of the L1014 F kdr mutation was determined in 14 field populations of house flies, Musca domestica L., with resistance factors at LD50 for pyrethrin/piperonyl butoxide and bioresmethrin/piperonyl butoxide from 4 to 29 and 2 to 98, respectively. A polymerase chain reaction test for identifying kdr homo- or heterozygote house flies was used to determine the frequency of kdr. The L1014 F allele was found in all populations tested. The frequency of kdr in the field populations was high and varied from 0.46 to 0.99. Eleven of the populations were in Hardy-Weinberg equilibrium, whereas two strains had higher number of heterozygotes than expected, indicating a possible heterozygote advantage. The frequency of kdr was strongly correlated with the reduced mortality observed in the bioassays with pyrethrum and bioresmethrin synergized by piperonyl butoxide. This indicates that kdr is a major mechanism for pyrethroid resistance in these field populations. Five field populations had resistance factors >25 and >10 for bioresmethrin/piperonyl butoxide and pyrethrin/piperonyl butoxide, respectively. The frequencies of kdr in these five populations varied from 0.89 to 0.99. The frequencies of kdr in the field populations showing no or a low level of resistance had frequencies of kdr from 0.46 to 0.75, which indicates that the L1014 F kdr allele is a fully recessive genetic trait in house flies. We have shown that the molecular diagnostic PASA method to determine the resistance phenotypes and the frequency of kdr is a powerful tool, which could be used to get information to make recommendations about pest and resistance management.     

Three years of insecticide resistance monitoring in Anopheles gambiae in Burkina Faso: resistance on the rise?

ABSTRACT: Background and methods A longitudinal Anopheles gambiae s.l. insecticide-resistance monitoring programme was established in four sentinel sites in Burkina Faso. For three years, between 2008 and 2010, WHO diagnostic dose assays were used to measure the prevalence of resistance to all the major classes of insecticides at the beginning and end of the malaria transmission season. Species identification and genotyping for target site mutations was also performed and the sporozoite rate in adults determined. RESULTS: At the onset of the study, resistance to DDT and pyrethroids was already prevalent in An. gambiae s.l. from the south-west of the country but mosquitoes from the two sites in central Burkina Faso were largely susceptible. Within three years, DDT and permethrin resistance was established in all four sites. Carbamate and organophosphate resistance remains relatively rare and largely confined to the south-western areas although a small number of bendiocarb survivors were found in all sites by the final round of monitoring. The ace-1R target site resistance allele was present in all localities and its frequency exceeded 20% in 2010 in two of the sites. The frequency of the 1014 F kdr mutation increased throughout the three years and by 2010, the frequency of 1014 F in all sites combined was 0.02 in Anopheles arabiensis, 0.56 in An. gambiae M form and 0.96 in An. gambiae S form. This frequency did not differ significantly between the sites. The 1014 S kdr allele was only found in An. arabiensis but its frequency increased significantly throughout the study (P = 0.0003) and in 2010 the 1014 S allele frequency was 0.08 in An. arabiensis. Maximum sporozoite rates (12%) were observed in Soumousso in 2009 and the difference between sites is significant for each year. CONCLUSION: Pyrethroid and DDT resistance is now established in An. gambiae s.l. throughout Burkina Faso. Results from diagnostic dose assays are highly variable within and between rounds of testing, and hence it is important that resistance monitoring is carried out on more than one occasion before decisions on insecticide procurement for vector control are made. The presence of 1014 S in An. gambiae s.l., in addition to 1014 F, is not unexpected given the recent report of 1014 S in Benin but highlights the importance of monitoring for both mutations throughout the continent. Future research must now focus on the impact that this resistance is having on malaria control in Burkina Faso.     

Multiple Origins of the Sodium Channel kdr Mutations in Codling Moth Populations

Resistance to insecticides is one interesting example of a rapid current evolutionary change. DNA variability in the voltage-gated sodium channel gene (trans-membrane segments 5 and 6 in domain II) was investigated in order to estimate resistance evolution to pyrethroid in codling moth populations at the World level. DNA variation among 38 sequences revealed a unique kdr mutation (L1014F) involved in pyrethroid resistance in this gene region, which likely resulted from several convergent substitutions. The analysis of codling moth samples from 52 apple orchards in 19 countries using a simple PCR-RFLP confirmed that this kdr mutation is almost worldwide distributed. The proportions of kdr mutation were negatively correlated with the annual temperatures in the sampled regions. Homozygous kdr genotypes in the French apple orchards showed lower P450 cytochrome oxidase activities than other genotypes. The most plausible interpretation of the geographic distribution of kdr in codling moth populations is that it has both multiple independent origins and a spreading limited by low temperature and negative interaction with the presence of alternative resistance mechanisms to pyrethroid in the populations.     

Distribution of a knockdown resistance mutation (L1014S) in Anopheles gambiae s.s. and Anopheles arabiensis in western and southern Kenya

In Kenya, insecticide-treated mosquito nets (ITNs) distributed to pregnant women and children under 5 years old through various programs have resulted in a significant reduction in malaria deaths. All of the World Health Organization-recommended insecticides for mosquito nets are pyrethroids, and vector mosquito resistance to these insecticides is one of the major obstacles to an effective malaria control program. Anopheles gambiae s.s. and Anopheles arabiensis are major malaria vectors that are widely distributed in Kenya. Two point mutations in the voltage-gated sodium channel (L1014F and L1014S) are associated with knockdown resistance (kdr) to DDT and pyrethroids in An. gambiae s.s. While the same point mutations have been reported to be rare in An. arabiensis, some evidence of metabolic resistance has been reported in this species. In order to determine the distribution of the point mutation L1014S in An. gambiae s.s. and An. arabiensis in southern and western Kenya, we collected larvae and screened for the mutation by DNA sequencing. We found high allelic and homozygous frequencies of the L1014S mutation in An. gambiae s.s. The L1014S mutation was also widely distributed in An. arabiensis, although the allelic frequency was lower than in An. gambiae s.s. The same intron sequence (length: 57 base) found in both species indicated that the mutation was introgressed by hybridization. The allelic frequency of L1014S was higher in both species in western regions, demonstrating the strong selection pressure imposed by long-lasting insecticide-treated nets (LLITN)/ITN on the An. gambiae s.s. and An. arabiensis populations in those areas. The present contribution of the L1014S mutation to pyrethroid resistance in An. arabiensis may be negligible. However, the homozygous frequency could increase with continuing selection pressure due to expanded LLITN coverage in the future.     

Mutations in the house fly Vssc1 sodium channel gene associated with super-kdr resistance abolish the pyrethroid sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes

The super-kdr insecticide resistance trait of the house fly confers resistance to pyrethroids and DDT by reducing the sensitivity of the fly nervous system. The super-kdr genetic locus is tightly linked to the Vssc1 gene, which encodes a voltage-sensitive sodium channel alpha subunit that is the principal site of pyrethroid action. DNA sequence analysis of Vssc1 alleles from several independent super-kdr fly strains identified two amino acid substitutions associated with the super-kdr trait: replacement of leucine at position 1014 with phenylalanine (L1014F), which has been shown to cause the kdr resistance trait in this species, and replacement of methionine at position 918 with threonine (M918T). We examined the functional significance of these mutations by expressing house fly sodium channels containing them in Xenopus laevis oocytes and by characterizing the biophysical properties and pyrethroid sensitivities of the expressed channels using two-electrode voltage clamp. House fly sodium channels that were specifically modified by site-directed mutagenesis to contain the M918T/L1014F double mutation gave reduced levels of sodium current expression in oocytes but otherwise exhibited functional properties similar to those of wildtype channels and channels containing the L1014F substitution. However, M918T/L1014F channels were completely insensitive to high concentrations of the pyrethroids cismethrin and cypermethrin. House fly sodium channels specifically modified to contain the M918T single mutation, which is not known to exist in nature except in association with the L1014F mutation, gave very small sodium currents in oocytes. Assays of these currents in the presence of high concentrations of cismethrin suggest that this mutation alone is sufficient to abolish the pyrethroid sensitivity of house fly sodium channels. These results define the functional significance of the Vssc1 mutations associated with the super-kdr trait of the house fly and are consistent with the hypothesis that the super-kdr trait arose by selection of a second-site mutation (M918T) that confers to flies possessing it even greater resistance than the kdr allele containing the L1014F mutation.     

First report of L1014F kdr mutation in Culex quinquefasciatus in Mexico

The L1014F mutation in the voltage‐sodium channel gene has been associated with resistance to DDT and pyrethroids in various arthropod species including mosquitoes. We determined the frequency of the L1014F kdr mutation in 16 field populations of Culex quinquefasciatus from Northeastern Mexico collected between 2008 and 2013. The L1014F was present in all populations analyzed with the lowest frequency (3.33%) corresponding to the population from Monclova collected in 2012, and the highest frequency (63.63%) from the Monterrey population collected in 2012. The presence of a kdr mutation in populations of Cx. quinquefasciatus from northeastern Mexico provides evidence of pyrethroid resistance. This requires a special attention, considering that pyrethroid‐based insecticides are commonly used in vector‐control campaigns, especially against Aedes aegypti (L.).     

Polymorphism of intron-1 in the voltage-gated sodium channel gene of Anopheles gambiae s.s. populations from Cameroon with emphasis on insecticide knockdown resistance mutations

Sequence variation at the intron-1 of the voltage-gated sodium channel gene in Anopheles gambiae M- and S-forms from Cameroon was assessed to explore the number of mutational events originating knockdown resistance ( kdr ) alleles. Mosquitoes were sampled between December 2005 and June 2006 from three geographical areas: (i) Magba in the western region; (ii) Loum, Tiko, Douala, Kribi, and Campo along the Atlantic coast; and (iii) Bertoua, in the eastern continental plateau. Both 1014S and 1014F kdr alleles were found in the S-form with overall frequencies of 14% and 42% respectively. Only the 1014F allele was found in the M-form at lower frequency (11%). Analysis of a 455 bp region of intron-1 upstream the kdr locus revealed four independent mutation events originating kdr alleles, here named MS1 -1014F, S1-1014S and S2-1014S kdr- intron-1 haplotypes in S-form and MS3-1014F kdr- intron-1 haplotype in the M-form. Furthermore, there was evidence for mutual introgression of kdr 1014F allele between the two molecular forms, MS1 and MS3 being widely shared by them. Although no M/S hybrid was observed in analysed samples, this wide distribution of haplotypes MS1 and MS3 suggests inter-form hybridizing at significant level and emphasizes the rapid diffusion of the kdr alleles in Africa. The mosaic of genetic events found in Cameroon is representative of the situation in the West–Central African region and highlights the importance of evaluating the spatial and temporal evolution of kdr alleles for a better management of insecticide resistance.     

Widespread pyrethroid resistance in Australian diamondback moth, Plutella xylostella (L.), is related to multiple mutations in the para sodium channel gene

Populations of Plutella xylostella, extending over 3800 km in southern Australia, show no genetic structure as assessed by microsatellite markers; yet outbreaks of pyrethroid resistance occur sporadically in cropping areas. Since mutations in the para voltage-gated sodium channel gene have been implicated in pyrethroid resistance, we looked for DNA sequence variation at this target among Australian moths. We found two resistance mutations previously reported for this species (L1014F and T929I), as well as a novel substitution (F1020S). Of the eight possible haplotypes formed by combinations of these three biallelic polymorphisms, only four were found in Australian populations: the wild-type allele (w), the kdr mutation allele (kdr) with only L1014F, the super-kdr-like combination of L1014F and T929I (skdrl), and the crashdown allele with only F1020S (cdr). Comparison of genotype frequencies among survivors of permethrin assays with those from untreated controls identified three resistant genotypes: skdrl homozygotes, cdr homozygotes and the corresponding heterozygote, cdr/skrdl - the heterozygote being at least as resistant as either homozygote. Spatial heterogeneity of allele frequencies was conspicuous, both across the continent and among local collections, consistent with reported spatial heterogeneity of pyrethroid resistance. Further, high resistance samples were sometimes associated with high frequency of cdr, sometimes high frequency of skdrl, or sometimes with a high combined cdr+skdrl frequency. The skdrl and cdr alleles explain a high proportion of the Australia-wide resistance variation. These data add to evidence that nerve insensitivity by mutations in the para-sodium channel gene is a common pyrethroid resistance mechanism in P. xylostella.     

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.     

Resistance to DDT and pyrethroids and increased kdr mutation frequency in An. gambiae after the implementation of permethrin-treated nets in Senegal

IntroductionThe aim of this study was to evaluate the susceptibility to insecticides of An. gambiae mosquitoes sampled in Dielmo (Senegal), in 2010, 2 years after the implementation of Long Lasting Insecticide-treated Nets (LLINs) and to report the evolution of kdr mutation frequency from 2006 to 2010.MethodsWHO bioassay susceptibility tests to 6 insecticides were performed on adults F0, issuing from immature stages of An. gambiae s.l., sampled in August 2010. Species and molecular forms as well as the presence of L1014F and L1014S kdr mutations were assessed by PCR. Longitudinal study of kdr mutations was performed on adult mosquitoes sampled monthly by night landing catches from 2006 to 2010.FindingsNo specimen studied presented the L1014S mutation. During the longitudinal study, L1014F allelic frequency rose from 2.4% in year before the implementation of LLINs to 4.6% 0–12 months after and 18.7% 13–30 months after. In 2010, An. gambiae were resistant to DDT, Lambda-cyhalothrin, Deltamethrin and Permethrin (mortality rates ranging from 46 to 63%) but highly susceptible to Fenitrothion and Bendiocarb (100% mortality). There was significantly more RR genotype among An. gambiae surviving exposure to DDT or Pyrethroids. An. arabiensis represented 3.7% of the sampled mosquitoes (11/300) with no kdr resistance allele detected. An. gambiae molecular form M represented 29.7% of the mosquitoes with, among them, kdr genotypes SR (18%) and SS (82%). An. gambiae molecular form S represented 66% of the population with, among them, kdr genotype SS (33.3%), SR (55.6%) and RR (11.1%). Only 2 MS hybrid mosquitoes were sampled and presented SS kdr genotype.ConclusionBiological evidence of resistance to DDT and pyrethroids was detected among An. gambiae mosquitoes in Dielmo (Senegal) within 24 months of community use of LLINs. Molecular identification of L1014F mutation indicated that target site resistance increased after the implementation of LLINs.     

Occurrence of L1014F and L1014S mutations in insecticide resistant Culex quinquefasciatus from filariasis endemic districts of West Bengal,India

IntroductionLymphatic filariasis causes long term morbidity and hampers the socio-economic status. Apart from the available treatments and medication, control of vector population Culex quinquefasciatus Say through the use of chemical insecticides is a widely applied strategy. However, the unrestrained application of these insecticides over many decades has led to resistance development in the vectors.MethodsIn order to determine the insecticide susceptibility/resistance status of Cx. quinquefasciatus from two filariasis endemic districts of West Bengal, India, wild mosquito populations were collected and assayed against six different insecticides and presence of L1014F; L1014S kdr mutations in the voltage-gated sodium channel gene was also screened along with the use of synergists to evaluate the role of major detoxifying enzymes in resistance development.ResultsThe collected mosquito populations showed severe resistance to insecticides and the two synergists used–PBO (piperonyl butoxide) and TPP (triphenyl phosphate), were unable to restore the susceptibility status of the vector thereupon pointing towards a minor role of metabolic enzymes. kdr mutations were present in the studied populations in varying percent with higher L1014F frequency indicating its association with the observed resistance to pyrethroids and DDT. This study reports L1014S mutation in Cx. quinquefasciatus for the first time.     

Temporal and spatial incidence of alleles conferring knockdown resistance to pyrethroids in the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae), and their association with other insecticide resistance mechanisms

The peach-potato aphid, Myzus persicae (sulzer), is an important arable pest species throughout the world. Extensive use of insecticides has led to the selection of resistance to most chemical classes including organochlorines, organophosphates, carbamates and pyrethroids. Resistance to pyrethroids is often the result of mutations in the para-type sodium channel protein (knockdown resistance or kdr). In M. persicae, knockdown resistance is associated with two amino-acid substitutions, L1014F (kdr) and M918T (super-kdr). In this study, the temporal and spatial distributions of these mutations, diagnosed using an allelic discriminating polymerase chain reaction assay, were investigated alongside other resistance mechanisms (modified acetylcholinesterase (MACE) and elevated carboxylesterases). Samples were collected from the UK, mainland Europe, Zimbabwe and south-eastern Australia. The kdr mutation and elevated carboxylesterases were widely distributed and recorded from nearly every country. MACE and super-kdr were widespread in Europe but absent from Australian samples. The detection of a strongly significant heterozygote excess for both kdr and super-kdr throughout implies strong selection against individuals homozygous for these resistance mutations. The pattern of distribution found in the UK seemed to indicate strong selection against the super-kdr (but not the kdr) mutation in any genotype, in the absence of insecticide pressure. There was a significant association (linkage disequilibrium) between different resistance mechanisms, which was probably promoted by a lack of recombination due to parthenogenesis.     

Detection and widespread distribution of sodium channel alleles characteristic of insecticide resistance in Culex pipiens complex mosquitoes in China

Culex pipiens complex mosquitoes are widely distributed throughout China and are known to be important disease vectors. Two pyrethroid resistance associated mutations have been identified in Cx. pipiens complex (Diptera: Culicidae), but there is little information on the diversity and distribution of kdr alleles in pyrethroid resistance in Cx. pipiens complex mosquitoes in China. In the present study, we report on a modified three tube allele-specific (AS)-PCR method for detecting the 1014F and 1014S alleles. The new technique was applied to identify the distribution of the two alleles in natural Cx. pipiens complex populations in China. The results confirmed that the new method is both sensitive and specific. The 1014F allele was found in all 14 of the field populations tested (frequency ranged from 6.8 to 76.2%) and the 1014S allele was found in almost two-thirds (frequency from 2.4 to 28.6%), indicating that the genotypes known to be associated with pyrethroid resistance are widespread in China. The resistance-associated alleles were more common in southern Chinese sampling sites than in northern sites. The coexistence of the two resistant mutations in individual mosquitoes was also observed in five of the field populations. Two alternative mutations within the L1014 codon were identified in Culex pipiens molestus Forskal, 1775, including a non-synonymous mutation resulting in a 1014C substitution.     

Distribution and Frequency of kdr Mutations within Anopheles gambiae s.l. Populations and First Report of the Ace.1G119S Mutation in Anopheles arabiensis from Burkina Faso (West Africa)

An entomological survey was carried out at 15 sites dispersed throughout the three eco-climatic regions of Burkina Faso (West Africa) in order to assess the current distribution and frequency of mutations that confer resistance to insecticides in An. gambiae s.l. populations in the country. Both knockdown (kdr) resistance mutation variants (L1014F and L1014S), that confer resistance to pyrethroid insecticides, were identified concomitant with the ace-1 G119S mutation confirming the presence of multiple resistance mechanisms in the An. gambiae complex in Burkina Faso. Compared to the last survey, the frequency of the L1014F kdr mutation appears to have remained largely stable and relatively high in all species. In contrast, the distribution and frequency of the L1014S mutation has increased significantly in An. gambiae s.l. across much of the country. Furthermore we report, for the first time, the identification of the ace.1 G116S mutation in An. arabiensis populations collected at 8 sites. This mutation, which confers resistance to organophosphate and carbamate insecticides, has been reported previously only in the An. gambiae S and M molecular forms. This finding is significant as organophosphates and carbamates are used in indoor residual sprays (IRS) to control malaria vectors as complementary strategies to the use of pyrethroid impregnated bednets. The occurrence of the three target-site resistance mutations in both An. gambiae molecular forms and now An. arabiensis has significant implications for the control of malaria vector populations in Burkina Faso and for resistance management strategies based on the rotation of insecticides with different modes of action.     

Insecticide resistance in Culex quinquefasciatus mosquitoes after the introduction of insecticide-treated bed nets in Macha, Zambia

Culex quinquefasciatus, an arboviral and filarial vector, is present in high numbers throughout sub-Saharan Africa, and insecticide-resistant populations have been reported worldwide. In order to determine the insecticide resistance status of Cx. quinquefasciatus in Macha, Zambia, adult mosquitoes reared from eggs collected from oviposition traps were tested by bioassay. High levels of resistance to DDT, pyrethroids, malathion, and deltamethrin-treated net material were detected, and molecular assays revealed that the knockdown resistance (kdr) allele was frequent in the Cx. quinquefasciatus population, with 7.0% homozygous for the kdr L1014 allele and 38.5% heterozygous (0.263 kdr frequency). The kdr frequency was significantly higher in mosquitoes that had successfully fed on human hosts, and screening archived specimens revealed that kdr was present at lower frequency prior to the introduction of ITNs, indicating that ITNs might be a selective force in this population. Additionally, metabolic detoxification enzyme activity assays showed upregulated glutathione S-transferases, α-esterases, and β-esterases. Continued monitoring and assessment of the Cx. quinquefasciatus population is necessary to determine levels of resistance.     

How the Malaria Vector Anopheles gambiae Adapts to the Use of Insecticide-Treated Nets by African Populations

BackgroundInsecticide treated bed nets have been recommended and proven efficient as a measure to protect African populations from malaria mosquito vector Anopheles spp. This study evaluates the consequences of bed nets use on vectors resistance to insecticides, their feeding behavior and malaria transmission in Dielmo village, Senegal, were LLINs were offered to all villagers in July 2008.MethodsAdult mosquitoes were collected monthly from January 2006 to December 2011 by human landing catches (HLC) and by pyrethroid spray catches (PCS). A randomly selected sub-sample of 15–20% of An. gambiae s.l. collected each month was used to investigate the molecular forms of the An. gambiae complex, kdr mutations, and Plasmodium falciparum circumsporozoite (CSP) rate. Malaria prevalence and gametocytaemia in Dielmo villagers were measured quarterly.ResultsInsecticide susceptible mosquitoes (wild kdr genotype) presented a reduced lifespan after LLINs implementation but they rapidly adapted their feeding behavior, becoming more exophageous and zoophilic, and biting earlier during the night. In the meantime, insecticide-resistant specimens (kdr L1014F genotype) increased in frequency in the population, with an unchanged lifespan and feeding behaviour. P. falciparum prevalence and gametocyte rate in villagers decreased dramatically after LLINs deployment. Malaria infection rate tended to zero in susceptible mosquitoes whereas the infection rate increased markedly in the kdr homozygote mosquitoes.ConclusionDramatic changes in vector populations and their behavior occurred after the deployment of LLINs due to the extraordinary adaptative skills of An. gambiae s. l. mosquitoes. However, despite the increasing proportion of insecticide resistant mosquitoes and their almost exclusive responsibility in malaria transmission, the P. falciparum gametocyte reservoir continued to decrease three years after the deployment of LLINs.     

Insecticide exposure impacts vector–parasite interactions in insecticide-resistant malaria vectors

Currently, there is a strong trend towards increasing insecticide-based vector control coverage in malaria endemic countries. The ecological consequence of insecticide applications has been mainly studied regarding the selection of resistance mechanisms; however, little is known about their impact on vector competence in mosquitoes responsible for malaria transmission. As they have limited toxicity to mosquitoes owing to the selection of resistance mechanisms, insecticides may also interact with pathogens developing in mosquitoes. In this study, we explored the impact of insecticide exposure on Plasmodium falciparum development in insecticide-resistant colonies of Anopheles gambiae s.s., homozygous for the ace-1 G119S mutation (Acerkis) or the kdr L1014F mutation (Kdrkis). Exposure to bendiocarb insecticide reduced the prevalence and intensity of P. falciparum oocysts developing in the infected midgut of the Acerkis strain, whereas exposure to dichlorodiphenyltrichloroethane reduced only the prevalence of P. falciparum infection in the Kdrkis strain. Thus, insecticide resistance leads to a selective pressure of insecticides on Plasmodium parasites, providing, to our knowledge, the first evidence of genotype by environment interactions on vector competence in a natural Anopheles–Plasmodium combination. Insecticide applications would affect the transmission of malaria in spite of resistance and would reduce to some degree the impact of insecticide resistance on malaria control interventions.     

Multiple Resistances and Complex Mechanisms of Anopheles sinensis Mosquito: A Major Obstacle to Mosquito-Borne Diseases Control and Elimination in China

Malaria, dengue fever, and filariasis are three of the most common mosquito-borne diseases worldwide. Malaria and lymphatic filariasis can occur as concomitant human infections while also sharing common mosquito vectors. The overall prevalence and health significance of malaria and filariasis have made them top priorities for global elimination and control programmes. Pyrethroid resistance in anopheline mosquito vectors represents a highly significant problem to malaria control worldwide. Several methods have been proposed to mitigate insecticide resistance, including rotational use of insecticides with different modes of action. Anopheles sinensis, an important malaria and filariasis vector in Southeast Asia, represents an interesting mosquito species for examining the consequences of long-term insecticide rotation use on resistance. We examined insecticide resistance in two An. Sinensis populations from central and southern China against pyrethroids, organochlorines, organophosphates, and carbamates, which are the major classes of insecticides recommended for indoor residual spray. We found that the mosquito populations were highly resistant to the four classes of insecticides. High frequency of kdr mutation was revealed in the central population, whereas no kdr mutation was detected in the southern population. The frequency of G119S mutation in the ace-1 gene was moderate in both populations. The classification and regression trees (CART) statistical analysis found that metabolic detoxification was the most important resistance mechanism, whereas target site insensitivity of L1014 kdr mutation played a less important role. Our results indicate that metabolic detoxification was the dominant mechanism of resistance compared to target site insensitivity, and suggests that long-term rotational use of various insecticides has led An. sinensis to evolve a high insecticide resistance. This study highlights the complex network of mechanisms conferring multiple resistances to chemical insecticides in mosquito vectors and it has important implication for designing and implementing vector resistance management strategies.     

Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT.