Kdr Frequency Research Articles

Knockdown resistance in Stomoxys calcitrans stable fly populations on German dairy farms: kdr alleles explain susceptibility of individual flies to deltamethrin

AbstractThe stable fly, Stomoxys calcitrans, is a globally important pest causing stress, economic losses and transmission of pathogens in livestock. Control on commercial farms relies predominantly on the use of insecticides, with pyrethroids being the most frequently used class of insecticides in industrialised countries. Here, laboratory isolates were obtained from four dairy farms in Brandenburg (Germany) and tested for phenotypic resistance to deltamethrin in comparison to a susceptible reference isolate using topical application. Individual flies were subsequently genotyped using allele-specific real-time PCRs. Phenotypic resistance was observed in all four field isolates with resistance ratios between 46 and 119 compared to the susceptible laboratory strain. At position 1014 of the voltage-sensitive sodium channel, allele-specific PCRs detected the wild-type, kdr-his and kdr genotypes encoding leucine, histidine and phenylalanine, respectively. In the susceptible laboratory isolate, only the wild-type was identified. On the farms with very high LD50 values, the kdr variant was most prevalent and logistic regression analysis revealed that the kdr variant increased the odds to survive exposure to deltamethrin more than the kdr-his genotype. Flies carrying two resistance alleles were less susceptible than flies that also carried one wild-type allele. In three out of four field isolates, the allele frequencies were significantly different from the expectations of the Hardy–Weinberg equilibrium suggesting ongoing selection. The data show that the phenotype can be largely explained by the kdr genotype and represent high frequencies of the L1014F kdr and L1014H kdr-his variants conferring high levels of resistance in northern Germany.

Insecticide Resistance and Target-Site Mutations kdr, N1575Y, and Ace-1 in Anopheles gambiae s.l. Populations in a Low-Malaria-Transmission Zone in the Sudanian Region of Senegal.

Background/Objectives: Significant progress in malaria control has been achieved through long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), raising hopes for malaria elimination. However, emerging insecticide resistance threatens these gains. This study assessed the susceptibility of Anopheles gambiae s.l. populations to public health insecticides, examined the frequencies of kdr, Ace-1, and N1575Y mutations, and explored their associations with phenotypic resistance in Dielmo and Ndiop, Senegal. Methods: Anopheles larvae collected between September and December 2022 were reared to adulthood. Adult mosquitoes were exposed to discriminating concentrations of various insecticides following WHO guidelines. Knockdown times (KDT50 and KDT95) for pyrethroids were calculated using the Probit model. RT-qPCR detected target-site mutations (kdr: L1014F and L1014S, Ace-1, N1575Y) and assessed correlations with phenotypic resistance. Species-specific PCR identified species within the An. gambiae complex. Results/Conclusions: The populations of Dielmo and Ndiop showed susceptibility to pirimiphos-methyl and bendiocarb, with no Ace-1 mutation detected. Resistance to DDT and pyrethroids was observed. The knockdown times indicated that alphacypermethrin and lambdacyhalothrin were more effective than permethrin and deltamethrin. The L1014F allele was widespread, while L1014S was absent in Ndiop and low in Dielmo. The N1575Y mutation occurred only in populations with L1014F. The L1014S mutation was significantly associated with resistance to lambdacyhalothrin in both villages and to deltamethrin in Ndiop.

First detection of kdr L1014F allele in Anopheles ziemanni and Anopheles pharoensis in Cameroon and distribution of the allele in members of the Anopheles gambiae complex

BackgroundKnockdown resistance (kdr) is one of the primary resistance mechanisms present in anopheline species. Although this mutation is largely spread across the Anopheles gambiae s.l. members, its prevalence in other species is still not well documented.MethodsThe present study investigated the distribution and allelic frequencies of kdr in An. gambiae s.l., An. pharoensis, and An. ziemanni samples collected in 2022 and 2023 in nine sites spread across five ecogeographical settings in Cameroon. Members of the An. gambiae complex were identified molecularly by polymerase chain reaction (PCR). kdr L1014F and L1014S alleles were screened by PCR and confirmed by sequencing.ResultsAn. gambiae (49.9%), An. coluzzii (36.5%), and An. arabiensis (13%) were identified, and the frequency of the kdr L1014F was high in both An. gambiae and An. coluzzii in all sites. The kdr L1014F allele was detected for the first time in 8 out of 14 An. ziemanni samples examined and in 5 out of 22 An. pharoensis samples examined. The kdr L1014S allele was scarce and found only in the heterozygote “RS” state in An. arabiensis and An. gambiae in Yangah and Santchou.ConclusionsThe present study sheds light on the rapid expansion of the kdr L1014F allele in malaria vectors in Cameroon and stresses the need for surveillance activities also targeting secondary malaria vectors to improve the control of malaria transmission.Graphical

Mutations and intron polymorphisms in voltage-gated sodium channel genes of different geographic populations of Culex pipiens pallens/Culex pipiens quinquefasciatus in China

BackgroundCulex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors. Long-term use of insecticides can cause mutations in the voltage-gated sodium channel (vgsc) gene of mosquitoes, but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations. Therefore, this study aimed to determine the current status of vgsc genes in Cx. p. pallens and Cx. p. quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution.MethodsSampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China. Genomic DNA was extracted from 1308 mosquitoes, the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction, and the sequence was used to evaluate allele frequency and knockdown resistance (kdr) frequency. MEGA 11 was used to construct neighbor-joining (NJ) tree. PopART was used to build a TCS network.ResultsThere were 6 alleles and 6 genotypes at the L1014 locus, which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F, TTC/F, TCT/S and TCA/S. The geographic populations with a kdr frequency less than 20.00% were mainly concentrated in the regions north of 38° N, and the geographic populations with a kdr frequency greater than 80.00% were concentrated in the regions south of 30° N. kdr frequency increased with decreasing latitude. And within the same latitude, the frequency of kdr in large cities is relatively high. Mutations were correlated with the number of introns. The mutant allele TCA/S has only one intron, the mutant allele TTT/F has three introns, and the wild-type allele TTA/L has 17 introns.ConclusionsCx. p. pallens and Cx. p. quinquefasciatus have developed resistance to insecticides in most regions of China. The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene. Mutations may originate from multiple mutation events rather than from a single origin, and populations lacking mutations may be genetically isolated.Graphical

The frequency of kdr and ace-1 alleles in Anopheles gambiae s.l. before and during indoor residual spraying (IRS) implementation and four years after IRS withdrawal in three districts in Atacora, Benin

BackgroundIndoor residual spraying (IRS) was first implemented in the Atacora department, Benin from 2011 to 2012 using bendiocarb (carbamate) followed by annual spraying with pirimiphos-methyl (organophosphate) from 2013 to 2018. Before and after IRS implementation in Atacora, standard pyrethroid insecticide-treated bed nets were the main method of vector control in the area. This study investigated the knockdown resistance (kdr) gene (L1014F) and the acetylcholinesterase (ace-1) gene (G119S), before and during IRS implementation, and 4-years after IRS withdrawal from Atacora. This was done to assess how changes in insecticide pressure from indoor residual spraying may have altered the genotypic resistance profile of Anopheles gambiae s.l.MethodIdentification of sibling species of An. gambiae s.l. and detection of the L1014F mutation in the kdr gene and G119S mutation in ace-1 genes was done using molecular analysis. Allelic and genotypic frequencies were calculated and compared with each other before and during IRS implementation and 4 years after IRS withdrawal. The Hardy–Weinberg equilibrium and genetic differentiation within and between populations were assessed.ResultsPrevalence of the L1014F mutation in all geographic An. gambiae s.l. (An. gambiae s.s., Anopheles. coluzzii, Anopheles. arabiensis, and hybrids of “An. gambiae s.s. and An. coluzzii”) populations increased from 69% before IRS to 87% and 90% during and after IRS. The G119S allele frequency during IRS (20%) was significantly higher than before IRS implementation (2%). Four years after IRS withdrawal, allele frequencies returned to similar levels as before IRS (3%). Four years after IRS withdrawal, the populations showed excess heterozygosity at the ace-1 gene and deficit heterozygosity at the kdr gene, whereas both genes had excess heterozygosity before and during IRS (FIS < 0). No genetic differentiation was observed within the populations.ConclusionsThis study shows that the withdrawal of IRS with bendiocarb and pirimiphos-methyl may have slowed down the selection of individual mosquitoes with ace-1 resistance alleles in contrast to populations of An. gambiae s.l. with the L1014F resistance allele of the kdr gene. This may suggest that withdrawing the use of carbamates or organophosphates from IRS or rotating alternative insecticides with different modes of action may slow the development of ace-1 insecticide-resistance mutations. The increase in the prevalence of the L1014F mutation of the kdr gene in the population, despite the cessation of IRS, could be explained by the growing use of pyrethroids and DDT in agriculture and for other domestic use. More observational studies in countries where carbamates or organophosphates are still being used as public health insecticides may provide additional insights into these associations.Graphical

Genetic structure and kdr mutations in Aedes aegypti populations along a road crossing the Amazon Forest in Amapá State, Brazil

Insecticide resistance in Aedes aegypti poses a significant threat to disease control. One form of resistance, caused by kdr mutations in the NaV gene, hinders vector control efforts in Brazil. Despite genetic differences typically accumulating among isolated populations, this mosquito can actively and passively disperse through human transportation. Our study investigated the genetic structure and spread of kdr mutations in Ae. aegypti populations across six localities in Amapá State, Brazil, within the Amazonian Forest. Using 12 microsatellite loci and qPCR methods, we assessed genetic structure and identified three common kdr mutations (V410L, V1016I, and F1534C). High prevalence of kdr alleles was observed in all localities, indicating widespread distribution in Amapá State. Microsatellite analysis revealed differentiation among mosquito populations, dividing them into two distinct clusters supported by Bayesian and DAPC analyses. Oiapoque, located along the northern border with French Guiana, exhibited the highest kdr frequencies and genetic differentiation compared to other localities. Our findings suggest genetic structure in Ae. aegypti populations in Amapá State, with some passive gene flow between clusters. The study underscores the need for continuous surveillance of Ae. aegypti populations to monitor the spread of insecticide resistance and inform effective vector control strategies.

Frequency and spatial distribution of knock-down resistance (kdr) to pyrethroids in multiple oilseed rape pest species of the genus Ceutorhynchus.

The protection of European oilseed rape (OSR) from damaging insects relies on pyrethroid insecticides, but the development of resistance in key coleopteran pests such as the pollen beetle (Brassicogethes aeneus) and the cabbage stem flea beetle (Psylliodes chrysocephala) has resulted in reduced effectiveness of these insecticides. The sodium channel gene mutation L1014F knock-down resistance (kdr) is a contributing factor in resistance to pyrethroids in B. aeneus and P. chrysocephala, but little is known about the status of resistance in weevils of the genus Ceutorhynchus (Coleoptera: Curculonidae). Therefore, the present study investigated pyrethroid susceptibility and the presence of the kdr mutation in four Ceutorhynchus species. The kdr mutation in either its heterozygous or homozygous form was found in all investigated Ceutorhynchus species (C. picitarsis, C. pallidactylus, C. napi and C. obstrictus). Samples where pyrethroids in bioassays still provided control at 100% field rate or below contained kdr at frequencies of ≤12.5%, whilst bioassays using 100% field rate that did not control Ceutorhynchus populations contained homozygous resistant individuals at frequencies of greater than 55%. Field sampling demonstrated that kdr frequencies in populations of C. picitarsis and C. obstrictus collected from across France and Germany ranged from 0 to 100%. The present study demonstrated the potential of all four Ceutorhynchus species tested to develop pyrethroid resistance via the L1014F (kdr) mutation. Although kdr frequency varies among species and geographic locations, the risk of loss of pyrethroid insecticide effectiveness is high. Integration of other control tools for resistance management is therefore needed. © 2023 Society of Chemical Industry.

Detection of pyrethroids resistance alleles in goat biting louse Bovicola caprae (Phthiraptera: Trichodectidae) in west and northwest of Iran.

Pyrethroid insecticides target voltage-gated sodium channels (VGSCs) that are essential for electrical signaling in the nervous system of insects. Three-point mutations at the corresponding amino acid sequence positions M815I, T917I, and L920F located in domain II conferring the knockdown resistance (kdr) are the most important mutations in pyrethroid-resistant lice worldwide. In addition, six new mutations have been reported in the extracellular loops IIS1-2 (H813P) and IIS5 (I927F, L928A, R929V, L930M, L932M) in the α- subunit of the sodium channel in lice. The aim of this study was to detect alleles resistant to pyrethroids in the domain II (S5-S6) of the VGSC gene in goat biting louse. Goat biting lice were collected from five provinces in the west and northwest of Iran. Genomic DNA was extracted from goat biting lice and Bovicola (Damalinia) caprae species was confirmed by amplifying the cytochrome oxidase subunit I (COXI) gene. A fragment in the domain II (S5-S6) of the VGSC gene was amplified using the specific primers and the resultant polymerase chain reaction products were sequenced. Substitutions T917I, L920F, I927F, L928A, R929V and L930M were identified in the examined sequences. The results showed that all the examined lice had at least one mutation in their VGSC gene associated with pyrethroid resistance or new mutations. The presence of these mutated alleles in the VGSC gene may be due to the long-term and multiple use of pyrethroids against arthropods. Thus, the molecular detection of resistance to pyrethroid insecticides in goat chewing lice can help plot a kdr frequency map to enact effective policies to control caprine pediculosis.

Entomological indicators of malaria transmission prior to a cluster-randomized controlled trial of a ‘lethal house lure’ intervention in central Côte d’Ivoire

BackgroundA study was conducted prior to implementing a cluster-randomized controlled trial (CRT) of a lethal house lure strategy in central Côte d’Ivoire to provide baseline information on malaria indicators in 40 villages across five health districts.MethodsHuman landing catches (HLC) were performed between November and December 2016, capturing mosquitoes indoors and outdoors between 18.00 and 08.00 h. Mosquitoes were processed for entomological indicators of malaria transmission (human biting, parity, sporozoite, and entomological inoculation rates (EIR)). Species composition and allelic frequencies of kdr-w and ace-1R mutations were also investigated within the Anopheles gambiae complex.ResultsOverall, 15,632 mosquitoes were captured. Anopheles gambiae sensu lato (s.l.) and Anopheles funestus were the two malaria vectors found during the survey period, with predominance for An. gambiae (66.2%) compared to An. funestus (10.3%). The mean biting rate for An. gambiae was almost five times higher than that for An. funestus (19.8 bites per person per night for An. gambiae vs 4.3 bites per person per night for An. funestus) and this was evident indoors and outdoors. Anopheles funestus was more competent to transmit malaria parasites in the study area, despite relatively lower number tested for sporozoite index (4.14% (63/1521) for An. gambiae vs 8.01% (59/736) for An. funestus; χ2 = 12.216; P < 0.0001). There were no significant differences between the proportions infected outdoors and indoors for An. gambiae (4.03 vs 4.13%; χ2 = 0.011; P = 0.9197) and for An. funestus (7.89 vs 8.16%; χ2 = 2.58e−29; P = 1). The majority of both infected vectors with malaria parasites harboured Plasmodium falciparum (93.65% for An. gambiae and 98. 31% for An. funestus). Overall, the EIR range for both species in the different districts appeared to be high (0.35–2.20 infected bites per human per night) with the highest value observed in the district of North-Eastern-Bouaké. There were no significant differences between transmission occurring outdoor and indoor for both species. Of the An. gambiae s.l. analysed, only An. gambiae sensu stricto (14.1%) and Anopheles coluzzii (85.9%) were found. The allelic frequencies of kdr and ace-1R were higher in An. gambiae (0.97 for kdr and 0.19 for ace-1R) than in An. coluzzii (0.86 for kdr and 0.10 for ace-1R) (P < 0.001).ConclusionDespite universal coverage with long-lasting insecticidal nets (LLINs) in the area, there was an abundance of the malaria vectors (An. gambiae and An. funestus) in the study area in central Côte d’Ivoire. Consistent with high insecticide resistance intensity previously detected in these districts, the current study detected high kdr frequency (> 85%), coupled with high malaria transmission pattern, which could guide the use of Eave tubes in the study areas.

Assessing Insecticide Susceptibility and Resistance Intensity of Anopheles gambiae s.l. Populations From Some Districts of Benin Republic, West Africa.

Pyrethroid resistance is widespread in sub-Saharan Africa. The objective of this study was to assess the insecticide resistance intensity in Anopheles gambiae s.l. (Diptera: Culicidae) in four districts of Benin in order to better understand how pyrethroid-only nets are likely to be effective. Thus, adult females of An. gambiae s.l., reared from field-collected larvae were used for assessing resistance intensity to permethrin and deltamethrin. They were tested at 1×, 5×, and 10× the diagnostic dose, using both WHO susceptibility tube testing and CDC bottle bioassays. Identification of molecular species, as well as of L1014F Kdr and Ace-1R mutations was performed using the PCR. The level of expression of biochemical enzymes was also evaluated. Overall, moderate to high resistance intensity to permethrin and deltamethrin was observed, irrespective of the testing method. While the L1014F Kdr frequency was high (>75%), Ace-1R was low (≤6%) in An. gambiaes.s. and Anopheles coluzzii, the two predominant species [52% (95% CI: 44.8-59.1) and 45% (95% CI: 38.0-52.2), respectively]. Anopheles arabiensis was found at very low frequency (3%, 95%CI: 1.1-6.4). For Biochemical analyses, α and β-esterases were over-expressed in all four districts, while mixed-function oxidases (MFOs) were over-expressed in only one. Overall, the two testing methods led to comparable conclusions, though there were a few inconsistencies between them. The moderate-high resistance intensity observed in the study area suggests that dual active-ingredient (AI) long-lasting insecticidal nets (LLINs) may provide better control of insecticide-resistant mosquitoes.

Frequency of Pyrethroid Insecticide Resistance kdr Gene and Its Associated Enzyme Modulation in Housefly, Musca domestica L. Populations From Jhang, Pakistan

Housefly, Musca domestica, is considered responsible for transmitting a wide variety of human and veterinary diseases. Mostly, insecticides are being used for their control and more commonly, pyrethroid insecticides worldwide. However, resistance has been reported against various pyrethroid insecticides. Houseflies become resistant by two major mechanisms, i.e., target site insensitivity through knockdown resistance gene mutation (kdr) and enzyme detoxification. Thus, the current study was designed to monitor the frequency of pyrethroid resistance gene kdr in housefly populations of District Jhang. The flies were collected from seven sampling sites and then reared in the lab for molecular and biochemical assays. The amplification of template DNA was performed for knockdown resistance gene through the outer primers kdr1 and kdr4, and the inner primers kdr1 and kdr2 using PASA (PCR Amplification of Specific Alleles) method which specifically amplify the domain-II of kdr gene. Three populations were found homozygous susceptible (+/+; 42.85%), whereas two populations were found genetically homozygous resistant (−/−; 28.57%) which are insensitive to pyrethroid insecticides. Similarly, two populations were found heterozygous (+/−; 28.57%) for kdr suggesting thereby that at least 1/4th homozygous-resistant (−/−) housefly populations with insensitivity to pyrethroids would be produced in the future keeping in view the Mendelian ratio. Biochemical assay showed that homozygous-resistant populations had increased activity of Acetylcholinesterase (AChE), α-Carboxylesterases (α-Carboxyl), β-Carboxylesterase (β-Carboxyl), Alkaline Phosphatase (AkP), and Acidic Phosphatase (AcP) enzymes. In addition, heterozygous populations also showed increased activities of these enzymes. The current results would not only help avoid the indiscriminate load of insecticides onto the environment but also serve as a hallmark for the management of housefly populations in target areas in the future.

Multiple insecticide resistance mechanisms in urban population of Anopheles coluzzii (Diptera: culicidae) from Lagos, South-West Nigeria

Malaria is a major public health challenge in Africa with Nigeria accounting for the highest burden of the disease in the world. Vector control has proved to be a highly effective component of malaria control, however, the development and spread of insecticide resistance in major vectors of malaria have been a major challenge. This study assessed resistance mechanisms in Anopheles coluzzii populations from Kosofe, Lagos mainland and Ojo Local Government Areas in Lagos, Nigeria where An. gambiae s.l is resistant to DDT and Permethrin. WHO susceptibility bioassay test was used in determining resistance status of An. coluzzii to discriminating doses of DDT and Permethrin while synergist assay was used to assess the involvement of monooxygenases in resistance development. Sub-species of An. gambiae s.l (An. gambiae and An. coluzzii) were identified using polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism (PCR-RFLP) while Allele-Specific Polymerase Chain Reaction (AS-PCR) assay was used to detect knockdown mutation (kdr-West; L1014F). Biochemical assays were used in determining the activities of metabolic enzymes. High DDT resistance was recorded in An. coluzzii populations from the three sites. Mortality rate of mosquitoes exposed confirmed Permethrin resistance in Kosofe (50%) and Lagos mainland (48%) but resistance was suspected in Ojo (96%). All specimens tested were confirmed as An. coluzzii with low kdr frequency; 11.6%, 16.4% and 6.7% in Kosofe, Lagos mainland and Ojo respectively. Pre-exposure to synergist (PBO) before exposure to Permethrin led to increased mortality in all populations. Esterase activity was insignificantly overexpressed in Kosofe (p = 0.849) and Lagos mainland (p = 0.229) populations. In contrast, GST activity was significantly lower in populations from Lagos mainland (63.650 ± 9.861; p = 0.007) and Ojo (91.765 ± 4.959; p = 0.042) than Kisumu susceptible strains (120.250 ± 13.972). Monooxygenase activity was higher in Lagos mainland (2.371 ± 0.261) and Ojo (1.361 ± 0.067) populations, albeit significantly in Lagos mainland (p = 0.007) only. Presence of target-site mutation in all populations, increased mortality with pre-exposure to PBO and elevated monooxygenase in Lagos mainland population were confirmed. Multiple resistance mechanisms in some urban populations of An. coluzzii from Lagos, Nigeria calls for appropriate resistance management strategies.

Changes in the genetic composition of Myzus persicae nicotianae populations in Chile and frequency of insecticide resistance mutations.

Myzus persicae is a cosmopolitan aphid that is highly polyphagous and an important agricultural pest. The subspecies M. persicae nicotianae has been described for highly specialized phenotypes adapted to tobacco (Nicotiana tabacum). In Chile, the population of M. persicae nicotianae was originally composed of a single red genotype that did not possess insecticide resistance mutations. However, in the last decade, variation in the colour of tobacco aphids has been observed in the field. To determine whether this variation stems from the presence of new genotypes, sampling was carried out across the entire distribution of tobacco cultivation regions in Chile. The aphids collected were genotyped, and the frequency of kdr (L1014F), super-kdr (M918T), modification of acetylcholinesterase (MACE) and nicotinic acetylcholine receptor β subunit (nAChRβ) mutations associated with insecticide resistance was determined. A total of 16 new genotypes of M. persicae nicotianae were detected in Chile: four of them possessed the MACE mutation, and none of them possessed the kdr, super-kdr or nAChRβ mutation. The previously described red genotype was not detected in any of the sampled fields over two seasons. These results raise questions about the mechanisms underlying changes in the genetic structure of M. persicae nicotianae populations in Chile. Future research aimed at addressing these questions could provide new insight into aphid evolution and agricultural practices.

Intensity and mechanisms of deltamethrin and permethrin resistance in Anopheles gambiae s.l. populations in southern Benin

BackgroundInsecticide resistance is threatening the effectiveness of efforts to control malaria vectors in Benin. This study explores the levels and mechanisms of insecticide resistance in An. gambiae s.l. to pyrethroids.MethodsLarvae were collected from August 2017 to July 2018 in five communes in southern Benin (Adjohoun, Allada, Bohicon, Cotonou, and Porto-Novo) representing diverse ecological regions, and were reared in Benin’s insectary. Two- to five-day-old female mosquitoes from each district were exposed to multiple doses of deltamethrin and permethrin (1×, 2×, 5×, and 10×) using the WHO insecticide resistance intensity bioassay. The effect of pre-exposure to the synergist, piperonyl butoxide (PBO), was also tested at different pyrethroid doses. Molecular allele frequencies of kdr (1014F) and ace-1R (119S) insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled from each study area.ResultsAn. gambiae s.l. were resistant to pyrethroid-only exposure up to 10× the diagnostic doses in all the study sites for both deltamethrin and permethrin. Mortality was significantly higher in An. gambiae s.l. pre-exposed to PBO followed by exposure to deltamethrin or permethrin compared to mosquitoes exposed to deltamethrin or permethrin only (p < 0.001). The difference in mortality between deltamethrin only and PBO plus deltamethrin was the smallest in Cotonou (16–64%) and the greatest in Bohicon (12–93%). The mortality difference between permethrin only and PBO plus permethrin was the smallest in Cotonou (44–75%) and the greatest in Bohicon (22–72%). In all the study sites, the kdr resistance allele (1014F) frequency was high (75–100%), while the ace-1 resistance allele (G119S) frequency was low (0–3%). Analysis of the metabolic enzymatic activity of An. gambiae s.l. showed overexpression of nonspecific esterases and glutathione S-transferases (GST) in all study sites. In contrast to the PBO results, oxidase expression was low and was similar to the susceptible An. gambiae s.s. Kisumu strain in all sites.ConclusionThere is high-intensity resistance to pyrethroids in southern Benin. However, pre-exposure to PBO significantly increased susceptibility to the pyrethroids in the different An. gambiae s.l. populations sampled. The use of PBO insecticide-treated bed nets may help maintain the gains in An. gambiae (s.l.) control in southern Benin.Graphical

Status of insecticide susceptibility in Anopheles arabiensis and detection of the knockdown resistance mutation (kdr) concerning agricultural practices from Northern Sudan state, Sudan

BackgroundChemical control has been the most efficient method in mosquito control, the development of insecticide resistance in target populations has a significant impact on vector control. The use of agricultural pesticides may have a profound impact on the development of resistance in the field populations of malaria vectors. Our study focused on insecticide resistance and knockdown resistance (kdr) of Anopheles arabiensis populations from Northern Sudan, related to agricultural pesticide usage. ResultsAnopheles arabiensis from urban and rural localities (Merowe and Al-hamadab) were fully susceptible to bendiocarb 0.1% and permethrin 0.75% insecticides while resistant to DDT 4% and malathion 5%. The population of laboratory reference colony F189 from Dongola showed a mortality of 91% to DDT (4%) and fully susceptible to others. GLM analysis indicated that insecticides, sites, site type, and their interaction were determinant factors on mortality rates (P < 0.01). Except for malathion, mortality rates of all insecticides were not significant (P > 0.05) according to sites. Mortality rates of malathion and DDT were varied significantly (P < 0.0001 and P < 0.05 respectively) by site types, while mortality rates of bendiocarb and permethrin were not significant (P >0.05). The West African kdr mutation (L1014F) was found in urban and rural sites. Even though, the low-moderate frequency of kdr (L1014F) mutation was observed. The findings presented here for An. arabiensis showed no correlation between the resistant phenotype as ascertained by bioassay and the presence of the kdr mutation, with all individuals tested except the Merowe site which showed a moderate association with DDT (OR= 6 in allelic test), suggesting that kdr genotype would be a poor indicator of phenotypic resistance. ConclusionThe results provide critical pieces of information regarding the insecticide susceptibility status of An. arabiensis in northern Sudan. The usage of the same pesticides in agricultural areas seemed to affect the Anopheles susceptibility when they are exposed to those insecticides in the field. The kdr mutation might have a less role than normally expected in pyrethroids resistance; however, other resistance genes should be in focus. These pieces of information will help to improve the surveillance system and The implication of different vector control programs employing any of these insecticides either in the treatment of bed nets or for indoor residual spraying would achieve satisfactory success rates.

Options for managing pesticide resistance in the redlegged earth mite (

Chemical strategies to manage pest populations that have evolved pesticide resistance can involve killing resistant individuals by using higher rates of the same pesticide or applying alternative pesticides for which resistance does not exist. However, the dilemma is that alternative pesticides available for many pest species are limited, while higher pesticide rates may lead to the evolution of higher resistance levels. Here, both strategies are tested in a resistant population of the redlegged earth mite, Halotydeus destructor, which contained resistance against two groups of chemicals: synthetic pyrethroids and organophosphates. Resistant mites were introduced into exclusion field plots, which were then treated with pyrethroid, organophosphate or alternative chemicals for 2 years at a low pressure (one spray at the registered field rate per year) or a high pressure (two sprays at the registered field rate per year). A single pyrethroid application suppressed mite numbers, but pyrethroid-resistant allele (kdr) frequencies quickly rose from ∼50% to nearly 100%. Thereafter, pyrethroid chemicals lost effectiveness. However, kdr frequencies declined across mite generations when pyrethroids were not used, regardless of other treatments. Organophosphates continued to suppress mite populations under both high and low pressures, irrespective of kdr allele frequencies, and laboratory bioassays showed no increase in organophosphate resistance levels. One of the alternative chemicals, diafenthiuron, applied once per year over two years, successfully suppressed mite numbers, whereas other treatments did not control mites. These findings demonstrate that different strategies are required to manage H. destructor with pyrethroid resistance and organophosphate resistance.

Insecticide resistance status of Aedes aegypti in Bangladesh

BackgroundArboviral diseases, including dengue and chikungunya, are major public health concerns in Bangladesh where there have been unprecedented levels of transmission reported in recent years. The primary approach to control these diseases is to control the vector Aedes aegypti using pyrethroid insecticides. Although chemical control has long been practiced, no comprehensive analysis of Ae. aegypti susceptibility to insecticides has been conducted to date. The aim of this study was to determine the insecticide resistance status of Ae. aegypti in Bangladesh and investigate the role of detoxification enzymes and altered target site sensitivity as resistance mechanisms.MethodsEggs of Aedes mosquitoes were collected using ovitraps from five districts across Bangladesh and in eight neighborhoods of the capital city Dhaka, from August to November 2017. CDC bottle bioassays were conducted for permethrin, deltamethrin, malathion, and bendiocarb using 3- to 5-day-old F0–F2 non-blood-fed female mosquitoes. Biochemical assays were conducted to detect metabolic resistance mechanisms, and real-time PCR was performed to determine the frequencies of the knockdown resistance (kdr) mutations Gly1016, Cys1534, and Leu410.ResultsHigh levels of resistance to permethrin were detected in all Ae. aegypti populations, with mortality ranging from 0 to 14.8% at the diagnostic dose. Substantial resistance continued to be detected against higher (2×) doses of permethrin (5.1–44.4% mortality). Susceptibility to deltamethrin and malathion varied between populations while complete susceptibility to bendiocarb was observed in all populations. Significantly higher levels of esterase and oxidase activity were detected in most of the test populations as compared to the susceptible reference Rockefeller strain. A significant association was detected between permethrin resistance and the presence of Gly1016 and Cys1534 homozygotes. The frequency of kdr (knockdown resistance) alleles varied across the Dhaka Aedes populations. Leu410 was not detected in any of the tested populations.ConclusionsThe detection of widespread pyrethroid resistance and multiple resistance mechanisms highlights the urgency for implementing alternate Ae. aegypti control strategies. In addition, implementing routine monitoring of insecticide resistance in Ae. aegypti in Bangladesh will lead to a greater understanding of susceptibility trends over space and time, thereby enabling the development of improved control strategies.Graphical

First report of N1575Y mutation in pyrethroid resistant Anopheles gambiae s.l. in Nigeria

In view of recent findings that codon 1575 mutation increases the L1014F-mediated resistance in Anopheles mosquitoes, we investigated the presence of this mutation in four States with intense pyrethroid resistance in Nigeria. Permethrin and deltamethrin susceptibility tests were carried out using 2–3 day old adult female Anopheles from larval collections. Based on the susceptibility test results, intensity tests with 5x and 10x concentrations were carried out at sites with high resistance (% mortality>85). Resistant specimen were identified by PCR and screened for the presence of L1014F and N1575Y mutations using allele specific PCR and Taqman assay respectively. The frequency of kdr mutation for permethrin varied from 0.71–0.75 for 5x and 0.73–0.75 for 10x. The kdr frequency for deltamethrin was not significantly different (P = 0.157): 0.54–0.60 for 5x and 0.66–0.71 for 10x. Percentage increase in kdr frequency between 1x and 5x permethrin doses ranges from 1.4% to 11.9% in all sites except Edo and Anambra; while only Lagos had percentage increase of 1.4% between 5x and 10x. For deltamethrin, Lagos and Niger had the same (3.8%) percentage increase in kdr frequency between 1x and 5x, while 5x and 10x gave 23.6% and 31.5% respectively. Resistant Anopheles gambiae at 5x and 10x concentrations of permethrin showed the presence of homozygous (Y) and heterozygous (NY) mutant allele in two sites. From this study, the mutation has no significant association with resistance intensity (χ2=0.8, P = 0.3711). There is need for frequent monitoring to forestall the effect this may have on national malaria control programs.

Impact of deltamethrin selection on kdr mutations and insecticide detoxifying enzymes in Aedes aegypti from Mexico

BackgroundInsecticide resistance is a serious problem for vector control programmes worldwide. Resistance is commonly attributed to mutations at the insecticide’s target site or increased activity of detoxification enzymes.MethodsWe determined the knockdown concentration (KC50) and lethal concentration (LC50) of deltamethrin in six natural populations of adult Aedes aegypti from southeastern Mexico. These populations were then selected over five generations using the LC50 from the preceding generation that underwent selection, and the heritability of deltamethrin resistance was quantified. For each generation, we also determined the frequency of the kdr alleles L410, I1016 and C1534, and the levels of activity of three enzyme families (α- and β-esterases, mixed-function oxidases and glutathione S-transferases (GST)) associated with insecticide detoxification.ResultsThere was an increase in KC50 and LC50 values in the subsequent generations of selection with deltamethrin (FS5vs FS0). According to the resistance ratios (RRs), we detected increases in LC50 ranging from 1.5 to 5.6 times the values of the parental generation and in KC50 ranging from 1.3–3.8 times the values of the parental generation. Triple homozygous mutant individuals (tri-locus, LL/II/CC) were present in the parental generations and increased in frequency after selection. The frequency of L410 increased from 1.18-fold to 2.63-fold after selection with deltamethrin (FS5vs FS0) in the populations analyzed; for I1016 an increase between 1.19-fold to 2.79-fold was observed, and C1534 was fixed in all populations after deltamethrin selection. Enzymatic activity varied significantly over the generations of selection. However, only α- esterase activity remained elevated in multiple populations after five generations of deltamethrin selection. We observed an increase in the mean activity levels of GSTs in two of the six populations analyzed.ConclusionsThe high levels of resistance and their association with high frequencies of kdr mutations (V410L, V1016I and F1534C) obtained through artificial selection, suggest an important role of these mutations in conferring resistance to deltamethrin. We highlight the need to implement strategies that involve the monitoring of kdr frequencies in insecticide resistance monitoring and management programmes.

Insecticides resistance status in Aedes aegypti and Aedes albopictus from Lahore, Pakistan

Aedes‐borne disease in Pakistan drew the attention after the dengue outbreak in Lahore in 2011, leaving thousands hospitalized and around 300 deaths. Since then many small and big outbreaks have been reported from various parts of the country. The only available and mostly used mosquito control strategy in Pakistan is the use of neurotoxic chemicals. Pyrethroid deltamethrin and permethrin were extensively used for adults’ control along with organophosphate temephos as a larvicide during and after the outbreaks. This study was designed to find the efficacy of the insecticides in use for Aedes control as well as other insecticides of the same or different classes. Preliminary results after WHO‐like tube tests or CDC‐bottle tests showed that both Aedes aegypti and Aedes albopictus are susceptible to larvicide OP temephos, IGR pyriproxifen and adulticide OP malathion while they are resistant to all classes of pyrethroids tested (deltamethrin, permethrin, etofenprox, alphacypermethrin). Genotyping assays showed low kdr frequencies while assays to find the expression of P450s and other enzymes related to resistance are underway. Sequencing of the voltage gated sodium channel gene will give us a better picture about the mutations in the IIS6 and IIIS6 regions which plays an important role in pyrethroids sensitivity. This study will assist the authorities to design future control strategies.Support or Funding InformationThe study is part of PhD thesis of the presenting author Rafi ur Rahman, who receives monthly scholarship from TWAS and CNPq. No additional funding is provided to the laboratory or the student.