Preliminary monitoring of knockdown resistance (kdr) mutation in Anopheles stephensi: insights from a malarious area in Southeastern Iran.

BACKGROUND: Anopheles stephensi is recognized as the main malaria vector in Iran. In recent years, resistance to several insecticide classes, including organochlorine, pyrethroids, and carbamate compounds, has been reported for this medically important malaria vector. The main objective of the present study was to evaluate the insecticide susceptibility status of An. stephensi collected from the southern part of Iran, and to clarify the mechanism of resistance, using bioassay tests and molecular methods comparing the sequence of susceptible and resistant mosquitoes. METHODS: Mosquito larvae were collected from various larval habitats across six different districts (Gabrik, Sardasht, Tidar, Dehbarez, Kishi and Bandar Abbas) in Hormozgan Provine, located in the southern part of Iran. From each district standing water areas with the highest densities of Anopheles larvae were selected for sampling, and adult mosquitoes were reared from them. Finally, the collected mosquito species were identified using valid keys. Insecticide susceptibility of An. stephensi was tested using permethrin 0.75%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, and DDT 4%, following the World Health Organization (WHO) test procedures for insecticide resistance monitoring. Additionally, knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene was sequenced and analysed among resistant populations to detect possible molecular mechanisms of observed resistance phenotypes. RESULTS: The susceptibility status of An. stephensi revealed that resistance to DDT and permethrin was found in all districts. Furthermore, resistance to all tested insecticides in An. stephensi was detected in Gabrik, Sardasht, Tidar, and Dehbarez. Analysis of knockdown resistance (kdr) mutations at the vgsc did not show evidence for the presence of this mutation in An. stephensi. CONCLUSION: Based on the results of the current study, it appears that in An. stephensi from Hormozgan Province (Iran), other resistance mechanisms such as biochemical resistance due to detoxification enzymes may be involved due to the absence of the kdr mutation or non-target site resistance. Further investigation is warranted in the future to identify the exact resistance mechanisms in this main malaria vector across the country.

Impact of the exposure of sublethal dose of mosquito coil on the development of insecticide resistance in Aedes aegypti (L.) (Diptera: Culicidae).

Mosquito coil is commonly used in many African households for protection against mosquito bites. The coil usually has semi-volatile pyrethroids as an active ingredient, which usually diffuse across open space, and the cloud either kills mosquitoes that are exposed, or mosquitoes can be exposed to sublethal doses of the insecticides. This study was conducted to assess the impact of sublethal doses of mosquito coil on the development of insecticide resistance in Aedes aegypti, a major vector for dengue fever and several other arboviral diseases. A laboratory colony of Ae. aegypti was exposed to sublethal doses of a meperfluthrin-based mosquito coil in a Peet-Grady chamber once per generation for 16 generations. The susceptibility of the exposed colony to a diagnostic dose of the mosquito coil as well as to three other insecticides was determined. Three different kdr mutations and five enzyme activities were evaluated in both the exposed and control colonies. After 16 generations of sublethal exposure to mosquito coils, the full diagnostic dose of the coil caused 68% mortality to the exposed colony compared to 100% mortality in the control colony. Mortality caused by deltamethrin (0.05%) was also significantly lower in the exposed colony. The frequency of 1016I kdr mutation as well as MFO and alpha esterase activities were higher in the exposed colony compared to the control colony. This study provides evidence of the development of pyrethroid resistance in an Ae. aegypti population due to sublethal exposure to mosquito coil for 16 generations. Given the large-scale use of mosquito coils in many African households, its role as a pyrethroid resistance selection source should be taken into consideration when designing resistance management strategies.

Unveiling fenpropathrin resistance levels in field populations of Tetranychus cinnabarinus (Boisduval): Insights, risks, and RNAi strategy.

Indoor cases of Tetranychus cinnabarinus displaying resistance have been documented, but the resistance level in field populations remains unexplored in China. This study delves into the resistance dynamics of T. cinnabarinus to fenpropathrin in various field populations across China, a pressing concern in contemporary agricultural pest control. The conventional bioassay and amplicon sequencing reveal a notable absence of significant fenpropathrin resistance in field populations, contrasting with known resistance in indoor cases. Current study highlights the limitations of traditional bioassays in detecting early-stage resistance and underscores the nuanced capabilities and constraints of amplicon sequencing in resistance gene frequency analysis. By employing an integrated approach, we combined dose-response bioassays, amplicon sequencing, and statistical modeling to assess resistance levels and investigate underlying genetic factors. The model with empirical data indicates that a 5% mutation frequency represents the threshold before resistance emerges. However, the detection of the kdr mutation in certain populations ranging from 0 to 1.2%, signals an early looming threat of future resistance emergence. Additionally, we further assessed a specific dsRNA targeting VGSC genes at two concentrations (10 ng/µL and 100 ng/µL), both inducing substantial mortality by silencing target genes effectively. The exploration of RNA interference (RNAi) as a novel, more environmentally friendly pest control measure opens new avenues, despite the ongoing challenge of resistance evolution. Overall, this study underscores the necessity for evolving pest management strategies, integrating advanced biotechnological approaches with traditional methods, to effectively counter pesticide resistance and ensure sustainable agricultural productivity.

Identification of breeding habitats and kdr mutations in Anopheles spp. in South Korea.

BACKGROUND: Malaria is still endemic in South Korea. However, limited information is available on the current Anopheles breeding sites and the occurrence of insecticide resistance-associated genetic mutations and their distribution needed to control the malaria vector efficiently. METHODS: This study explored breeding sites of Anopheline adults in Gimpo-si, near the demilitarized zone (DMZ) in Gyeonggi-do province, South Korea, from 2022 to 2023. Genetic diversity was investigated based on the internal transcribed spacer (ITS2), cytochrome c oxidase subunit I (COI), and knockdown resistance (kdr) genes of Anopheles mosquitoes. A natural environment associated with the seasonal abundance of Anopheles larvae was characterized. RESULTS: Two breeding sites of Anopheles larvae and adults were found at a stream margin or shallow freshwater near the forest in Wolgot-myeon in Gimpo-si without cattle shed within 1 km and in Naega-myeon in Ganghwa-gun with cow shed within 100 m in 2022 and 2023, respectively. Both sites were located between the newly cultivated lands and the forest. Besides, both breeding sites were in the valley at a slight elevation of 60-70 m from ground lands and maintained the shadow all day. Overall, the Wolgot-myeon breeding site showed various Anopheles spp. larvae, including Anopheles sinensis. Naega-myeon, an additional breeding site found in 2023, had Anopheles sineroides larvae, and approximately 59.7% (89/149) of An. sinensis adults inhabited within a 100-m distance. The total collection, including larvae and adults, revealed that An. sinensis, Anopheles pullus, Anopheles kleini, An. sineroides, Anopheles belenrae, and Anopheles lindesayi accounted for 44.2% (118/267), 0.7% (2/267), 0.7% (2/267), 22.1% (59/267), 1.9% (5/267), and 30.3% (81/267), respectively. Furthermore, various kdr mutant genotypes (F/F, C/C, L/F, L/C and F/C) in An. sinensis, and the first kdr allele mutant (L/F1014) in An. belenrae were identified in South Korea. CONCLUSIONS: Two breeding sites of Anopheles larvae were studied in Wolgot-myeon and Naega-myeon. Various Anopheles spp. larvae were detected in both habitats, but overall, An. sinensis was the most prevalent adults in both study sites. The occurrence of kdr allele mutant of An. belenrae in South Korea was reported. Rigorous larvae monitoring of Anopheles spp., continuously updating information on Anopheles breeding sites, and understanding the environmental conditions of Anopheles habitats are required to develop an effective malaria control programme in South Korea.

First report of classical knockdown resistance (kdr) mutation, L1014F, in human head louse Pediculus humanus capitis (Phthiraptera: Anoplura).

There are at least three known knockdown resistance (kdr) mutations reported globally in the human head louse Pediculus humanus capitis De Geer (Phthiraptera: Anoplura) that are associated with reduced sensitivity to pyrethroids. However, the prevalence of kdr mutation in head lice is not known in the Indian subcontinent. To identify kdr mutations in the Indian head lice population, the genomic region of the voltage-gated sodium channel gene encompassing IIS1-2 linker to IIS6 segments was PCR-amplified and sequenced from P. humanus capitis samples collected from different geographic localities of India. DNA sequencing revealed the presence of four kdr mutations: M827I, T929I, L932F and L1014F. The presence of a classical kdr mutation L1014F, the most widely reported mutation across insect-taxa associated with the kdr-trait, is being reported for the first time in P. humanus capitis.

Leucine to tryptophane substitution in the pore helix IIP1 confer sodium channel resistance to pyrethroids and DDT.

Aedes aegypti is responsible for transmitting a variety of arboviral infectious diseases such as dengue and chikungunya. Insecticides, particularly pyrethroids, are used widely for mosquito control. However, intensive used of pyrethroids has led to the selection of kdr mutations on sodium channels. L982W, locating in the PyR1 (Pyrethroid receptor site 1), was first reported in Ae. aegypti populations collected from Vietnam. Recently, the high frequency of L982W was detected in pyrethroid-resistant populations of Vietnam and Cambodia, and also concomitant mutations L982W + F1534C was detected in both countries. However, the role of L982W in pyrethroid resistance remains unclear. In this study, we examined the effects of L982W on gating properties and pyrethroid sensitivity in Xenopus oocytes. We found that mutations L982W and L982W + F1534C shifted the voltage dependence of activation in the depolarizing direction, however, neither mutations altered the voltage dependence of inactivation. L982W significantly reduced channel sensitivity to Type I pyrethroids, permethrin and bifenthrin, and Type II pyrethroids, deltamethrin and cypermethrin. No enhancement was observed when synergized with F1534C. In addition, L982W and L982W + F1534C mutations reduced the channel sensitivity to DDT. Our results illustrate the molecular basis of resistance mediates by L982W mutation, which will be helpful to understand the interacions of pyrethroids or DDT with sodium channels and develop molecular markers for monitoring pest resistance to pyrethroids and DDT.

Novel real-time PCR assay detects widespread distribution of knock down resistance (kdr) mutations associated with pyrethroid resistance in the mosquito, Culex quinquefasciatus, in Thailand.

Susceptibility to pyrethroids in the mosquito Culex quinquefasciatus, the major vector of lymphatic filariasis, is being seriously threatened worldwide. Knockdown resistance (kdr), caused by mutations in the voltage gated sodium channel (VGSC) gene, particularly the L1014F mutation, is an important resistance mechanism. Our aim was to develop a real-time PCR with melt curve analysis to evaluate the distribution of the L1014F mutation in Cx. quinquefasciatus throughout Thailand and to determine the polymorphism pattern of a VGSC gene fragment spanning the L1014F mutation. A total of 3760 females from 18 localities across five regions of Thailand were bio-assayed by exposure to 0.05% deltamethrin WHO papers, showing mortality rates ranging from 2.4% to 83.0%. Genotyping of 753 dead and surviving mosquitoes using our novel real-time PCR assay with melt curve analysis and tetra-primer allele-specific PCR revealed the mutant F1014 allele is closely associated with the deltamethrin resistance phenotype. The L1014F mutation was found at high frequency throughout Thailand, particularly in the North. However, some survivors were homozygous for wild type L1014 allele, which were further sequenced for the IIP-IIS6 region of VGSC gene. The haplotype network of phenotypically characterized individuals indicated the presence of other possible kdr alleles/resistance mechanisms at play including two novel mutations, V978E and D992E. The finding of new putative kdr alleles and widespread distribution of the F1014 allele emphasizes the significant role of kdr mutations in pyrethroid resistance in Thai Cx. quinquefasciatus populations. Monitoring kdr variations and phenotypic resistance is critical for managing resistance in Cx. quinquefasciatus.

Genetic markers associated with insecticide resistance and resting behaviour in Anopheles gambiae mosquitoes in selected sites in Kenya.

BACKGROUND: Molecular diagnostic tools have been incorporated in insecticide resistance monitoring programmes to identify underlying genetic basis of resistance and develop early warning systems of vector control failure. Identifying genetic markers of insecticide resistance is crucial in enhancing the ability to mitigate potential effects of resistance. The knockdown resistance (kdr) mutation associated with resistance to DDT and pyrethroids, the acetylcholinesterase-1 (ace-1R) mutation associated with resistance to organophosphates and carbamates and 2La chromosomal inversion associated with indoor resting behaviour, were investigated in the present study. METHODS: Anopheles mosquitoes sampled from different sites in Kenya and collected within the context of malaria vector surveillance were analysed. Mosquitoes were collected indoors using light traps, pyrethrum spray and hand catches between August 2016 and November 2017. Mosquitoes were identified using morphological keys and Anopheles gambiae sensu lato (s.l.) mosquitoes further identified into sibling species by the polymerase chain reaction method following DNA extraction by alcohol precipitation. Anopheles gambiae and Anopheles arabiensis were analysed for the presence of the kdr and ace-1R mutations, while 2La inversion was only screened for in An. gambiae where it is polymorphic. Chi-square statistics were used to determine correlation between the 2La inversion karyotype and kdr-east mutation. RESULTS: The kdr-east mutation occurred at frequencies ranging from 0.5 to 65.6% between sites. The kdr-west mutation was only found in Migori at a total frequency of 5.3% (n = 124). No kdr mutants were detected in Tana River. The ace-1R mutation was absent in all populations. The 2La chromosomal inversion screened in An. gambiae occurred at frequencies of 87% (n = 30), 80% (n = 10) and 52% (n = 50) in Baringo, Tana River and Migori, respectively. A significant association between the 2La chromosomal inversion and the kdr-east mutation was found. CONCLUSION: The significant association between the 2La inversion karyotype and kdr-east mutation suggests that pyrethroid resistant An. gambiae continue to rest indoors regardless of the presence of treated bed nets and residual sprays, a persistence further substantiated by studies documenting continued mosquito abundance indoors. Behavioural resistance by which Anopheles vectors prefer not to rest indoors may, therefore, not be a factor of concern in this study's malaria vector populations.

The polymorphism and geographical distribution of knockdown resistance of adult Anopheles sinensis populations in eastern China.

BACKGROUND: Anopheles sinensis is one of the major malaria vectors in China and other southeast Asian countries, including Vietnam, Cambodia, Thailand. Vector control is considered to be the critical measure for malaria control, while the increasing prevalence of insecticide resistance caused by long-term use of insecticides, especially pyrethroids, is threatening the successful control of An. sinensis. In order to understand the underlying resistance mechanisms involved and molecular basis, the principal malaria vector, An. sinensis from Jiangsu and Anhui provinces, Southeast China, was investigated. METHODS: The adult Anopheles mosquitoes were sampled from multiple sites across Jiangsu and Anhui provinces, and sufficient mosquitoes collected from eleven sites for insecticide susceptibility bioassays. The DIIS4-DIIS6 region of the para-type sodium channel gene was amplified and sequenced, then multiple PCR and Taqman assays were used to assess the frequencies of kdr mutations at the target gene. RESULTS: In the present study, most of the adult An. sinensis populations were pyrethroids resistant, which indicated the presence of kdr resistance mutations in the para-type sodium channel gene. Sequence analyses demonstrated the kdr mutation existed at codon 1014 in Jiangsu and Anhui provinces. In adult An. sinensis, three mutant types (TTT L1014F, TTC L1014F, and TGT L1014C) of kdr alleles were detected, while no wild type (TTG L1014) was observed. The TTC L1014F mutation was first reported in Anhui province. CONCLUSIONS: The highly polymorphic kdr alleles were observed in all the adult An. sinensis populations, which suggested that in-depth studies are required for carrying on insecticide resistance monitoring and specific resistance mechanisms studying into establish effective long-term malaria vector control program in eastern China.

First report of target site insensitivity to pyrethroids in human flea, Pulex irritans (Siphonaptera: Pulicidae).

The human flea, Pulex irritans, is the most important ectoparasite of humans. Intensive use of pyrethroids for its control has led to insecticide resistance. Monitoring pyrethroid resistance and its underlying mechanisms is essential for flea control. The aims of this study were to identify the susceptibility status of human flea to permethrin and to detect the presence of knockdown resistance (kdr) mutation and its frequency in populations of P. irritans. Adults of P. irritans were collected from Zanjan Province, northwest of Iran, during 2013-2017. Different populations of this flea were exposed to permethrin 0.75% for one and 8 h and then the mortality rate, as well as KD50 and KD95 times were calculated. Total RNA and gDNA of samples were extracted, and the fragments of cDNA encoding the partial voltage-gated sodium channel (VGSC) peptides were amplified using degenerated primers. Specific PCR and TaqMan real-time assays were conducted to characterize the vgsc gene and to detect the presence of mutation and genotyping of the populations. Mortality rates were in the range from 32% to 67% for one-hour and 73% to 90% for eight-hour exposure to permethrin 0.75%. KD50 and KD95 times varied in a range from 46 to 241 and 177 to 899 min, respectively. Sequencing of 70 amplified fragments of gDNA resulted in a 578-bp product. These fragments contained two introns (92 and 63 bp) and three exons (141, 189, and 92 bp) encoding 138 amino acids that encompassed IIS4-IIS6 and the partial linker between domains II and III of VGSC. All the studied populations showed L1014F mutation, substitution of CTT for TTT at the 1014 allele. The result of TaqMan assay for 624 samples showed 96.6% homogenous and 3.36% heterozygous mutant. The development of permethrin resistance and the presence of the L1014F mutation at high frequency in flea populations indicate that pyrethroids are likely ineffective in controlling human flea. Therefore, novel alternative control methods are needed to combat this human ectoparasite.

Susceptibility of Anopheles gambiae to insecticides used for malaria vector control in Rwanda.

BACKGROUND: The widespread emergence of resistance to pyrethroids is a major threat to the gains made in malaria control. To monitor the presence and possible emergence of resistance against a variety of insecticides used for malaria control in Rwanda, nationwide insecticide resistance surveys were conducted in 2011 and 2013. METHODS: Larvae of Anopheles gambiae sensu lato mosquitoes were collected in 12 sentinel sites throughout Rwanda. These were reared to adults and analysed for knock-down and mortality using WHO insecticide test papers with standard diagnostic doses of the recommended insecticides. A sub-sample of tested specimens was analysed for the presence of knockdown resistance (kdr) mutations. RESULTS: A total of 14,311 mosquitoes were tested and from a sample of 1406 specimens, 1165 (82.9%) were identified as Anopheles arabiensis and 241 (17.1%) as Anopheles gambiae sensu stricto. Mortality results indicated a significant increase in resistance to lambda-cyhalothrin from 2011 to 2013 in 83% of the sites, permethrin in 25% of the sites, deltamethrin in 25% of the sites and DDT in 50% of the sites. Mosquitoes from 83% of the sites showed full susceptibility to bendiocarb and 17% of sites were suspected to harbour resistance that requires further confirmation. No resistance was observed to fenitrothion in all study sites during the entire survey. The kdr genotype results in An. gambiae s.s. showed that 67 (50%) possessed susceptibility (SS) alleles, while 35 (26.1%) and 32 (23.9%) mosquitoes had heterozygous (RS) and homozygous (RR) alleles, respectively. Of the 591 An. arabiensis genotyped, 425 (71.9%) possessed homozygous (SS) alleles while 158 (26.7%) and 8 (1.4%) had heterozygous (RS) and homozygous (RR) alleles, respectively. Metabolic resistance involving oxidase enzymes was also detected using the synergist PBO. CONCLUSION: This is the first nationwide study of insecticide resistance in malaria vectors in Rwanda. It shows the gradual increase of insecticide resistance to pyrethroids (lambda-cyhalothrin, deltamethrin, permethrin) and organochlorines (DDT) and the large presence of target site insensitivity. The results demonstrate the need for Rwanda to expand monitoring for insecticide resistance including further metabolic resistance testing and implement an insecticide resistance management strategy to sustain the gains made in malaria control.

The origin and insecticide resistance of Aedes albopictus mosquitoes established in southern Mozambique.

BACKGROUND: The Aedes albopictus mosquito is of medical concern due to its ability to transmit viral diseases, such as dengue and chikungunya. Aedes albopictus originated in Asia and is now present on all continents, with the exception of Antarctica. In Mozambique, Ae. albopictus was first reported in 2015 within the capital city of Maputo, and by 2019, it had become established in the surrounding area. It was suspected that the mosquito population originated in Madagascar or islands of the Western Indian Ocean (IWIO). The aim of this study was to determine its origin. Given the risk of spreading insecticide resistance, we also examined relevant mutations in the voltage-sensitive sodium channel (VSSC). METHODS: Eggs of Ae. albopictus were collected in Matola-Rio, a municipality adjacent to Maputo, and reared to adults in the laboratory. Cytochrome c oxidase subunit I (COI) sequences and microsatellite loci were analyzed to estimate origins. The presence of knockdown resistance (kdr) mutations within domain II and III of the VSSC were examined using Sanger sequencing. RESULTS: The COI network analysis denied the hypothesis that the Ae. albopictus population originated in Madagascar or IWIO; rather both the COI network and microsatellites analyses showed that the population was genetically similar to those in continental Southeast Asia and Hangzhou, China. Sanger sequencing determined the presence of the F1534C knockdown mutation, which is widely distributed among Asian populations, with a high allele frequency (46%). CONCLUSIONS: These results do not support the hypothesis that the Mozambique Ae. albopictus population originated in Madagascar or IWIO. Instead, they suggest that the origin is continental Southeast Asia or a coastal town in China.

Resistance to pyrethroids and the relationship between adult resistance and knockdown resistance (kdr) mutations in Aedes albopictus in dengue surveillance areas of Guizhou Province, China.

The Ae. albopictus mosquito has gained global attention due to its ability to transmit viruses, including the dengue and zika. Mosquito control is the only effective way to manage dengue fever, as no effective treatments or vaccines are available. Insecticides are highly effective in controlling mosquito densities, which reduces the chances of virus transmission. However, Ae. albopictus has developed resistance to pyrethroids in several provinces in China. Pyrethroids target the voltage-gated sodium channel gene (VGSC), and mutations in this gene may result in knockdown resistance (kdr). Correlation studies between resistance and mutations can assist viruses in managing Ae. albopictus, which has not been studied in Guizhou province. Nine field populations of Ae. albopictus at the larval stage were collected from Guizhou Province in 2022 and reared to F1 to F2 generations. Resistance bioassays were conducted against permethrin, beta-cypermethrin, and deltamethrin for both larvae and adults of Ae. albopictus. Kdr mutations were characterized by PCR and sequencing. Additionally, the correlation between the kdr allele and pyrethroid resistance was analyzed. All nine populations of Ae. albopictus larvae and adults were found to be resistant to three pyrethroid insecticides. One kdr mutant allele at codon 1016, one at 1532 and three at 1534 were identified with frequencies of 13.86% (V1016G), 0.53% (I1532T), 58.02% (F1534S), 11.69% (F1534C), 0.06% (F1534L) and 0.99% (F1534P), respectively. Both V1016G and F1534S mutation mosquitoes were found in all populations. The kdr mutation F1534S was positively correlated with three pyrethroid resistance phenotypes (OR > 1, P < 0.05), V1016G with deltamethrin and beta-cypermethrin resistance (OR > 1, P < 0.05) and F1534C only with beta-cypermethrin resistance (OR > 1, P < 0.05). Current susceptibility status of wild populations of Ae. albopictus to insecticides and a higher frequency of kdr mutations from dengue-monitored areas in Guizhou Province are reported in this paper. Outcomes of this study can serve as data support for further research and development of effective insecticidal interventions against Ae. albopictus populations in Guizhou Province.

Metabolic Resistance and Not Voltage-Gated Sodium Channel Gene Mutation Is Associated with Pyrethroid Resistance of Aedes albopictus (Skuse, 1894) from Cambodia.

(1) Background: In Cambodia, Aedes albopictus is an important vector of the dengue virus. Vector control using insecticides is a major strategy implemented in managing mosquito-borne diseases. Resistance, however, threatens to undermine the use of insecticides. In this study, we present the levels of insecticide resistance of Ae. albopictus in Cambodia and the mechanisms involved. (2) Methods: Two Ae. albopictus populations were collected from the capital, Phnom Penh city, and from rural Pailin province. Adults were tested with diagnostic doses of malathion (0.8%), deltamethrin (0.03%), permethrin (0.25%), and DDT (4%) using WHO tube assays. Synergist assays using piperonyl butoxide (PBO) were implemented before the pyrethroid assays to detect the potential involvement of metabolic resistance mechanisms. Adult female mosquitoes collected from Phnom Penh and Pailin were tested for voltage-gated sodium channel (VGSC) kdr (knockdown resistance) mutations commonly found in Aedes sp.-resistant populations throughout Asia (S989P, V1016G, and F1534C), as well as for other mutations (V410L, L982W, A1007G, I1011M, T1520I, and D1763Y). (3) Results: The two populations showed resistance against all the insecticides tested (<90% mortality). The use of PBO (an inhibitor of P450s) strongly restored the efficacy of deltamethrin and permethrin against the two resistant populations. Sequences of regions of the vgsc gene showed a lack of kdr mutations known to be associated with pyrethroid resistance. However, four novel non-synonymous mutations (L412P/S, C983S, Q1554STOP, and R1718L) and twenty-nine synonymous mutations were detected. It remains to be determined whether these mutations contribute to pyrethroid resistance. (4) Conclusions: Pyrethroid resistance is occurring in two Ae. albopictus populations originating from urban and rural areas of Cambodia. The resistance is likely due to metabolic resistance specifically involving P450s monooxygenases. The levels of resistance against different insecticide classes are a cause for concern in Cambodia. Alternative tools and insecticides for controlling dengue vectors should be used to minimize disease prevalence in the country.

The Effect of Plasmodium falciparum (Welch) (Haemospororida: Plasmodiidae) Infection on the Susceptibility of Anopheles gambiae s.l. and Anopheles funestus (Diptera: Culicidae) to Pyrethroid Insecticides in the North-Western and South-Eastern, Tanzania.

The rapid development of insecticide resistance in malaria vectors threatens insecticide-based interventions. It is hypothesized that infection of insecticide-resistant vectors with Plasmodium parasites increases their vulnerability to insecticides, thus assuring the effectiveness of insecticide-based strategies for malaria control. Nonetheless, there is limited field data to support this. We investigated the effect of the Plasmodium falciparum infection on the susceptibility of Anopheles gambiae s.l. and Anopheles funestus to pyrethroids in south-eastern (Kilombero) and north-western (Muleba), Tanzania. The wild-collected mosquitoes were tested against 0.05% deltamethrin and 0.75% permethrin, then assessed for sporozoite rate and resistant gene (kdr) mutations. All Anopheles gambiae s.l. from Kilombero were An. arabiensis (Patton, 1905) while those from Muleba were 87% An. gambiae s.s (Giles, 1902) and 13% An. Arabiensis. High levels of pyrethroid resistance were observed in both areas studied. The kdr mutation was only detected in An. gambiae s.s. at the frequency of 100% in survivors and 97% in dead mosquitoes. The P. falciparum sporozoite rates were slightly higher in susceptible than in resistant mosquitoes. In Muleba, sporozoite rates in An. gambiae s.l. were 8.1% and 6.4% in dead mosquitoes and survivors, respectively (SRR = 1.28, p = 0.19). The sporozoite rates in Kilombero were 1.3% and 0.7% in the dead and survived mosquitoes, respectively (sporozoite rate ratio (SRR) = 1.9, p = 0.33). In An. funestus group sporozoite rates were 6.2% and 4.4% in dead and survived mosquitoes, respectively (SRR = 1.4, p = 0.54). These findings indicate that insecticides might still be effective in malaria control despite the rapid development of insecticide resistance in malaria vectors.

Status of Acaricide Resistance and Detecting the Knockdown Resistance Mutation T2134A in the Cattle Tick Rhipicephalus microplus (Acari: Ixodidae) from Northeastern Mexico.

Rhipicephalus microplus is the most important tick in veterinary medicine, given its repercussions on animal production. The principal strategy to avoid adverse effects associated with R. microplus is the chemical control of tick populations through organosynthetic acaricides. Therefore, monitoring susceptibility to acaricides is paramount in any control program. This study aimed to analyze the resistance status of 2 populations of R. microplus from northeastern Mexico to the organochlorine (OC) lindane, organophosphates (OP) coumaphos, chlorfenvinphos, diazinon, and chlorpyrifos, and the synthetic pyrethroids (SPs) flumethrin, deltamethrin, and cypermethrin. Discriminating doses (DD) of each acaricide were used in the larval packet bioassay (LPT). Additionally, the presence of the knockdown resistance (kdr) mutation T2134A associated with pyrethroid resistance was evaluated using allele-specific polymerase chain reaction (PCR). The populations of R. microplus showed a high frequency of resistance to SP, with mortality rates of less than 5%; they also showed resistance to the OPs (diazinon and chlorpyrifos) with mortality rates ranging from 1.29% to 34.62%; meanwhile, they were susceptible to coumaphos and chlorfenvinphos. Mortality rates higher than 66% were observed for lindane, indicating susceptibility. The mutant allele of the kdr mutation T2134A was detected in 75% and 100% of the pools analyzed. The populations studied presented a highly resistant profile to pyrethroids, with the presence of the kdr mutant allele A2134. The susceptibility to the organophosphates such as coumaphos and chlorfenvinphos of R. microplus from northeastern Mexico should be noted.

Co-occurrence of multiple kdr mutations (F1534C, V1016I, V410L) in Aedes aegypti from coastal areas in Ghana and assessment of the role of mosquito coil in causing pyrethroid resistance.

The rapid spread of knockdown-resistance (kdr) mutations in Africa calls for monitoring and investigation into the cause of pyrethroid resistance to inform management strategies. This study investigated the pyrethroid resistance profile of Aedes aegypti from coastal towns in Ghana and the impact of mosquito coil, a popular household pyrethroid-based anti-mosquito tool, on the development of pyrethroid resistance. Susceptibility to deltamethrin and the presence of kdr mutations was determined in adult female mosquitoes reared from larvae. Furthermore, the LT50 of a mosquito coil (0.08% meperfluthrin) against a laboratory colony was determined, and the value was used as a sublethal dose in an experimental study. The laboratory colony of Ae. aegypti was exposed to the sublethal dose of the coil once per generation for six generations (F6). The susceptibility of the exposed colony to deltamethrin (0.05%) was determined. The Ae. aegypti populations from the coastal towns were resistant to deltamethrin with co-occurrence of F1534C, V1016I and V410L kdr mutations. In the experimental study, the LT50 (95% CI) of the selected colony against the coil rose from 8 minutes (95% CI; 6-9) at F0 to 28 minutes (95% CI; 23-34) at F6. Nonetheless, deltamethrin caused similar mortalities in the selected and control colonies. The mutant allele frequencies of 1534C and 410L were similar but 1016I was higher in the selected colony (17%) than in the control (5%). However, the increased tolerance to the coil and high mutant allele frequency of 1016I in the selected colony did not affect the mosquito's resistance level to deltamethrin insecticide. Further study is needed to elucidate the role of pyrethroid-based mosquito coils in the development of insecticide resistance in mosquito vectors.

Mutations of voltage-gated sodium channel contribute to pyrethroid resistance in Panonychus citri.

Insecticide resistance in Panonychus citri is a major obstacle to mite control in citrus orchards. Pyrethroid insecticides are continually used to control mites in China, although resistance to pyrethroids has evolved in some populations. Here, the resistance to the pyrethroid fenpropathrin was investigated and 7 out of 8 field-collected populations of P. citri exhibited a high level of resistance, ranging from 171-fold to 15 391-fold higher than the susceptible (SS) comparison strain. Three voltage-gated sodium channel (VGSC) mutations were identified in the tested populations: L1031V, F1747L, and F1751I. Amplicon sequencing was used to evaluate the frequency of these mutations in the 19 field populations. L1031V and F1751I were present in all populations at frequencies of 11.6%-82.1% and 0.5%-31.8%, respectively, whereas the F1747L mutation was only present in 12 populations from Chongqing, Sichuan, Guangxi, and Yunnan provinces. Introduction of these mutations singly or in combination into transgenic flies significantly increased their resistance to fenpropathrin and these flies also exhibited reduced mortality after exposure to the pyrethroids permethrin and ß-cypermethrin. Panonychus citri VGSC homology modeling and ligand docking indicate that F1747 and F1751 form direct binding contacts with pyrethroids, which are lost with mutation, whereas L1031 mutation may diminish pyrethroid effects through an allosteric mechanism. Overall, the results provide molecular markers for monitoring pest resistance to pyrethroids and offer new insights into the basis of pyrethroid actions on sodium channels.

Community structure and insecticide resistance of malaria vectors in northern-central Myanmar.

BACKGROUND: Myanmar is one of the six countries in the Greater Mekong Subregion (GMS) of Southeast Asia. Malaria vectors comprise many Anopheles species, which vary in abundance and importance in malaria transmission among different geographical locations in the GMS. Information about the species composition, abundance, and insecticide resistance status of vectorial systems in Myanmar is scarce, hindering our efforts to effectively control malaria vectors in this region. METHODS: During October and November 2019, larvae and adult females of Anopheles mosquitoes were collected in three sentinel villages of Banmauk township in northern Myanmar. Adult female mosquitoes collected by cow-baited tent collection (CBTC) and adults reared from field-collected larvae (RFCL) were used to determine mortality rates and knockdown resistance (kdr) against deltamethrin using the standard WHO susceptibility test. Molecular species identification was performed by multiplex PCR and ITS2 PCR, followed by DNA sequencing. The kdr mutation at position 1014 of the voltage-gated sodium channel gene was genotyped by DNA sequencing for all Anopheles species tested. RESULTS: A total of 1596 Anopheles mosquitoes from seven morphologically identified species groups were bioassayed. Confirmed resistance to deltamethrin was detected in the populations of An. barbirostris (s.l.), An. hyrcanus (s.l.), and An. vagus, while possible resistance was detected in An. annularis (s.l.), An. minimus, and An. tessellatus. Anopheles kochi was found susceptible to deltamethrin. Compared to adults collected by CBTC, female adults from RFCL had significantly lower mortality rates in the four species complexes. A total of 1638 individuals from 22 Anopheles species were molecularly identified, with the four most common species being An. dissidens (20.5%) of the Barbirostris group, An. peditaeniatus (19.4%) of the Hyrcanus group, An. aconitus (13.4%) of the Funestus group, and An. nivipes (11.5%) of the Annularis group. The kdr mutation L1014F was only detected in the homozygous state in two An. subpictus (s.l.) specimens and in a heterozygous state in one An. culicifacies (s.l.) specimen. CONCLUSIONS: This study provides updated information about malaria vector species composition and insecticide resistance status in northern Myanmar. The confirmed deltamethrin resistance in multiple species groups constitutes a significant threat to malaria vector control. The lack or low frequency of target-site resistance mutations suggests that other mechanisms are involved in resistance. Continual monitoring of the insecticide resistance of malaria vectors is required for effective vector control and insecticide resistance management.

Detection and population genetic analysis of kdr L1014F variant in eastern Ethiopian Anopheles stephensi.

Anopheles stephensi is a malaria vector that has been recently introduced into East Africa, where it threatens to increase malaria disease burden. The use of insecticides, especially pyrethroids, is still one of the primary malaria vector control strategies worldwide. The knockdown resistance (kdr) mutation in the IIS6 transmembrane segment of the voltage-gated sodium channel (vgsc) is one of the main molecular mechanisms of pyrethroid resistance in Anopheles. Extensive pyrethroid resistance in An. stephensi has been previously reported in Ethiopia. Thus, it is important to determine whether or not the kdr mutation is present in An. stephensi populations in Ethiopia to inform vector control strategies. In the present study, the kdr locus was analyzed in An. stephensi collected from ten urban sites (Awash Sebat Kilo, Bati, Dire Dawa, Degehabur, Erer Gota, Godey, Gewane, Jigjiga, Semera, and Kebridehar) situated in Somali, Afar, and Amhara regions, and Dire Dawa Administrative City, to evaluate the frequency and evolution of kdr mutations and the association of the mutation with permethrin resistance phenotypes. Permethrin is one of the pyrethroid insecticides used for vector control in eastern Ethiopia. DNA extractions were performed on adult mosquitoes from CDC light trap collections and those raised from larval and pupal collections. PCR and targeted sequencing were used to analyze the IIS6 transmembrane segment of the vgsc gene. Of 159 An. stephensi specimens analyzed from the population survey, nine (5.7%) carried the kdr mutation (L1014F). An. stephensi with kdr mutations were only observed from Bati, Degehabur, Dire Dawa, Gewane, and Semera. We further selected randomly twenty resistant and twenty susceptible An. stephensi mosquitoes from Dire Dawa post-exposure to permethrin and investigated the role of kdr in pyrethroid resistance by comparing the vgsc gene in the two populations. We found no kdr mutations in the permethrin-resistant mosquitoes. Population genetic analysis of the sequences, including neighboring introns, revealed limited evidence of non-neutral evolution (e.g., selection) at this locus. The low kdr mutation frequency detected and the lack of kdr mutation in the permethrin-resistant mosquitoes suggest the existence of other molecular mechanisms of pyrethroid resistance in eastern Ethiopian An. stephensi.