Target Site Insensitivity Research Articles

Evidence of Permethrin Resistance and Fipronil Tolerance in Rhipicephalus sanguineus s.l. (Acari: Ixodidae) Populations From Florida and California.

Rhipicephalus sanguineus s.l. (Latreille), is a vector of multiple disease-causing pathogens to humans and dogs. Permethrin and fipronil are two acaricides used to manage R. sanguineus s.l. infestations. Homeowners have reported treatment failures in managing brown dog ticks using permethrin and fipronil based products. Previous studies demonstrated that high permethrin resistance in some R. sanguineus s.l. populations was due to metabolic detoxification and target site insensitivity. In this study, three R. sanguineus s.l. strains, one from a laboratory colony (NC) and two colonies originally collected from Florida (FL) and California (CA), were evaluated for resistance expression against permethrin and fipronil. Metabolic detoxification mechanisms were evaluated in the FL strain using three synergists, while a polymerase chain reaction assay was used to detect a resistance mutation in all strains. The NC strain was susceptible to both permethrin and fipronil, while both the FL and CA strains exhibited high resistance to permethrin and tolerance to fipronil. The synergist tests and PCR results indicated that the FL strain utilized both metabolic resistance and target site insensitivity against permethrin, while the CA strain was documented to have the target-site insensitivity resistant allele. This study confirmed permethrin resistance in both California and Florida populations and its persistence in Florida populations, although its susceptibility can potentially be increased by adding a synergist. Fipronil resistance was not detected suggesting this acaricide may provide suitable tick control.

Global Pattern of kdr-Type Alleles in Musca domestica (L.).

Houseflies, Musca domestica L., are an important sanitary pest that affects human and domesticated animals. They are mechanical carriers of more than 100 human and animal diseases including protozoan, bacterial, helminthic, and viral infections. Recently, it was demonstrated that houseflies acquired, harbored, and transmitted SARS-CoV-2 (COVID-19) for up to 1day post-exposure. The most widely used control strategy relays on the application of pyrethroid insecticides due to their effectiveness, low mammalian toxicity, low cost, and environmental safety. The main mechanism of action of pyrethroids is to exert their toxic effects through affecting the voltage-sensitive sodium channel (VSSC) modifying the transmission of the nerve impulse and leading to the death of the insects. Target site insensitivity of the VSSC is due to the presence of single nuclear polymorphisms (SNPs) named knockdown mutations (kdr). In this review, we synthetize recent data on the type and distribution of these mutations globally. Housefly resistance is reported in several countries. Increased applications of pyrethroids to control housefly populations led to the emergence of multiple evolutionary origins of resistance determined by five amino acid substitutions or specific mutations in the VSSC: kdr (L1014F), kdr-his (L1014H), super-kdr (M918T + L1014F), type N (D600N + M918T + L1014F), and 1B (T929I + L1014F). According to the global map obtained, high levels of resistance to pyrethroids are associated with the L1014F mutation found mostly in North America, Europe, and Asia, while the super-kdr mutation was mostly found in the American continent. The level of protection conferred by these alleles against pyrethroids was generally kdr-his < kdr < Type N ≤ super-kdr ≤ 1B. The relative fitness of the alleles under laboratory conditions was susceptible ≅ kdr-his > kdr > super-kdr suggesting that the fitness cost of an allele was relative to the presence of other alleles in a population and that the reversion of resistance in a free insecticide environment might be quite variable from one region to another. An adequate integrated pest management program should consider monitoring susceptibility to pyrethroids to detect early levels of resistance and predict the spread and evolution of resistant phenotypes and genotypes. From this review, the pyrethroid resistance status of housefly population was determined in very few countries and has evolved independently in different areas of the world affecting chemical control programs.

Defence mitigation by predators of chemically defended prey integrated over the predation sequence and across biological levels with a focus on cardiotonic steroids.

Predator–prey interactions have long served as models for the investigation of adaptation and fitness in natural environments. Anti-predator defences such as mimicry and camouflage provide some of the best examples of evolution. Predators, in turn, have evolved sensory systems, cognitive abilities and physiological resistance to prey defences. In contrast to prey defences which have been reviewed extensively, the evolution of predator counter-strategies has received less attention. To gain a comprehensive view of how prey defences can influence the evolution of predator counter-strategies, it is essential to investigate how and when selection can operate. In this review we evaluate how predators overcome prey defences during (i) encounter, (ii) detection, (iii) identification, (iv) approach, (v) subjugation, and (vi) consumption. We focus on prey that are protected by cardiotonic steroids (CTS)—defensive compounds that are found in a wide range of taxa, and that have a specific physiological target. In this system, coevolution is well characterized between specialist insect herbivores and their host plants but evidence for coevolution between CTS-defended prey and their predators has received less attention. Using the predation sequence framework, we organize 574 studies reporting predators overcoming CTS defences, integrate these counter-strategies across biological levels of organization, and discuss the costs and benefits of attacking CTS-defended prey. We show that distinct lineages of predators have evolved dissecting behaviour, changes in perception of risk and of taste perception, and target-site insensitivity. We draw attention to biochemical, hormonal and microbiological strategies that have yet to be investigated as predator counter-adaptations to CTS defences. We show that the predation sequence framework will be useful for organizing future studies of chemically mediated systems and coevolution.

Metabolic detoxification and ace-1 target site mutations associated with acetamiprid resistance in Aedes aegypti L.

Despite the continuous use of chemical interventions, Aedes-borne diseases remain on the rise. Neonicotinoids are new, safer, and relatively effective pharmacological interventions against mosquitoes. Neonicotinoids interact with the postsynaptic nicotinic acetylcholine receptors (nAChRs) of the insect central nervous system, but the absence of nAChR polymorphism in resistant phenotypes makes their involvement in neonicotinoid resistance uncertain. Thus, an investigation was carried out to understand the role of metabolic detoxification and target site insensitivity in imparting acetamiprid resistance in Aedes aegypti larvae. Studies were conducted on the parent susceptible strain (PS), acetamiprid-larval selected strain for five generations (ACSF-5; 8.83-fold resistance) and 10 generations (ACSF-10; 19.74-fold resistance) of Ae. aegypti. The larval selection raised α-esterase and β-esterase activities by 1.32-fold and 1.34-fold, respectively, in ACSF-10 as compared to PS, while the corresponding glutathione-S-transferase and acetylcholinesterase activity increased by 22.5 and 2%. The ace-1 gene in PS and ACSF-10 showed four mismatches in the 1312—1511 bp region due to mutations in the Y455C codon (tyrosine to cysteine) at the 1367th position (TAC→TGC); I457V codon (isoleucine to valine) at 1372 bp and 1374 bp (ATA→GTG); and R494M codon (arginine to methionine) at 1484 bp (AGG→ATG). The R494M mutation was the novel and dominant type, observed in 70% ACSF-10 population, and has not been reported so far. The studies evidenced the combination of metabolic detoxification and target site mutation in imparting acetamiprid resistance in Ae. aegypti.

Overexpression of glutamate-gated chloride channel in the integument is mainly responsible for emamectin benzoate resistance in the western flower thrips Frankliniella occidentalis.

Western flower thrips Frankliniella occidentalis is a serious polyphagous pest worldwide. In this study, we investigated the potential mechanisms of resistance including enhanced metabolism and target site insensitivity in an emamectin benzoate (EB)-resistant (EB-R) strain. The EB-R strain of F. occidentalis showed 356-fold increased resistance compared to a susceptible RDA strain. Analysis of cross-resistance to four other insecticides confirmed that EB resistance is highly specific to the contact toxicity of EB. Synergistic bioassay and quantitative PCR of cytochrome P450 monooxygenase (CYP) genes revealed that three overexpressed Cyps were likely involved in resistance. Among three putative glutamate-gated chloride channel (GluCl) genes identified, FoGluClc showed four radical amino acid substitutions and 3.8-fold and 31-fold transcription level in the head and integument in the EB-R strain when compared to the RDA strain. Backcrossing analysis and RNA interference confirmed that both amino acid substitution and overexpression of FoGluClc are responsible for EB resistance. In situ hybridization revealed that FoGluClc is mainly distributed in the integument in the EB-R strain. Cross-comparison of known genomes and transcriptomes of thrips species revealed that FoGluClc is unique to the Frankliniella genus. While mutations and overexpression of FoGluClc play major roles in EB resistance, the overexpressed Cyps are partially involved as metabolic factors. Higher expression of FoGluClc in the integument may suggest its role in the first-line defense against EB in the EB-R strain. Unique distribution of FoGluClc in the Frankliniella genus but not in other thrips species further suggests that FoGluClc may be a surplus channel not having an essential endogenous function and is thus recruited as a defense barrier against xenobiotics. © 2022 Society of Chemical Industry.

Combined metabolic and target-site resistance mechanisms confer fipronil and deltamethrin resistance in field-collected German cockroaches (Blattodea: Ectobiidae)

Despite insecticide resistance issues, pyrethroids and fipronil have continued to be used extensively to control the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae) for more than two decades. We evaluated the physiological insecticide resistance in five German cockroach populations collected from 2018 to 2020 and measured the extent of metabolic detoxification and target-site insensitivity resistance mechanisms. Topically applied doses of the 3 x LD95 of deltamethrin, fipronil, DDT, or dieldrin of a susceptible strain (UCR, Diagnostic Dose) failed to cause >23% mortality, and the 10 x LD95 of deltamethrin or fipronil failed to cause >53% mortality. All field-collected strains possessed a combination of metabolic and target-site insensitivity mechanisms that cause reduced susceptibility. Elevated activities of esterase and glutathione S-transferase were measured, and the synergists piperonyl butoxide or S,S,S-tributyl phosphorotrithioate increased topical mortality up to 100% for deltamethrin and 93% for fipronil 10 x LD95. The target-site mutations L993F of the para-homologous sodium channel and A302S of the GABA-gated chloride channel associated with pyrethroid and fipronil resistance, respectively, were found at ~80–100% frequency in field populations. Pyrethroid and fipronil spray formulations also were ineffective in a choice box assay against field-collected strains suggesting that these treatments would fail to control cockroaches under field conditions.

Pyrethroid-linked resistance allelic mutations by molecular analysis in wild human head louse (Phthiraptera: Pediculidae) populations from schoolgirls of South Iran

BackgroundHuman head louse, Pediculus humanus capitis De Geer, 1767 (Anoplura: Pediculidae), is one of the most frequent ectoparasites infesting Homo sapiens worldwide. Reduced sensitivity to treatment due to genetic mutations, in particular knockdown resistance (kdr) (or target site insensitivity) allele, has led to this infestation prevalence. Molecular characterization of this resistance has a crucial impact on selecting appropriate treatment protocol. The aim of this study was to investigate kdr gene mutations on voltage-sensitive sodium channel (VSSC) among wild head lice samples from Fars province, southern Iran. MethodsHead lice were collected using plastic detection combs on girls enrolled in public schools from 10 counties in Fars province. The specimens were screened in 10 pools (each pool per county containing 35 specimens), with three pools (30%) being positive. Following species identification with valid entomological keys, 350 (68%) out of 514 randomly collected adult head lice were analyzed after their somatic genomic DNA extraction using Sinaclon kit. Samples were investigated by polymerase chain reactions (PCR), and the amplicon was subsequently sequenced. ResultsSequence analysis showed that the sodium channel genes in the pooled ectoparasites had two intron and three exon regions. Single (L840F), double (I836L, E837K), and triple novel point mutations (V875L, Q876P, S879V); the last involving two concomitant allelic substitutions; were discovered in the second and third exon regions of head louse DNA on chromosome II from three (30%) counties. Other exon or intron regions remained non-mutated from the remaining seven counties. ConclusionsThe detection of six amino acid substitutions from 30% of examined head lice among infested schoolgirls reveal that mutants are minutely developing. These findings provide further incentive to recapitulate the legitimacy of current control measures and resolve dynamics of resistance in human head louse populations.

Demonstrating the role of symbionts in mediating detoxification in herbivores

Plant toxins constitute an effective defense against herbivorous animals. However, many herbivores have evolved adaptations to cope with dietary toxins through detoxification, excretion, sequestration, target site insensitivity and/or via behavioral avoidance. While these adaptations are often directly encoded in herbivore genomes, evidence is accumulating that microbial symbionts can reduce the dose of plant toxins by metabolizing or sequestering them prior to absorption by the herbivore. Here, we describe a few well-studied examples to assess such symbiont-mediated detoxification and showcase different approaches that have been used for their analyses. These include: (i) a host phenotypic route in which the symbiotic association is manipulated to reveal host fitness costs upon toxin exposure in the presence/absence of detoxifying symbionts, including function restoration after symbiont re-infection, (ii) a molecular microbiological approach that focuses on the identification and characterization of microbial genes involved in plant toxin metabolism, and (iii) an analytical chemical route that aims to characterize the conversion of the toxin to less harmful metabolites in vivo and link conversion to the activities of a detoxifying symbiont. The advantages and challenges of each approach are discussed, and it is argued that a multi-pronged strategy combining phenotypic, molecular, and chemical evidence is needed to unambiguously demonstrate microbial contributions to plant toxin reduction and the importance of these processes for host fitness. Given the interdisciplinary nature of the topic, we aim to provide a guideline to researchers interested in symbiont-mediated detoxification and hope to encourage future studies that contribute to a more comprehensive and mechanistic understanding of detoxification in herbivores and their symbionts.

Characterization of the glutathione S-transferase genes in the sand flies Phlebotomus papatasi and Lutzomyia longipalpis shows expansion of the novel glutathione S-transferase xi (X) class.

Leishmaniasis control often relies upon insecticidal control of phlebotomine sandfly vector populations. Such methods are vulnerable to the evolution of insecticide resistance via a range of molecular mechanisms. There is evidence that two major resistance mechanisms, target site insensitivity and metabolic resistance, have evolved in some sandfly populations and further genetic characterization of resistance would be useful to understand and combat it. To facilitate the study of the mechanisms of metabolic resistance, here we improved the annotation and characterized a major detoxification gene family, the glutathione‐s‐transferases (GST), in the genomes of two sand fly species: Phlebotomus papatasi and Lutzomyia longipalpis. The compositions of the GST gene family differ markedly from those of Aedes and Anopheles mosquitoes. Most strikingly, the xi (X) class of GSTs appears to have expanded in both sand fly genomes. Our results provide a basis for further studies of metabolic resistance mechanisms in these important disease vector species.

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.

Trade-offs between cost of ingestion and rate of intake drive defensive toxin use.

Animals that ingest toxins can become unpalatable and even toxic to predators and parasites through toxin sequestration. Because most animals rapidly eliminate toxins to survive their ingestion, it is unclear how populations transition from susceptibility and toxin elimination to tolerance and accumulation as chemical defence emerges. Studies of chemical defence have generally focused on species with active toxin sequestration and target-site insensitivity mutations or toxin-binding proteins that permit survival without necessitating toxin elimination. Here, we investigate whether animals that presumably rely on toxin elimination for survival can use ingested toxins for defence. We use the A4 and A3 Drosophila melanogaster fly strains from the Drosophila Synthetic Population Resource (DSPR), which respectively possess high and low metabolic nicotine resistance among DSPR fly lines. We find that ingesting nicotine increased A4 but not A3 fly survival against Leptopilina heterotoma wasp parasitism. Further, we find that despite possessing genetic variants that enhance toxin elimination, A4 flies accrued more nicotine than A3 individuals, likely by consuming more medium. Our results suggest that enhanced toxin metabolism can allow greater toxin intake by offsetting the cost of toxin ingestion. Passive toxin accumulation that accompanies increased toxin intake may underlie the early origins of chemical defence.

Kdr genotyping (V1016I, F1534C) of the Nav channel of Aedes aegypti (L.) mosquito populations in Harris County (Houston), Texas, USA, after Permanone 31-66 field tests and its influence on probability of survival.

Aedes aegypti (L.) is an important mosquito vector of emerging arboviruses such as Zika, dengue, yellow fever, and chikungunya. To quell potential disease outbreaks, its populations are controlled by applying pyrethroid insecticides, which selection pressure may lead to the development of insecticide resistance. Target site insensitivity to pyrethroids caused by non-synonymous knockdown resistance (kdr) mutations in the voltage-gated sodium (NaV) channel is a predominant mechanism of resistance in mosquitoes. To evaluate the potential impact of pyrethroid resistance on vector control, Ae. aegypti eggs were collected from eight mosquito control operational areas in Harris County, Texas, and emerged females were treated in field tests at four different distances from the pyrethroid Permanone 31–66 source. The females were genotyped by melting curve analyses to detect two kdr mutations (V1016I and F1534C) in the NaV channel. Harris County females had higher survivorship rates at each distance than the pyrethroid-susceptible Orlando strain females. Survivorship increased with distance from the pyrethroid source, with 39% of field-collected mosquitoes surviving at 7.62 m and 82.3% at 22.86 m from the treatment source. Both the V1016I and F1534C pyrethroid resistant genotypes were widely distributed and at high frequency, with 77% of the females being double homozygous resistant (II/CC), this being the first report of kdr mutations in Ae. aegypti in Harris County. Analysis of the probability of survival for each mutation site independently indicated that the CC genotype had similar probability of survival as the FC heterozygous, while the II genotype had higher survival than both the VI and VV, that did not differ. The double homozygous resistant genotype (II/CC) had the highest probability of survival. A linear model estimated probability of survival for areas and genotypes. The high frequency and widespread distribution of double-homozygote pyrethroid-resistant Ae. aegypti may jeopardize disease vector control efforts in Harris County.

Sequence variation and regulatory variation in acetylcholinesterase genes contribute to insecticide resistance in different populations of Leptinotarsa decemlineata.

Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos‐methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance‐associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos‐methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides.

Convergent evolution of cardiac-glycoside resistance in predators and parasites of milkweed herbivores

The community of plant-feeding insects (herbivores) that specialize on milkweeds (Apocynaceae) form a remarkable example of convergent evolution across levels of biological organization1. In response to toxic cardiac glycosides produced by these plants, the monarch butterfly (Danaus plexippus) and other specialist herbivores have evolved parallel substitutions in the alpha subunit (ATPA) of the Na+/K+-ATPase. These substitutions render the pump insensitive to cardiac glycosides2,3, allowing the monarch and other specialists, from aphids to beetles, to sequester cardiac glycosides, which in turn provide defense against attacks by enemies from the third trophic level4. The evolution of 'target-site-insensitivity' substitutions in these herbivores poses a fundamental biological question: have predators and parasitoids that feed on cardiac-glycoside-sequestering insects also evolved Na+/K+-ATPases that are similarly insensitive to cardiac glycosides (as predicted by Whiteman and Mooney)5? In other words, can plant toxins cause evolutionary cascades that reach the third trophic level? Here we show that at least four enemies of the monarch and other milkweed herbivores have indeed evolved amino-acid substitutions associated with target-site insensitivity to cardiac glycosides. These attackers represent four major animal clades, implicating cardiac glycosides as keystone molecules6 and establishing ATPalpha, which encodes ATPA, as a keystone gene with effects that reverberate within ecological communities7.

Preliminary characterisation of known pesticide resistance alleles in Spodoptera frugiperda (Lepidoptera: Noctuidae) in its invasive Australian range

AbstractFall armyworm (Spodoptera frugiperda) has recently been detected in Australia. Globally, S. frugiperda is reported to be resistant to insecticides, including those permitted for its control by the Australian Pesticides and Veterinary Medicines Authority. Consequently, an understanding of the insecticide resistance status of newly migrated S. frugiperda into Australia, as well as an ability to accurately identify newly hatched larvae, would facilitate sustainable management. To aid identification, we developed a real‐time polymerase chain reaction diagnostic assay that can identify S. frugiperda species, irrespective of life stage, and distinguish between the rice and corn strains. We then screened S. frugiperda individuals from Queensland, Western Australia, the Northern Territory and New South Wales for mutations causing target site insensitivity in the acetylcholinesterase (AChE) gene, the voltage‐gated sodium channel gene and the ryanodine receptor gene. In the populations tested, we found that mutation at positions A210S and F290V in AChE were common. These mutations are known to be associated with carbamate and organophosphate resistance in S. frugiperda outside Australia. In contrast, no mutation was found in gene voltage‐gated sodium channel causing pyrethroid resistance or the ryanodine receptor gene associated with diamide resistance. As S. frugiperda is new to Australia, the wide distribution of mutations in the AChE gene associated with organophosphate/carbamate resistance suggests that the original migrated S. frugiperda moths may have carried the mutations from outside Australia. As resistance genes have been detected, it is important to continue to monitor the resistance status of Australian S. frugiperda and further integrate complementary bioassay when available. Finally, as pesticide resistance is possible, it should be additionally considered when making pest management decisions and any spray failure should be followed by a pesticide from a different chemical group.

Analysis of the insecticide resistance mechanism in Anopheles culicifacies sensu lato from a malaria-endemic state in India.

Understanding the dynamics and mechanisms of insecticide resistance in malaria vectors is crucial for vector control activities. The present study investigates the level of insecticide resistance in Anopheles culicifacies and explores the role of two main mechanisms in conferring resistance target site insensitivity and metabolic resistance. A. culicifacies mosquitoes were collected and the voltage-gated sodium channel (VGSC) gene was amplified and sequenced to analyse the knockdown resistance (kdr) mutations. Further, a non-experimental homology model was generated to investigate the effect of kdr mutations on the conformation of protein. Metabolic resistance was determined using bioassay-based resistant and susceptible mosquitoes and the expression levels of the genes CYP6Z1 and GSTe2 were compared between the two groups. Sequence analysis of the VGSC gene revealed the presence of L1014F (n=48 [17%]), L1014S and V1010L (n=5 [1.7%]) mutations in the study area. In gene expression studies, a significant upregulation of CYP6Z1 in deltamethrin-resistant (fold change 243.62; p=0.02) mosquitoes and that of GSTe2 in dichlorodiphenyltrichloroethane (fold change 403.45; p=0.01) and alpha-cypemethrin resistant (fold change 217.51; p=0.0005) mosquitoes was observed. The study revealed that expression of the genes (CYP6Z1 and GSTe2) conferring metabolic resistance play a key role in insecticide resistance in A. culicifacies populations in central India. However, mutations L101F, L10104S and V10101L also have a role to some extent in spreading resistance. GeneBank accession numbers: MW559058, MW559059 and MW559060 Cover Image: Workflow of Chimera-Modeller interface. In the top window of Chimera's multi-align viewer the sequence alignment of VGSC proteins of human (pdb id_6AGF), cockroach (pdb id 5XOM) and A. culicifacies (ACT176122.1) is shown. The dialog box in the middle is of the comparative modelling tool of Modeller. The A. culicifacies sequence is designated as the target while human and cockroach sequences are templates. Upon selection of the template sequences in the dialog box, the structures of the respective proteins are displayed in the Chimera window. As the run is completed, the results are displayed in the form of a list of models with their scores in a table.

Cuticle thickening associated with fenitrothion and imidacloprid resistance and influence of voltage-gated sodium channel mutations on pyrethroid resistance in the tropical bed bug, Cimex hemipterus.

The common bed bug, Cimex lectularius L., and the tropical bed bug, Cimex hemipterus (F.), are now widely regarded as important public health pests following their rapid global resurgence, largely due to insecticide resistance and an increased rate of global travel. The insecticide resistance mechanisms are well documented in C. lectularius, however, only one mechanism is validated in C. hemipterus thus far. This demands further understanding on the resistance mechanisms involved in C. hemipterus. Here, we identified differences in resistance to fenitrothion (organophosphate) and imidacloprid (neonicotinoid) related cuticle thickness in C. hemipterus. There is evidence of a possible association between cuticle thickness and resistance, but the association can be tenuous, likely because resistance is multifactorial in C. hemipterus. We also discovered a novel T1011 residue in domain IIS6 of the voltage-gated sodium channel that likely enhanced susceptibility to deltamethrin (pyrethroid) despite the presence of a L1014F mutation known to confer pyrethroid resistance in C. hemipterus. Our findings also confirmed that the M918I mutation enhanced resistance to pyrethroid when present with the L1014F mutation, which was consistent with a super-kdr phenotype, as reported previously. Multiple resistance mechanisms can be found within a single C. hemipterus population, and the presence of both M918I + L1014F mutations likely masked the influence of cuticle thickness in conferring resistance against deltamethrin. The elevated metabolic enzyme activities in some strains were not necessarily associated with increased insecticide resistance. This study has enhanced our understanding on the penetration resistance mechanism and target site insensitivity of sodium channels in C. hemipterus.

Mechanisms of amitraz resistance in a Rhipicephalus microplus strain from southern Brazil

Amitraz is one of the most used acaricides for the control of ticks of domestic animals, however, extensive use of this active ingredient has favored the development of resistant populations of Rhipicephalus microplus worldwide. The possible mechanisms of metabolic and/or target-site alterations mechanisms of amitraz resistance were investigated in a Brazilian field population of R. microplus (São Gabriel strain). Bioassays with the synergists piperonylbutoxide, triphenylphosphate and diethyl-maleate were used to evaluate the metabolic mechanisms involved. Target-site insensitivity was investigated by amplification and sequencing of a fragment of the octopamine/tyramine (OCT/TYR) receptor gene. Piperonylbutoxide synergism (synergism ratio = 2.8) indicated the participation of the P450 pathway in the detoxification of amitraz. Previously reported single nucleotide polymorphisms that confer amino acid changes in the OCT/TYR receptor, threonine to proline (T8P) and leucine to serine (L22S), were found in the amitraz-resistant strain but not in the susceptible reference strain. The results suggest that amitraz resistance in the studied strain is multi-factorial and may result from cytochrome P450 detoxification and mutations in octopamine receptors.

Resistance to Fipronil in the Common Bed Bug (Hemiptera: Cimicidae).

Cimex lectularius L. populations have been documented worldwide to be resistant to pyrethroids and neonicotinoids, insecticides that have been widely used to control bed bugs. There is an urgent need to discover new active ingredients with different modes of action to control bed bug populations. Fipronil, a phenylpyrazole that targets the GABA receptor, has been shown to be highly effective on bed bugs. However, because fipronil shares the same target site with dieldrin, we investigated the potential of fipronil resistance in bed bugs. Resistance ratios in eight North American populations and one European population ranged from 1.4- to >985-fold, with highly resistant populations on both continents. We evaluated metabolic resistance mechanisms mediated by cytochrome P450s, esterases, carboxylesterases, and glutathione S-transferases using synergists and a combination of synergists. All four detoxification enzyme classes play significant but variable roles in bed bug resistance to fipronil. Suppression of P450s and esterases with synergists eliminated resistance to fipronil in highly resistant bed bugs. Target-site insensitivity was evaluated by sequencing a fragment of the Rdl gene to detect the A302S mutation, known to confer resistance to dieldrin and fipronil in other species. All nine populations were homozygous for the wild-type genotype (susceptible phenotype). Highly resistant populations were also highly resistant to deltamethrin, suggesting that metabolic enzymes that are responsible for pyrethroid detoxification might also metabolize fipronil. It is imperative to understand the origins of fipronil resistance in the development or adoption of new active ingredients and implementation of integrated pest management programs.

Multiplex allele-specific polymerase chain reaction to detect kdr and rdl mutations in the cattle tick Rhipicephalus microplus (Acari: Ixodidae)

The resistance of Rhipicephalus microplus to pyrethroids is widely dispersed worldwide and has been associated with several nucleotide substitutions in its target site, the para-sodium ion channel (Na-channel) gene. The resistance of the tick to fipronil has been increasing in South America, and mutations in the GABA-gated chloride channel (GABA-Cl) have been described in fipronil-resistant tick strains. We developed a multiplex allele-specific PCR (mAS-PCR) to screen for single-nucleotide polymorphisms (SNPs) associated with the resistance to pyrethroids (knockdown resistance or kdr) and fipronil (resistance to dieldrin or rdl) in susceptible tick populations from Uruguay (n = 11) and the Rio Grande do Sul state in Southern Brazil (n = 15). Toxicological in vitro assays with larvae and adults were used to confirm the resistance to cypermethrin, flumethrin, and fipronil. Three SNPs in the Na-channel gene were investigated (C190A, G215 T, and T2134A), and the mAS-PCR included the detection of an SNP (G858 T) coding a non-synonymous mutation in the GABA-Cl gene. C190A was present in all pyrethroid-resistant populations from Uruguay and Brazil, most frequently homozygous. The SNPs G215 T and T2134A were not found. Of the seventeen fipronil-resistant populations, fourteen presented at least one mutant GABA-Cl gene allele, more frequently in heterozygosis. Other mechanisms apart from target site insensitivity may be involved in fipronil resistance since in some resistant populations, the SNP G858 T was not detected. Sixteen (61,5%) of the populations presented individuals with simultaneous mutations in the Na-channel and GABA-Cl genes. This could be a significant problem for the future control of R. microplus. This study shows the wide dispersion of a pyrethroid resistance–associated SNP in high frequency in the region. Fipronil resistance mutations are also dispersed across the region and increasing.