Para-type Sodium Channel Gene Research Articles

Molecular identification of allelic genotypes of pyrethroid-insecticide resistance in housefly, Iraq

According to global-wide presence of insecticides resistance to pyrethroids, the current study identified the purpose to detect the allelic genotypes regarding this issue in house flies in Iraq. From the governorate of Al-Qadisiyah, Iraq, 60 morphologically and molecularly recognized house flies were caught from 6 different regions. Using a technique called polymerase chain reaction (PCR) amplification of specific allele (PASA), PCR was employed to reveal the presence of allele-genetic variations in the para-type sodium channel (para) gene to recognize knockdown resistance (kdr) mutation from the homozygous-wild type of complete susceptibility (sus/sus) to the mutated-homozygous type of complete resistance (kdr/kdr) or to the mutated-heterozygous type (kdr/sus). Here, these genotypes were targeted using specific primers to identify these genetic variations. The results have declared the presence of the sus/sus at 100%-frequency rate in all flies, and none of the other genotypes were detected (0%) in all flies. This valued piece of result indicates the reality of resistance persistence due to lack of insecticide-spraying programs in the governorate. This study provides high-quality information about the current status of insecticide resistance in house flies in Iraq about supporting the fact of genetic-base development of such resistance via frequent use of insecticides.

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

BackgroundAnopheles 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.MethodsThe 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.ResultsIn 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.ConclusionsThe 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.

Modification of contact avoidance behaviour associated with pyrethroid resistance in Anopheles sinensis (Diptera: Culicidae)

BackgroundAnopheles sinensis is the primary vector of vivax malaria in China and its control is under great threat as the development of insecticide resistance. In contrast to physiological resistance, there is no report of behavioural modifications of resistant An. sinensis after long-term insecticide use, despite their huge potential impact on malaria transmission.MethodsLarvae or pupae of An. sinensis were collected from Yuanyang, Bishan, and Wuhe counties from southwestern to eastern China. Resistance to deltamethrin was assayed using the standard World Health Organization (WHO) susceptibility test. The frequency distribution of the kdr allele of the para-type sodium channel gene was determined by polymerase chain reaction (PCR) amplification and DNA sequencing. Contact repellency to deltamethrin-impregnated bed nets was evaluated using a modified WHO cone bioassay.ResultsAll contemporary field populations for all three geographic locations were resistant to deltamethrin, with mortality ranging from 6.00 to 26.79%. Three kdr genotypes with either an L1014F or L1014C substitution with frequencies of 76.10–100% were identified in the Bishan and Wuhe populations, but no kdr mutations were detected in the Yuanyang samples despite high phenotypic resistance. The susceptible mosquitoes exhibited significantly longer flying time and more takeoffs on deltamethrin-treated bed nets (DTN) than on untreated bed nets (UTN), suggestive of robust avoidance behaviour. However, no significant increases in the frequency of takeoffs or flying time were observed in deltamethrin-resistant An. sinensis populations when exposed on DTNs, regardless of the presence of a kdr mutation. Moreover, the first takeoff from DTNs by resistant mosquitoes significantly lagged behind compared to susceptible mosquitoes.ConclusionThe An. sinensis populations were highly resistant to deltamethrin and exhibited decreased avoidance behaviour. Behavioural modification significantly associated with deltamethrin resistance, but not directly related to the presence of kdr mutations, indicating that there are additional factors contributing to the changes.

Bioassay and Molecular Study for Detection of Insecticide Resistance Dengue Causing Mosquito Vectors

Nowadays, dengue infection creates a major problem across the country. The vector species carrying dengue infection has progressively started to developed resistance against most of the currently used insecticides. Hence, a study was carried out in dengue-endemic areas of Assam and Arunachal Pradesh to find the current situation of insecticide susceptibility status of dengue vectors. Based on the previous history of dengue incidence, Aedes mosquitoes were collected from Dibrugarh, Kamrup, Sivasagar, Tezpur and Tinsukia districts in Assam and Pasighat district in Arunachal Pradesh to test the insecticide resistance status through bioassay and molecular methods. The WHO standard bioassay test kits were used to detect insecticide susceptibility status among dengue vectors. In molecular study, allele-specific polymerase chain reaction (PCR) method was done for the detection of mutations in paratype voltage-gated sodium channel gene of Aedes aegypti and Aedes albopictus mosquitoes. In bioassay method, 100% A. aegypti mosquitoes were found to be resistant towards dichlorodiphenyltrichloroethane (DDT), 8% towards pyrethroid and 4% towards malathion. Similarly, 92% A. albopictus mosquitoes have shown resistance competency towards DDT, 12% towards pyrethroid and 8% towards malathion. In allele-specific PCR methods, V1016G heterozygous mutations were detected from the field collected A. aegypti and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. Similarly, F1534C heterozygous mutations were observed from A. aegypti mosquitoes of Tezpur, Tinsukia and Sivasagar district and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. From the study, it was concluded that the Aedes mosquitoes have progressively started to developed resistance towards commonly used insecticides.

Impact of pest management practices on the frequency of insecticide resistance alleles in Bemisia tabaci (Hemiptera: Aleyrodidae) populations in three countries of West Africa

In West Africa, the use of organophospates and pyrethroid insecticides to control cotton pests has led to the evolution of resistance in field populations of Bemisia tabaci Gennadius. Three pest management programs have been commonly recommended: the Conventional Program (CP) where 6 treatments are applied, the use of Bt cotton plants for which only 2 applications of neonicotinoids are required and that has been adopted in many countries, and a biological program (BP) without any chemical treatment. The present study aimed to determine the influence of these practices on the frequency of mutations that confer resistance to pyrethroids (mutation L925I in the para-type voltage-gated sodium channel gene) and organophosphates (mutation F331W in the acetylcholinesterase enzyme ace1: allele Ace1R) in B. tabaci populations using Bt cotton and CP areas in Pô and Saria (Burkina Faso), CP and BP areas in Kandi (Benin) and only CP areas in Tové and Infa (Togo). All individuals sampled belonged to the MED (biotypes MED-Q1) and Africa Silver Leafing (ASL) species. MED-Q1 was found in sympatry with ASL in Burkina Faso both on CP and Bt cotton areas at variable frequencies. In Togo and Benin, only ASL was found, except in Tové where MED-Q1 was also detected, but at low frequency. Frequencies of mutations that confer resistance varied between localities and species but we did not find any strong evidence of a relationship between the pest management program and these frequencies except for the allele Ace1R in Burkina Faso for which the frequencies decrease when chemical applications are reduced. This study provides valuable information for the development of efficient integrated pest management programs.

Insecticide resistance in Trialeurodes vaporariorum populations and novel diagnostics for kdr mutations.

Neonicotinoids, pyrethroids and ketoenols are currently used for the control of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). In this study, insecticide resistance status and mechanisms were investigated using classical bioassays and molecular techniques. Dose-response bioassays were performed on 19 Greek populations, among the 35 different whitefly populations used for the whole analysis. Resistance factors scaled up to 207-, 4657- and 59-fold for imidacloprid, bifenthrin and spiromesifen, respectively. Molecular assays were used to investigate the frequency of known resistance mutations. A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for detecting the pyrethroid-resistant alleles r1 (mutation L925I) and r2 (mutation T929I) of the para-type voltage-gated sodium channel gene (VGSC). Both alleles were present at high frequencies (on average 65% and 33%, respectively) in 14 populations from Greece. The M918 L pyrethroid resistance mutation was not detected in any of the Greek populations. Sequencing and a Taqman allelic discrimination were used to monitor the frequency of the mutation E645K of the acetyl-coenzyme A carboxylase gene (ACC) recently linked to spiromesifen resistance. This mutation was detected in 20 of the 24 populations examined in ∼38% frequency among the 433 individuals tested. However, its association with the spiromesifen resistance phenotype was not confirmed in the Greek populations. Finally, two homologues of the CYP6CM1 Bemisia tabaci P450, the known neonicotinoid metabolizer, were found upregulated in two T. vaporariorum neonicotinoid-resistant populations; they were both functionally expressed in Escherichia coli, but the recombinant proteins encoded did not metabolize those neonicotinoid insecticides tested. The development of simple diagnostics and their use alongside classical and molecular techniques for the early detection of resistant populations are of great importance for pest management strategies. The practical implications of our results are discussed in light of whitefly control. © 2017 Society of Chemical Industry.

Insecticide resistance status of malaria vectors in Lao PDR.

Knowledge on insecticide resistance in Anopheles species is a basic requirement to guide malaria vector control programs. In Lao PDR, vector control relies on insecticide residual spraying (IRS) and impregnated bed-nets (ITNs) with the use of pyrethroids. Here, the susceptibility of Anopheles species, including several malaria vectors (An. maculatus and An. minimus), to various insecticides was investigated in ten provinces of Lao PDR through a north-south transect. Bioassays were performed on field caught female mosquitoes using the standard WHO susceptibility tests with DDT (4%), deltamethrin (0.05%) and permethrin (0.75%). In addition, the DIIS6 region of the para-type sodium channel gene was amplified and sequenced to identify knockdown resistance mutations (kdr). Resistance to DDT and permethrin was detected in suspected malaria vectors, such as An. nivipes and An. philippinensis in Lao PDR. Resistance to the formerly used DDT was found in a population of An. maculatus s.l. from Luang Prabang province. No resistance to pyrethroids was found in primary vectors, indicating that these insecticides are still adequate for malaria vector control. However, high resistance levels to pyrethroids was found in-vector species and reduced susceptibility to permethrin in An. minimus and An. maculatus was reported in specific localities which raises concerns for pyrethroid-based control in the future. No kdr mutation was found in any of the resistant populations tested hence suggesting a probable role detoxification enzymes in resistance. This study highlights the necessity to continue the monitoring of insecticide susceptibility to early detect potential occurrence and/or migration of insecticide resistance in malaria vectors in Lao PDR.

Landscape genetic structure and evolutionary genetics of insecticide resistance gene mutations in Anopheles sinensis.

BackgroundAnopheles sinensis is one of the most abundant vectors of malaria and other diseases in Asia. Vector control through the use of insecticides is the front line control method of vector-borne diseases. Pyrethroids are the most commonly used insecticides due to their low toxicity to vertebrates and low repellency. However, the extensive use of insecticides has imposed strong selection pressure on mosquito populations for resistance. High levels of resistance to pyrethroid insecticides and various mutations and haplotypes in the para sodium channel gene that confers knockdown resistance (kdr) have been detected in An. sinensis. Despite the importance of kdr mutations in pyrethroid resistance, the evolutionary origin of the kdr mutations is unknown. This study aims to examine the evolutionary genetics of kdr mutations in relation to spatial population genetic structure of An. sinensis.MethodsAdults or larvae of Anopheles sinensis were collected from various geographic locations in China. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of the para-type sodium channel gene were conducted to analyse kdr allele frequency distribution, kdr codon upstream and downstream intron polymorphism, population genetic diversity and kdr codon evolution. The mitochondrial cytochrome c oxidase COI and COII genes were amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion and gene flow patterns.ResultsThree non-synonymous mutations (L1014F, L1014C, and L1014S) were detected at the kdr codon L1014 of para-type sodium channel gene. A patchy distribution of kdr mutation allele frequencies from southern to central China was found. Near fixation of kdr mutation was detected in populations from central China, but no kdr mutations were found in populations from southwestern China. More than eight independent mutation events were detected in the three kdr alleles, and at least one of them evolved multiple times subsequent to their first divergence. Based on sequence analysis of the mitochondrial COI and COII genes, significant and large genetic differentiation was detected between populations from southwestern China and central China. The patchy distribution of kdr mutation frequencies is likely a consequence of geographic isolation in the mosquito populations and the long-term insecticide selection.ConclusionOur results indicate multiple origins of the kdr insecticide-resistant alleles in An. sinensis from southern and central China. Local selection related to intense and prolonged use of insecticide for agricultural purposes, as well as frequent migrations among populations are likely the explanations for the patchy distribution of kdr mutations in China. On the contrary, the lack of kdr mutations in Yunnan and Sichuan is likely a consequence of genetic isolation and absence of strong selection pressure. The present study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1513-6) contains supplementary material, which is available to authorized users.

Knockdown resistance of Anopheles sinensis in Henan province, China.

BackgroundVivax malaria was historically epidemic in Henan Province of China and Anopheles sinensis was the main vectors and poor farming communities bare the greatest burden of disease. Knockdown resistance in An. sinensis is one of the mechanisms of resistance against pyrethroids. In the present study, the frequency of mutations from An. sinensis was examined in Henan province, China.MethodsAnopheles was collected from Kaifeng, Tongbai, Tanghe, Pingqiao, Shihe, and Yongcheng counties of Henan province in 2013. Molecular identification of Anopheles species was conducted by polymerase chain reaction (PCR) amplifying the internal transcribed spacer 2 (ITS2). Part of the IIS6 domain of the para-type sodium channel protein gene was polymerase chain reaction-amplified and directly sequenced. Frequency and geographic difference of kdr gene mutant types were analysed.Results208 Anopheles were received molecular identification, of which 169 (81.25%) were An. sinensis, 25 (12.02%) were Anopheles yatsushiroensis, and 12 (5.77%) were Anopheles lesteri. A 325 bp fragment of the para-type sodium channel gene including position 1014 was successfully sequenced from 139 Anopheles, of which 125 (89.93%) were An. sinensis, 12 (8.63%) were An. yatsushiroensis, 2 (1.44%) were An. lesteri. The molecular analyses revealed that mutations existed at codon 1014 in An. sinensis but not in An. yatsushiroensis and An. lesteri. Frequency of kdr mutation was 73.60% (92/125) from population of An. sinensis in Henan province, of which L1014F (TTT + TTC) allele frequencies accounted for 46.40% (58/125), and was higher than that of L1014C(TGT) which accounted for 27.20% (34/125) ( χ2 = 55.423, P < 0.001). The frequency of kdr mutation in Kaifeng county was 100% (49/49), and was higher than that of 37.93% (11/29) in Tongbai, 54.55% (6/11) in Pingqiao, 50.00% (3/3) in Shihe, and 62.50% (10/16) in Yongcheng county, respectively (χ2 = 39.538, P < 0.001; χ2 = 24.298, P < 0.001; χ2 = 25.913, P < 0.001; χ2 = 20.244, P < 0.001). While 92.86% (13/14) frequency of kdr mutation was found in Tanghe county, which was higher than that in Tongbai county (χ2 = 11.550, P = 0.0018).ConclusionsA high frequency of kdr gene mutations from population of An. sinensis in Henan province was found.

Molecular Detection of Knockdown Resistance (kdr) inBlattella germanica(Blattodea: Blattellidae) From Northwestern Iran

Pyrethroid insecticides are highly insecticidal compounds that are widely used against the German cockroach, a significant household insect pest. In several insect species, there is a point mutation in the para-type sodium channel gene associated with knockdown resistance (kdr). In the current study, genomic DNA was analyzed in the region where the kdr and super-kdr (an enhanced form of pyrethroid resistance) mutations reside in Blatella germanica (L., 1767) (Blattodea: Blattellidae) collected from Iran. Studies on the extracted DNA from hand-captured German cockroach specimens were conducted by polymerase chain reaction and sequencing to detect related mutations. The kdr mutation, substitution of G for C (L1014F), which results in amino acid replacement (leucine with phenylalanine), was detected in all 18 sequenced specimens from three different locations. However, the super-kdr mutation (M918T), which is detected in super-kdr house flies, was not found in the sequences of the current study. The high ratio of the kdr mutation in a field population of B. germanica in Urmia confirms that the individuals are homozygous. These data should be helpful in designing and implementing a control program and resistance management.

복숭아혹진딧물 야외개체군의 살충제 저항성 마커 선발

2009~2011년 동안 국내 주요 배추 재배지 5개 지역(평창, 홍천, 봉화, 무주, 제주)에서 살충제에 대한 복숭아혹진딧물의 저항성 발달 정도를 조사하고, 야외 개체군에 적용 가능한 살충제 저항성 마커를 개발하기 위해 본 연구를 수행하였다. 조사된 5개 지역 개체군 모두 여러 살충제 종류에 대하여 다양한 저항성을 보였다. 다양한 저항성을 보인 5개 지역 야외 개체군으로부터 여라 살충제에 대하여 복합적으로 저항성을 보이는 복합저항성 계통(MR)을 선발하였고, 이 MR과 모든 지역 채집 개체군에 대해 등전점전기영동과 정량염기서열분석(quantitative sequencing, QS)을 통하여 에스터레이즈 과발현과 살충제 작용점 내 돌연변이를 확인하였다. MR을 포함한 모든 야외 개체군에서 에스터레이즈의 과발현과 아세틸콜린에스터레이즈 1 유전자(ace1)의 StoF 돌연변이를 확인할 수 있었다. 넉다운 저항성 돌연변이로 잘 알려진 파라 타입 나트륨 채널 유전자(para)의 LtoF 돌연변이는 모든 지역 채집 개체군은 물론 비펜스린에 대해 3,461배 저항성을 보이는 MR에서도 발견되지 않았다. 그 외에 MtoL 돌연변이를 발견하였는데, 생물검정 결과 저항성 수준과 돌연변이 발생 빈도가 일치하였다. 따라서 생물검정 대신, 이러한 분자 마커를 활용 한다면 더 효율적으로 살충제 저항성 평가가 가능할 것이다. 이러한 분자 마커들(ace1의 StoF, para의 MtoL)은 정량염기서열분석, PCR amplification of specific alleles (PASA) 등의 진단 방법에 쉽게 응용이 가능 하고, 이러한 방법은 야외 복숭아혹진딧물 저항성 관리에 적용이 가능 할 것이다. The resistance levels of the green peach aphid, Myzus persicae (Sulzer), against 10 insecticides was checked and selected the applicable insecticide resistance markers. We conducted our study in 5 cabbage cultivation regions (Pyeongchang, Hongcheon, Bongwha, Muju, and Jeju) of Korea, over 3 successive years (2009-2011). We selected a multi-resistant (MR) strain from among the 5 field-collected populations. We analyzed esterase over-expression and mutation(s) in the target sites, by using native isoelectric focusing (IEF) and quantitative sequencing (QS). We detected esterase over-expression and StoF mutation in the acetylcholinesterase 1 gene (ace1) in all of the field-collected populations, including the MR strain. We did not detect the LtoF mutation, which is a well-known knockdown resistance (kdr) mutation in the para-type sodium channel gene (para), in the MR strain; however, the value of the MR strain for bifenthrin was 3,461-fold higher than that of the susceptible strain. Our results indicate that insecticide resistance is more effectively evaluated using molecular markers than by conducting a bioassay. The molecular markers StoF in ace1 and MtoL in para can easily be applied in diagnostic methods such as QS or PCR amplification of specific alleles (PASA). These methods may be extended to management of M. persicae resistance in the field.

Target-site resistance to pyrethroid insecticides in German populations of the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae)

Cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) is a major pest of winter oilseed rape in several European countries particularly attacking young emerging plants in autumn. Over the last several decades, pyrethroid insecticides have been foliarly applied to control flea beetle outbreaks. Recent control failures in northern Germany suggested pyrethroid resistance development in cabbage stem flea beetles, which were confirmed by resistance monitoring bioassays using lambda-cyhalothrin in an adult vial test. The purpose of this study was to investigate the presence of polymorphisms in the para-type voltage-gated sodium channel gene of P. chrysocephala known to be involved in knock-down resistance (kdr). By using a degenerate primer approach we PCR amplified part of the para-type sodium channel gene and identified in resistant flea beetles a single nucleotide polymorphism resulting in an L1014F (kdr) mutation within domain IIS6 of the channel protein, known as one of the chief pyrethroid target-site resistance mechanisms in several other pest insects. Twenty populations including four archived museum samples collected between 1945 and 1958 were analyzed using a newly developed pyrosequencing diagnostic assay. The assay revealed a kdr allele frequency of 90–100% in those flea beetle populations expressing high-level cross-resistance in discriminating dose bioassays against different pyrethroids such as lambda-cyhalothrin, tau-fluvalinate, etofenprox and bifenthrin. The presence of target-site resistance to pyrethroids in cabbage stem flea beetle is extremely worrying considering the lack of effective alternative modes of action to control this pest in Germany and other European countries, and is likely to result in major control problems once it expands to other geographies. The striking fact that cabbage stem flea beetle is next to pollen beetle, Meligethes aeneus the second coleopteran pest in European winter oilseed rape resisting pyrethroid treatments by expressing a target-site mutation, underpins the importance of diversity in available chemistry for resistance management tactics based on mode of action rotation in order to guarantee sustainable winter oilseed rape cultivation in Europe.

Relationship between knockdown resistance, metabolic detoxification and organismal resistance to pyrethroids in Anopheles sinensis.

Anopheles sinensis is the most important vector of malaria in Southeast Asia, including China. Currently, the most effective measure to prevent malaria transmission relies on vector control through the use of insecticides, primarily pyrethroids. Extensive use of insecticides poses strong selection pressure on mosquito populations for resistance. Resistance to insecticides can arise due to mutations in the insecticide target site (target site resistance), which in the case of pyrethroids is the para-type sodium channel gene, and/or the catabolism of the insecticide by detoxification enzymes before it reaches its target (metabolic detoxification resistance). In this study, we examined deltamethrin resistance in An. sinensis from China and investigated the relative importance of target site versus metabolic detoxification mechanisms in resistance. A high frequency (>85%) of nonsynonymous mutations in the para gene was found in populations from central China, but not in populations from southern China. Metabolic detoxification as measured by the activity of monooxygenases and glutathione S-transferases (GSTs) was detected in populations from both central and southern China. Monooxygenase activity levels were significantly higher in the resistant than the susceptible mosquitoes, independently of their geographic origin. Stepwise multiple regression analyses in mosquito populations from central China found that both knockdown resistance (kdr) mutations and monooxygenase activity were significantly associated with deltamethrin resistance, with monooxygenase activity playing a stronger role. These results demonstrate the importance of metabolic detoxification in pyrethroid resistance in An. sinensis, and suggest that different mechanisms of resistance could evolve in geographically different populations.

Target-site resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus F.

Pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae) is a major univoltine pest of oilseed rape in many European countries. Winter oilseed rape is cultivated on several million hectares in Europe and the continuous use of pyrethroid insecticides to control pollen beetle populations has resulted in high selection pressure and subsequent development of resistance. Resistance to pyrethroid insecticides in this pest is now widespread and the levels of resistance are often sufficient to result in field control failures at recommended application rates. Recently, metabolic resistance mediated by cytochrome P450 monooxygenases was implicated in the resistance of several pollen beetle populations from different European regions. Here, we have also investigated the possible occurrence of a target-site mechanism caused by modification of the pollen beetle para-type voltage-gated sodium channel gene. We detected a single nucleotide change that results in an amino acid substitution (L1014F) within the domain IIS6 region of the channel protein. The L1014F mutation, often termed kdr, has been found in several other insect pests and is known to confer moderate levels of resistance to pyrethroids. We developed a pyrosequencing-based diagnostic assay that can detect the L1014F mutation in individual beetles and tested more than 350 populations collected between 2006 and 2010 in 13 European countries. In the majority of populations tested the mutation was absent, and only samples from two countries, Denmark and Sweden, contained pollen beetles heterozygous or homozygous for the L1014F mutation. The mutation was first detected in a sample from Denmark collected in 2007 after reports of field failure using tau-fluvalinate, and has since been detected in 7 out of 11 samples from Denmark and 25 of 33 samples from Sweden. No super-kdr mutations (e.g. M918T) known to cause resistance to pyrethroids were detected. The implications of these results for resistance management strategies of pollen beetle populations in oilseed rape crops are discussed.

Survey of pyrethroid and organophosphate resistance in Brazilian field populations of Rhipicephalus (Boophilus) microplus: Detection of C190A mutation in domain II of the para-type sodium channel gene

The cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Brazil and other countries. Its control is becoming more difficult due to the development of resistance in populations. Early detection of resistance can help in developing effective control strategies. This study evaluated the susceptibility of R. microplus to cypermethrin and chlorpyriphos and was the first attempt to identify the mechanism of resistance (target site insensitivity) in cattle tick populations from Minas Gerais state (Southeastern Brazil). Engorged female ticks were collected from 10 ranches within the state of Minas Gerais, and susceptibility was evaluated with the larval packet test (LPT) using technical grade cypermethrin and chlorpyriphos. It was possible to analyze LPT results of seven populations. Target site insensitivity was investigated in all 10 isolates by using molecular approaches for detection of the T2134A substitution within the domain III S6 segment and the C190A in the domain II S4-5 linker from the para-type sodium channel gene. LPT showed that all seven populations were resistant to cypermethrin with resistance ratio (RR) ranging from 16.0 to 25.0 and 85.7% were resistant to chlorpyriphos (RR=2.2–15.6). Although the T2134A mutation was not detected, the C190A mutation was highly prevalent, being present in 82–100% of the alleles sampled in field populations. A significant correlation was found between the LC50 values for cypermethrin and the frequency of the C190A mutation suggesting that it might be responsible for the phenotypic resistance detected.

Knockdown resistance in Anopheles vagus, An. sinensis, An. paraliae and An. peditaeniatus populations of the Mekong region

BackgroundIn the Mekong region (Vietnam, Cambodia and Laos), a large investigation was conducted to assess the susceptibility of Anopheles species against DDT and pyrethroids. In this study, the resistance status of the potential malaria vectors An. vagus, An. sinensis, An. paraliae and An. peditaeniatus was assessed.MethodsBioassays were performed on field collected unfed female mosquitoes using the standard WHO susceptibility tests. In addition, the DIIS6 region of the para-type sodium channel gene was amplified and sequenced and four allele-specific PCR assays were developed to assess the kdr frequencies.ResultsIn Southern Vietnam all species were DDT and pyrethroid resistant, which might suggest the presence of a kdr resistance mechanism. Sequence-analysis of the DIIS6 region of the para-type sodium channel gene revealed the presence of a L1014S kdr mutation in An. vagus, An. sinensis and An. paraliae. In An. peditaeniatus, a low frequency L1014S kdr mutation was found in combination with a high frequency L1014F kdr mutation. For pyrethroids and DDT, no genotypic differentiation was found between survivors and non-survivors for any of these species. In the two widespread species, An. vagus and An. sinensis, kdr was found only in southern Vietnam and in Cambodia near the Vietnamese border.ConclusionsDifferent levels of resistance were measured in Laos, Cambodia and Vietnam. The kdr mutation in different Anopheles species seems to occur in the same geographical area. These species breed in open agricultural lands where malaria endemicity is low or absent and vector control programs less intensive. It is therefore likely that the selection pressure occurred on the larval stages by insecticides used for agricultural purposes.

IDENTIFICATION OF MUTATION IN THE BEMISIA TABACI (GENN.) PARA SODIUM CHANNEL GENE ASSOCIATED WITH RESISTANCE TO PYRETHROIDS

Mechanism of resistance to lambda-cyhalothrin in highly resistant Bemisia tabaci strain can be explained by the voltage-gated sodium channel which is the primary target site of pyrethroid insecticides. The super knockdown resistance (super-kdr) to pyrethroids is caused by changes at specific sites on the para-type sodium channel protein domain II (IIS4-6). The B. tabaci para-type sodium channel gene was RT-PCR amplified from lab-strain (reference), Parent (first generation after treatment with the insecticide) and generation thirteen after treatment was considered. The mechanisms of resistance to Lambda-cyhalothrin (Karat 20% EC) in a Q biotype, highly resistant Bemisia tabaci strain. Analysis of the sequence of the IIS4-IIS6 region of the para sodium channel gene of lab-strain, Parent and resistant strain (G13) revealed two amino acid replacements compared to that of the SUD-S susceptible strain. One is the leucine to isoleucine substitution at position 925 (L925I) and allele r1-Q1 (GenBank accession no. DQ205206) has identical intron sequences with samples of parent and lab-strain, other is a novel kdr resistant mutation for B. tabaci, a threonine to valine substitution at position (T929V) and alleles r2-Q1 (GenBank accession no DQ205207) has identical intron sequence with G13..

Biochemical and molecular diagnosis of insecticide resistance conferred by esterase, MACE, kdr and super- kdr based mechanisms in Italian strains of the peach potato aphid, Myzus persicae (Sulzer)

In this paper we analysed the basis of insecticide resistance in 59 Italian strains of the peach potato aphid Myzus persicae using both molecular and biochemical assays. Our data as a whole clearly indicate that most M. persicae strains (76.3%) have high or extremely high production of an esterase enzyme which sequester and detoxify insecticides with esteric group. Kdr genotypes conferring resistance towards pyrethoids are present in 57.7% of the analysed populations. Moreover, 26.5% of the kdr positive strains possess also the M918T mutation conferring super- kdr phenotype. Strains with modified AChE (MACE) are not so numerous (27.1%), although they can be found almost everywhere in Italy. Considering all the strains analysed, both MACE and kdr phenotypes are associated with high levels of esterase activity. In Central–Southern regions, kdr and MACE resistance mechanisms resulted in linkage disequilibrium. Bioassays performed in order to evaluate the efficacy of a pyrethroid insecticide against a strain possessing a F979S mutation within its para-type sodium channel gene suggests that this amino acid substitution could affect the sodium channel responsivity to pyrethroids.

Metabolic detoxication and the kdr mutation in pyrethroid resistant house flies, Musca domestica (L.)

Two house fly strains, ALHF and SeALHF, were collected from Alabama after control failures with pyrethroids. While pyrethroid resistance in ALHF partially conferred by P450 monooxygenase- and hydrolase-mediated metabolism has been reported, no studies have been conducted on resistance of SeALHF. The current study was carried out to investigate mechanisms of pyrethroid resistance in SeALHF and the possible role of target site insensitivity, due to kdr mutation, in pyrethroid resistance of ALHF. Resistance to permethrin in SeALHF was dramatically and partially suppressed by PBO and DEF, respectively, suggesting that P450 monooxygenase-mediated metabolism plays a major role in permethrin resistance in this strain, while hydrolytic metabolism has a minor contribution to resistance. Incomplete suppression of permethrin resistance by PBO and DEF suggests that one or more additional minor mechanisms are involved in overall resistance of SeALHF. Injection did not decrease levels of resistance to permethrin in SeALHF, implying that a decreased rate of cuticular penetration ( pen) does not play a role in permethrin resistance in this strain. A 392 bp para-type sodium channel gene fragment, where kdr (L1014F) and super-kdr (M918T) mutations reside, was generated by RT-PCR from ALHF and SeALHF. The M918T mutation was not detected in ALHF or SeALHF, suggesting that the super-kdr mutation is not important in permethrin resistance of these two house fly strains even though ALHF possesses a much higher level of resistance than SeALHF. The L1014F mutation was present in ALHF, but not in SeALHF, suggesting that the kdr mutation is an important factor in pyrethroid resistance in ALHF. A leucine to histidine (L1014H) substitution at the position corresponding to kdr mutation was detected in SeALHF. The importance of this mutation in resistance is discussed.

Voltage-dependent Na+channels in pyrethroid-resistantCulex pipiensL mosquitoes

In some insect species, knockdown resistance (kdr) to pyrethroids and DDT is linked to point mutations in the sequence of the para-type voltage-dependent sodium channel gene. The effects of pyrethroids were assayed on six Culex pipiens strains: two were susceptible to pyrethroids and the four others displayed various levels of resistance, but, in each case, a kdr-type mechanism was strongly suggested. Degenerate primers were designed on the basis of the corresponding sequences of the para orthologous gene reported from several orders of insects. These primers were used to amplify the region of the sodium channel gene which includes the positions where the kdr and super-kdr mutations have been found in Musca domestica. As expected, the amplified fragment was highly homologous to the para sequences. The super-kdr-like mutation (methionine to threonine at position 918 of the M domestica para sequence) was never detected in any strain. In contrast, the same kdr mutation (leucine to phenylalanine at position 1014) was present in some Culex pyrethroid-resistant samples. An alternative substitution of the same leucine to a serine was detected in one strain slightly resistant to pyrethroids but highly resistant to DDT. These data have allowed us to design a PCR-based diagnostic test on genomic DNA to determine the presence or the absence of the kdr allele in single C pipiens collected in several countries. The validity of this test was checked by comparing the frequency of the resistance allele and the toxicological data. © 1999 Society of Chemical Industry