Distribution and dynamics of esterase alleles in Culex pipiens complex in China

To investigate insecticide resistance and dynamic changes of carboxylesterase polymorphism in mosquitoes with time in the Culex pipiens complex (Diptera: Culicidae), nine field mosquito populations were collected in China. The resistance levels of fourth-instar larvae to organophosphate (dichlorvos, parathion, and chlorpyrifos), carbamate (fenobucarb and propoxur), and pyrethroid (permethrin, deltamethrin and tetramethrin) insecticides were determined by bioassay. Larvae had more resistance to organophosphate insecticides than to carbamate insecticides. A low but significant resistance was observed for carbamate insecticides. The resistance to pyrethroid insecticides varied from sensitive to high. Starch gel electrophoresis revealed the presence of the overproduced esterases B1, A2B2, A8B8, A9B9, B10 and A11B11. The frequency of each overproduced esterases varied depending on its regional localities. Compared with published surveys, the C. pipiens complex, which exhibited a high polymorphism of applied esterase alleles in China, showed dynamic evolution over time under local specific insecticide selection. The results are discussed in the context of recent alterations to insecticide campaigns, and in the evolution of resistance genes in Chinese C. pipiens populations.     

Implications of gene amplification for evolution and management of insecticide resistance

Simulations were used to compare evolution of insecticide resistance predicted by a conventional two-allele model with predictions from three- and four-allele models that assume resistance is based on gene amplification. Results were similar between models when insecticide concentration was low or moderate. In contrast, when 10% of the population was not exposed to insecticide each generation, high insecticide concentrations slowed resistance development in the two-allele model, but caused rapid development of high levels of resistance in the three- and four-allele models. The presence of a third allele at an initial frequency as low as 10-7 doubled or tripled the rate of resistance development in some cases. Attempts to slow evolution of resistance by overwhelming it with high concentrations of insecticides are not likely to succeed if gene amplification or other mechanisms generate alleles that confer high levels of resistance.     

Does insecticide resistance alone account for the low genetic variability of asexually reproducing populations of the peach-potato aphid Myzus persicae?

The typical life cycle of aphids includes several parthenogenetic generations and a single sexual generation (cyclical parthenogenesis), but some species or populations are totally asexual (obligate parthenogenesis). Genetic variability is generally low in these asexually reproducing populations, that is, few genotypes are spread over large geographic areas. Both genetic drift and natural selection are often invoked to account for this low genetic variability. The peach-potato aphid, Myzus persicae, which encompasses both cyclical and obligate parthenogens, has developed several insecticide resistance mechanisms as a consequence of intense insecticide use since the 1950s. We collected asexually reproducing M. persicae from oilseed rape and examined genetic variability at eight microsatellite loci and three insecticide resistance genes to determine whether their genetic structure was driven by drift and/or selection. We identified only 16 multilocus microsatellite genotypes among 255 individuals. One clone, which combined two insecticide resistance mechanisms, was frequently detected in all populations whatever their location over a large geographical area (the northern half of France). These unexpected findings suggest that drift is not the unique cause of this low variability. Instead, the intensification of both insecticide treatments and oilseed rape cultivation may have favored a few genotypes. Thus, we propose that selective pressures resulting from human activities have considerably modified the genetic structure of M. persicae populations in northern France in a relatively short period of time.     

Landscape genomics of Colorado potato beetle provides evidence of polygenic adaptation to insecticides

The ability of insect pests to rapidly and repeatedly adapt to insecticides has long challenged entomologists and evolutionary biologists. Since Crow's seminal paper on insecticide resistance in 1957, new data and insights continue to emerge that are relevant to the old questions about how insecticide resistance evolves: such as whether it is predominantly mono‐ or polygenic, and evolving from standing vs. de novo genetic variation. Many studies support the monogenic hypothesis, and current management recommendations assume single‐ or two‐locus models. But inferences could be improved by integrating data from a broader sample of pest populations and genomes. Here, we generate evidence relevant to these questions by applying a landscape genomics framework to the study of insecticide resistance in a major agricultural pest, Colorado potato beetle, Leptinotarsa decemlineata (Say). Genome–environment association tests using genomic variation from 16 populations spanning gradients of landscape variables associated with insecticide exposure over time revealed 42 strong candidate insecticide resistance genes, with potentially overlapping roles in multiple resistance mechanisms. Measurements of resistance to a widely used insecticide, imidacloprid, among 47 L. decemlineata populations revealed heterogeneity at a small (2 km) scale and no spatial signature of origin or spread throughout the landscape. Analysis of nucleotide diversity suggested candidate resistance loci have undergone varying degrees of selective sweeps, often maintaining similar levels of nucleotide diversity to neutral loci. This study suggests that many genes are involved in insecticide resistance in L. decemlineata and that resistance likely evolves from both de novo and standing genetic variation.     

The role of gene splicing, gene amplification and regulation in mosquito insecticide resistance

The primary routes of insecticide resistance in all insects are alterations in the insecticide target sites or changes in the rate at which the insecticide is detoxified. Three enzyme systems, glutathione S-transferases, esterases and monooxygenases, are involved in the detoxification of the four major insecticide classes. These enzymes act by rapidly metabolizing the insecticide to non-toxic products, or by rapidly binding and very slowly turning over the insecticide (sequestration). In Culex mosquitoes, the most common organophosphate insecticide resistance mechanism is caused by co-amplification of two esterases. The amplified esterases are differentially regulated, with three times more Est beta 2(1) being produced than Est alpha 2(1). Cis-acting regulatory sequences associated with these esterases are under investigation. All the amplified esterases in different Culex species act through sequestration. The rates at which they bind with insecticides are more rapid than those for their non-amplified counterparts in the insecticide-susceptible insects. In contrast, esterase-based organophosphate resistance in Anopheles is invariably based on changes in substrate specificities and increased turnover rates of a small subset of insecticides. The up-regulation of both glutathione S-transferases and monooxygenases in resistant mosquitoes is due to the effects of a single major gene in each case. The products of these major genes up-regulate a broad range of enzymes. The diversity of glutathione S-transferases produced by Anopheles mosquitoes is increased by the splicing of different 5' ends of genes, with a single 3' end, within one class of this enzyme family. The trans-acting regulatory factors responsible for the up-regulation of both the monooxygenase and glutathione S-transferases still need to be identified, but the recent development of molecular tools for positional cloning in Anopheles gambiae now makes this possible.     

THE CUMULATIVE TOXICITY OF γ-BHC AND DIAZINON APPLIED IN SMALL DOSES TO LOCUSTS

The toxicity of γ-BHC and Diazinon when applied over a period of time to adults of the desert locust, Schistocerca gregaria (Forsk.), has been determined. The insecticides were applied in oil solutions, by means of the micro-drop syringe, to the ventral surface of the abdomen.
When doses were applied in two equal portions with a 72 hr. interval, or in four equal portions at 24 hr. intervals, no significant decrease in toxicity, in comparison with a single dose, could be detected with either insecticide. It is concluded, therefore, that if similar effects occur when locusts are sprayed with these insecticides in the field, successive spraying will be fully cumulative over a period of 72 hr. Previous work using DNC has shown that doses applied over 4 days were only half as effective as equivalent single doses and that continued dosing after 4 days brought about very little increase in kill.
The sexes were similar in resistance to γ-BHC when the doses were measured in μg./g., but females were considerably more resistant to Diazinon than males.     

天敌昆虫抗药性研究进展

天敌昆虫抗药性研究在协调害虫化学防治和生物防治中有着重要的理论和现实意义,其研究的最终目的在于更好地推进抗性天敌在害虫综合治理(IPM)中的应用。抗药性天敌昆虫具有潜在的巨大价值。鉴于此,本文系统地综述了天敌昆虫抗药性最新研究进展,包括杀虫剂对天敌昆虫的影响、天敌昆虫抗药性现状、抗药性机理和限制天敌昆虫抗药性发展因素等。文章最后还对抗药性天敌昆虫的应用前景进行了展望。     

The molecular basis of two contrasting metabolic mechanisms of insecticide resistance

The esterase-based insecticide resistance mechanisms characterised to date predominantly involve elevation of activity through gene amplification allowing increased levels of insecticide sequestration, or point mutations within the esterase structural genes which change their substrate specificity. The amplified esterases are subject to various types of gene regulation in different insect species. In contrast, elevation of glutathione S-transferase activity involves upregulation of multiple enzymes belonging to one or more glutathione S-transferase classes or more rarely upregulation of a single enzyme. There is no evidence of insecticide resistance associated with gene amplification in this enzyme class. The biochemical and molecular basis of these two metabolically-based insecticide resistance mechanisms is reviewed.     

Relating fitness to long‐term environmental variations in natura

Quantifying links between ecological processes and adaptation dynamics in natura remains a crucial challenge. Many studies have documented the strength, form and direction of selection, and its variations in space and time, but only a few managed to link these variations to their proximal causes. This step is, however, crucial, if we are to understand how the variation in selective pressure affects adaptive allele dynamics in natural settings. We used data from a long‐term survey (about 30 years) monitoring the adaptation to insecticides of Culex pipiens mosquitoes in Montpellier area (France), focusing on three resistance alleles of the Ester locus. We used a population genetics model taking temporal and spatial variations in selective pressure into account, to assess the quantitative relationships between variations in the proximal agent of selection (amounts of insecticide sprayed) and the fitness of resistance alleles. The response to variations in selective pressure was fast, and the alleles displayed different fitness‐to‐environment relationships: the analyses revealed that even slight changes in insecticide doses could induce changes in the strength and direction of selection, thus changing the fitness ranking of the adaptive alleles. They also revealed that selective pressures other than the insecticides used for mosquito control affected the resistance allele dynamics. These fitness‐to‐environment relationships, fast responses and continuous evolution limit our ability to predict the outcome of adaptive allele dynamics in a changing environment, but they clearly contribute to the maintenance of polymorphism in natural populations. Our study also emphasizes the necessity of long‐term surveys in evolutionary ecology.     

The presence of knockdown resistance mutations reduces male mating competitiveness in the major arbovirus vector,Aedes aegypti

BackgroundThe development of insecticide resistance in mosquitoes can have pleiotropic effects on key behaviours such as mating competition and host-location. Documenting these effects is crucial for understanding the dynamics and costs of insecticide resistance and may give researchers an evidence base for promoting vector control programs that aim to restore or conserve insecticide susceptibility.Methods and findingsWe evaluated changes in behaviour in a backcrossed strain of Aedes aegypti, homozygous for two knockdown resistance (kdr) mutations (V1016G and S989P) isolated in an otherwise fully susceptible genetic background. We compared biting activity, host location behaviours, wing beat frequency (WBF) and mating competition between the backcrossed strain, and the fully susceptible and resistant parental strains from which it was derived. The presence of the homozygous kdr mutations did not have significant effects on blood avidity, the time to locate a host, or WBF in females. There was, however, a significant reduction in mean WBF in males and a significant reduction in estimated male mating success (17.3%), associated with the isolated kdr genotype.ConclusionsOur results demonstrate a cost of insecticide resistance associated with an isolated kdr genotype and manifest as a reduction in male mating success. While there was no recorded difference in WBF between the females of our strains, the significant reduction in male WBF recorded in our backcrossed strain might contribute to mate-recognition and mating disruption. These consequences of resistance evolution, especially when combined with other pleiotropic fitness costs that have been previously described, may encourage reversion to susceptibility in the absence of insecticide selection pressures. This offers justification for the implementation of insecticide resistance management strategies based on the rotation or alternation of different insecticide classes in space and time.     

Dieldrin-resistant cabbage root fly in England

The existence of dieldrin resistance in Erioischia brassicae has been investigated in the main areas in England and Wales where brassicas are grown for human consumption. Dieldrin-susceptible and dieldrin-resistant flies were collected and bred as separate strains in the laboratory. In toxicity tests adult flies were treated with topically applied doses of insecticide in microdrops of cellosolve, and larvae were dipped in solutions of insecticide in 70% acetone and 30% water. Adults of the laboratory resistant strain showed a high level of resistance to dieldrin and aldrin and a lower level of resistance to γ-BHC. There was no cross-resistance with chlorfenvinphos or diazinon. Larvae of the resistant strain were shown to be resistant to dieldrin. Dieldrin resistance has been found in four areas in southern England, but not in a fifth major area, and resistant flies did not increase as rapidly in some areas as others. Sufficient migration of flies appeared to take place to produce similar percentages of resistant flies over areas of several square miles, but considerable differences were found in fly populations about 10 miles apart. It is suggested that the speed of development of insecticide resistance is affected by the proportion of brassica crops which are treated in an area, the amounts of insecticide applied to individual crops, and hence the insecticidal pressure applied to the E. brassicae population.     

Multiple Origins of Cyclodiene Insecticide Resistance in Tribolium castaneum (Coleoptera: Tenebrionidae)

The number of origins of pesticide resistance-associated mutations is important not only to our understanding of the evolution of resistance but also in modeling its spread. Previous studies of amplified esterase genes in a highly dispersive Culex mosquito have suggested that insecticide resistance-associated mutations (specifically a single-gene duplication event) can occur a single time and then spread throughout global populations. In order to provide data for resistance-associated point mutations, which are more typical of pesticide mechanisms as a whole, we studied the number of independent origins of cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum. Target-site insensitivity to cyclodienes is conferred by single point mutations in the gene Resistance to dieldrin (Rdl), which codes for a subunit of a γ-aminobutyric acid (GABA) receptor. These point mutations are associated with replacements of alanine 302 which render the receptor insensitive to block by the insecticide. We collected 141 strains of Tribolium worldwide and screened them for resistance. Twenty-four strains contained resistant individuals. After homozygosing 23 of these resistance alleles we derived a nucleotide sequence phylogeny of the resistant strains from a 694-bp section of Rdl, encompassing exon 7 (which contains the resistance-associated mutation) and part of a flanking intron. The phylogeny also included six susceptible alleles chosen at random from a range of geographical locations. Resistance alleles fell into six clades and three clades contained both resistant and susceptible alleles. Although statistical analysis provided support at only the 5–6% level, the pattern of variation in resistance alleles is more readily explained by multiple independent origins of resistance than by spread of a single resistance-associated mutation. For example, two resistance alleles differed from two susceptible alleles only by the resistance-associated mutation itself, suggesting that they form the susceptible ancestors and that resistance arose independently in several susceptible backgrounds. This suggests that in Tribolium Rdl, de novo mutations for resistance have arisen independently in several populations. Identical alleles were found in geographically distant regions as well, also implying that some Rdl alleles have been exported in stored grain. These differences from the Culex study may stem both from differences in the population genetics of Tribolium versus that of mosquitoes and differences in mutation rates associated with point mutations versus gene duplication events. The Tribolium data therefore suggest that multiple origins of insecticide resistance (associated with specific point mutations) may be more common than the spread of single events. These findings have implications for the way in which we model the evolution and spread of insecticide resistance genes and also suggest that parallel adaptive substitutions may not be uncommon in phyletic evolution. Received: 14 October 1998 / Accepted: 4 January 1999     

Modeling the development of resistance by stalk-boring lepidopteran insects (Crambidae) in areas with transgenic corn and frequent insecticide use

We simulated the population dynamics and population genetics of two bivoltine species of corn borers, the European corn borer, Ostrinia nubilalis (Hübner), and the southwestern corn borer, Diatraea grandiosella Dyar, in a hypothetical region of irrigated transgenic and nontransgenic corn where insecticide was applied only to the nontransgenic refuge crop. Over the 100-yr time horizon, resistance developed quickly in both species and to both transgenic corn and the insecticide when the allele for resistance to the respective toxin was dominant. When the allele for transgenic resistance was not dominant and the refuge location was constant over the time horizon, spraying the refuge to control southwestern corn borer had no effect on how quickly resistance to the transgenic corn developed. In contrast, the European corn borer developed resistance to transgenic corn much sooner when the refuge was sprayed once per year, and the time to 3% resistance allele frequency decreased as efficacy of the insecticide increased. Only when the refuge was treated less than once every 5 yr (10 generations) did the frequency of application decline enough to permit resistance management for the European corn borer to approximate the effectiveness of an unsprayed refuge. A consistently sprayed refuge <40% of the corn acreage was an inadequate resistance management strategy for the European corn borer even when a low efficacy insecticide (70% mortality) was used. When assumptions about European corn borer adult behavior were changed and the adults behaved similarly to adult southwestern corn borer, the development of resistance to the transgenic crop was slowed significantly.     

Effectiveness of odor-baited trap trees for plum curculio (Coleoptera: Curculionidae) monitoring in commercial apple orchards in the northeast

The plum curculio, Conotrachelus nenuphar (Herbst), is a key pest of pome and stone fruit in eastern and central North America. For effective management of this insect pest in commercial apple (Malus spp.) orchards in the northeastern United States and Canada, one of the greatest challenges has been to determine the need for and timing of insecticide applications that will protect apple fruit from injury by adults. In a 2004-2005 study, we assessed the efficacy and economic viability of a reduced-risk integrated pest management strategy involving an odor-baited trap tree approach to determine need for and timing of insecticide use against plum curculio based on appearance of fresh egg-laying scars. Evaluations took place in commercial apple orchards in seven northeastern U.S. states. More specifically, we compared the trap-tree approach with three calendar-driven whole-block sprays and with heat-unit accumulation models that predict how long insecticide should be applied to orchard trees to prevent injury by plum curculio late in the season. Trap tree plots received a whole-plot insecticide spray by the time of petal fall, and succeeding sprays (if needed) were applied to peripheral-row trees only, depending on a threshold of one fresh plum curculio egg-laying scar out of 25 fruit sampled from a single trap tree. In both years, level of plum curculio injury to fruit sampled from perimeter-row, the most interior-row trees and whole-plot injury in trap tree plots did not differ significantly from that recorded in plots subject to conventional management or in plots managed using the heat-unit accumulation approach. The amount of insecticide used in trap tree plots was reduced at least by 43% compared with plots managed with the conventional approach. Advantages and potential pitfalls of the bio-based trap tree approach to plum curculio monitoring in apple orchards are discussed.     

First report on co-occurrence knockdown resistance mutations and susceptibility to beta-cypermethrin in Anopheles sinensis from Jiangsu Province, China

The increasing prevalence of insecticide resistance in Anopheles sinensis, a major vector of malaria in Jiangsu province in eastern China, threatens to compromise the successful use of insecticides in malaria control strategies. It is therefore vital to understand the insecticide resistance status of An. sinensis in the region. This study examined the nucleotide diversity of the para-sodium channel and knockdown resistance (kdr) in five field populations of adult An. sinensis mosquitoes collected in Jiangsu province, identifying the L1014F and L1014C substitutions for the first time. Competitive polymerase chain reaction (PCR) amplification of specific allele (cPASA) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) for resistance diagnosis were developed and validated. Comparing the results with direct sequencing revealed that the PCR-RFLP method was more sensitive and specific whereas the cPASA method was more convenient and suitable. The significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014C substitutions in the kdr gene provides a useful molecular marker for monitoring beta-cypermethrin resistance in natural populations of An. sinensis. Our results point to the L1014F substitution as the key mutation associated with beta-cypermethrin resistance. The high resistance and mutation frequency detected in the five populations also suggest cross-resistance with other pyrethroids may occur in An. sinensis, highlighting the need for further surveys to map insecticide resistance in China and the adoption of a rational management of insecticide application for resistance management and mosquito vector control.     

Constraints on adaptive mutations in the codling moth Cydia pomonella (L.): measuring fitness trade-offs and natural selection

Adaptive changes in populations encountering a new environment are often constrained by deleterious pleiotropic interactions with ancestral physiological functions. Evolutionary responses of populations can thus be limited by natural selection under fluctuating environmental conditions, if the adaptive mutations are associated with pleiotropic fitness costs. In this context, we have followed the evolution of the frequencies of insecticide-resistant mutants of Cydia pomonella when reintroduced into an untreated environment. The novel set of selective forces after removal of insecticide pressure led to the decline of the frequencies of resistant phenotypes over time, suggesting that the insecticide-adapted genetic variants were selected against the absence of insecticide (with a selective coefficient estimated at 0.11). The selective coefficients were also estimated for both the major cytochrome P450-dependent monooxygenase (MFO) and the minor glutathione S-transferase (GST) systems (0.17 and negligible, respectively), which have been previously shown to be involved in resistance. The involvement of metabolic systems acting both through xenobiotic detoxification and biosynthetic pathways of endogenous compounds may be central to explaining the deleterious physiological consequences resulting from pleiotropy of adaptive changes. The estimation of the magnitude of the fitness cost associated with insecticide resistance in C. pomonella suggests that resistance management strategies exclusively based on insecticide alternations would be unlikely to delay such a selection process.     

Costly Inheritance and the Persistence of Insecticide Resistance in Aedes aegypti Populations

Global emergence of arboviruses is a growing public health concern, since most of these diseases have no vaccine or prevention treatment available. In this scenario, vector control through the use of chemical insecticides is one of the most important prevention tools. Nevertheless, their effectiveness has been increasingly compromised by the development of strong resistance observed in field populations, even in spite of fitness costs usually associated to resistance. Using a stage-structured deterministic model parametrised for the Aedes aegypti—the main vector for dengue—we investigated the persistence of resistance by studying the time for a population which displays resistance to insecticide to revert to a susceptible population. By means of a comprehensive series of in-silico experiments, we studied this reversal time as a function of fitness costs and the initial presence of the resistance allele in the population. The resulting map provides both a guiding and a surveillance tool for public health officers to address the resistance situation of field populations. Application to field data from Brazil indicates that reversal can take, in some cases, decades even if fitness costs are not small. As by-products of this investigation, we were able to fit very simple formulas to the reversal times as a function of either cost or initial presence of the resistance allele. In addition, the in-silico experiments also showed that density dependent regulation plays an important role in the dynamics, slowing down the reversal process.     

Sodium channel genes and their differential genotypes at the L-to-F kdr locus in the mosquito Culex quinquefasciatus

The para-type sodium channel in insects is the primary target of pyrethroid and DDT insecticides. However, modifications in the target protein structure such as point mutations or substitutions, resulting from single nucleotide polymorphisms (SNP), cause insensitivity of the insect’s nervous system to pyrethroids and DDT and, in turn, result in insecticide resistance. Among these mutations, substitution of leucine to phenylalanine (L to F) in the 6th segment of domain II (IIS6) has been clearly associated with pyrethroid and DDT resistance in many insect species, including mosquitoes. Here, multiple copies of the sodium channel gene were identified in the mosquito Culex quinquefasciatus by Southern blot analysis and polymerase chain reaction (PCR) analysis. Two genomic DNA fragments of the mosquito sodium channel gene (509 and 181 bp) were detected by a single PCR primer pair. Sequence analysis indicated the lack of an intron sequence in the 181 bp sodium channel fragment. Single nucleotide polymorphism (SNP) analysis revealed a strong correlation among the frequencies of L-to-F allelic (T) expression at the RNA level, the frequencies and resistance allele (T) at the L-to-F site of the 509 bp genomic DNA fragment, which did include an intron sequence, and the levels of insecticide resistance. Taking together, this study, for the first time, not only revealed multiple copies of the sodium channel gene presented in the Culex mosquito genome but also suggested that the one with the intro sequence may be a functional copy of the sodium channel gene in the Culex mosquitoes.     

辛硫磷和溴氰菊酯混剂对家蝇抗性发展的影响

以家蝇（ＭｕｓｃａｄｏｍｅｓｔｉｃａｖｉｃｉｎａＭａｃｑｕａｒｔ）为试虫,用辛硫磷溴氰菊酯单剂及不同配比的混剂进行汰选试验。所有混剂选育的家蝇抗性发展都很缓慢,而单剂抗性发展都很快。增效试验表明,辛硫磷与溴氰菊酯混配有明显增效作用,特别是对抗性品系。生化分析结果表明,对溴氰菊酯的抗性发展与酯酶的酶活升高有关。对辛硫磷抗性发展与多功能氧化酶的酶活升高和乙酰胆碱酯酶敏感性降低有关。混剂选育的家蝇其对单剂的敏感性的变化及酶系的变化,随着混剂的配比而变化