Dominance of Insecticide Resistance Presents a Plastic Response

Dominance level of insecticide resistance provided by one major gene (an insensitive acetylcholinesterase) in the mosquito Culex pipiens was studied in two distinct environments. Dominance level was found to be very different between environments, varying from almost complete dominance to almost recessive when either propoxur (a carbamate insecticide) or chlorpyrifos (an organophosphorus insecticide) was used. To better understand this plastic response, three environmental parameters were manipulated and their interactions studied. For chlorpyrifos, each parameter had a small effect, but when all parameters were changed, the dominance level was greatly affected. For propoxur, one environmental parameter had a large effect by itself. It was further studied to understand the causal relationship of this plasticity. Recessivity of resistance was associated with more demanding environments. These results are discussed in the context of the various theories of the evolution of dominance. It appears that dominance of insecticide resistance cannot be directly predicted by Wright's physiological theory.     

棉铃虫抗药性的生理生化机制研究

本文报道了棉铃虫Helicoverpa armigera田间抗性种群对杀虫剂抗药性的生理生化机制。抗性种群(HJ-R)5龄幼虫羧酸酯酶、谷胱甘肽转移酶、多功能氧化酶活力均明显高于相对敏感种群(HD-S)。两种群乙酰胆碱酯酶对杀虫剂敏感性没有显著差异。HJ-R种群的腹神经索对氰戊菊酯表现了2-3倍的神经不敏感性。HJ-R种群对氨基甲酸酯类杀虫剂的抗性主要是由代谢机制引起,其中多功能氧化酶可能起主导作用;对菊酯的抗性是由多功能氧化酶、酯酶、以及神经不敏感性几个因子综合作用的结果。     

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     

The genetic basis of malathion resistance in housefly (Musca domestica L.) strains from Turkey

Organophosphate insecticide (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdalphaE7 gene is probably playing a role in detoxification of xenebiotic esters. In our research, we have isolated, cloned and sequenced the MdalphaE7 gene from 5 different Turkish housefly strains. High doses of malathion (600 microg/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana and Ankara strains while no insecticide treatment was performed in the laboratory to Kirazli strain. Trp251 --> Ser substitution was found in the product of MdalphaE7 gene in all malathion resistant and Kirazli stocks. In addition, we checked the malathion carboxylesterase (MCE), percent remaining activities in acetylcholinesterase (AChE), glutathion-S-transferase (GST), and general esterase activities in all 5 strains used in this study. In comparing with universal standard sensitive control WHO, a high level of MCE and GST activities were observed while lower level of general esterase activities was detected in the tested strains. In addition, a higher percent remaining activities in AChE than WHO susceptible strain were observed in all malathion resistant strains.     

Amino-acid substitutions in acetylcholinesterase 1 involved in insecticide resistance in mosquitoes

In natural populations of mosquitoes, high level of resistance to carbamates (CX) and organophosphates (OP) is provided by insensitive acetylcholinesterase (AChE1). Different alleles conferring resistance have been identified at the ace1 locus. They differ from the wild-type by only one amino-acid substitution. The comparison of the biochemical characteristics of mutated recombinant proteins and AChE1 in resistant mosquito extracts confirmed the role of each substitution in insensitivity. Selection of these different resistant alleles in field populations depends likely on the insecticides used locally. Theoretical modelling studies are initiated to develop novel strategies of mosquito control.     

Geographical distribution and evolutionary history of organophosphate-resistant Ace alleles in the olive fly (Bactrocera oleae)

Acetylcholinesterase (Ace) is the molecular target of organophosphate (OP) insecticides, and two mutations that confer different levels of OP insensitivity have previously been identified in the olive fly, Bactrocera oleae. Numerous sensitive and two insensitive alleles (including one convergent acquisition) are described from the entire worldwide distribution of the fly. Most of the variation is harbored in the native range of the species and in the Middle East and consists of numerous low-frequency sensitive alleles. The insensitive alleles likely came to high frequency more recently in the Mediterranean region or in the Middle East, reaching frequencies as high as 100% in some populations, and determined a corresponding decline in overall genetic variation. We hypothesize that the major force that shaped the current distribution of resistant and non-resistant acetylcholinesterase alleles is natural selection, likely responsible for the high frequency of insensitive alleles in areas where organophosphates have been used extensively. We also discuss a role for historical contingency, that can explain why sensitive alleles are absent altogether in the species ancestral range and present in areas of recent expansion, such as California, despite the limited use of OPs.     

Biochemical characterization of insecticide resistance in the German cockroach, Blattella germanica, from Malaysia

The possible insecticide resistance mechanisms of four Malaysian field-collected strains of the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), were characterized with biochemical assays and native polyacrylamide gel electrophoresis (PAGE). Elevated esterase activity (at low to moderate frequency) and altered acetylcholinesterase (low frequency) were detected in all field strains, while elevated glutathione S-transferase levels were present in only two strains. Seven esterase bands were separated by native PAGE; a greater intensity occurred in three bands in the resistant strains compared to the susceptible strain. Inhibition studies using specific inhibitors on polyacrylamide gels suggested that the slowest of these three esterases is a cholinesterase, while the other two are carboxylesterases with a preference for beta- over alpha-naphthyl acetate.     

Inheritance of partial resistance to powdery mildew in spring wheat

Summary Four spring wheat (Triticum aestivum L.) cultivars exhibiting partial resistance to powdery mildew induced by Erysiphe graminis f.sp. tritici were crossed to a common susceptible cultivar to study the inheritance of resistance. The genetic parameters contributing to resistance were estimated by generation means analyses. Additive gene action was the most important genetic component of variation among generation means in all four crosses. Additive by additive effects were significant in one cross and both additive by additive and additive by dominance effects were significant in another. Dominance effects were not significant. The F2/F3 correlations in three crosses ranged from 0.27 to 0.43. Three additional crosses among resistant cultivars were employed to study the effectiveness of selection in improving resistance. By selecting the most resistant plants from the F2 and evaluating the progenies in the F4, increases in resistance ranging from 21% to 31% were obtained. In all crosses, there was transgressive segregation in both directions indicating that the genes conferring resistance to these cultivars differ and exhibit additive effects.     

Biochemical genetics of altered acetylcholinesterase resistance to insecticides in the house fly

Resistance to the organophosphate insecticide tetrachlorvinphos was examined in a house fly (Musca domestica L.) strain with an altered acetylcholinesterase (AChE) of decreased sensitivity to inhibition by the insecticide. Genetic tests showed that both resistance and the altered AChE were controlled by semidominant gene(s) on chromosome II. The gene for resistance was five crossover units from the mutant marker stubby wing (stw). A house fly strain was prepared in which resistance was introduced in to a susceptible stw strain by recombination. Biochemical assays revealed that the altered AChE was introduced along with resistance. Assays of the AChE of resistant and susceptible stw strains by two independent methods showed that the enzyme from resistant flies was 30 times more slowly inhibited by tetrachlorvinphos than the enzyme from susceptible flies.This work was supported in part by NIH Grant ES 00901.Technical Article 13340, Texas Agricultural Experiment Station.     

Acetylcholinesterase alterations reveal the fitness cost of mutations conferring insecticide resistance

Background

Insecticide resistance is now common in insects due to the frequent use of chemicals to control them, which provides a useful tool to study the adaptation of eukaryotic genome to new environments. Although numerous potential mutations may provide high level of resistance, only few alleles are found in insect natural populations. Then, we hypothesized that only alleles linked to the highest fitness in the absence of insecticide are selected.

Results

To obtain information on the origin of the fitness of resistant alleles, we studied Drosophila melanogaster acetylcholinesterase, the target of organophosphate and carbamate insecticides. We produced in vitro 15 possible proteins resulting from the combination of the four most frequent mutations and we tested their catalytic activity and enzymatic stability. Mutations affected deacetylation of the enzyme, decreasing or increasing its catalytic efficiency and all mutations diminished the stability of the enzyme. Combination of mutations result to an additive alteration.

Conclusion

Our findings suggest that the alteration of activity and stability of acetylcholinesterase are at the origin of the fitness cost associated with mutations providing resistance. Magnitude of the alterations was related to the allelic frequency in Drosophila populations suggesting that the fitness cost is the main driving force for the maintenance of resistant alleles in insecticide free conditions.

     

Molecular and Biochemical Diagnosis of Esterase-Mediated Pyrethroid Resistance in a Mexican Strain of Boophilus microplus (Acari: Ixodidae)

We examined pyrethroid resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to determine the mechanisms conferring resistance. Permethrin hydrolysis assays and esterase activity gels indicated enhanced esterase-mediated metabolic detoxification in the Cz strain, while one other pyrethroid resistant strain, SF, and two pyrethroid susceptible strains had lower levels of permethrin hydrolysis. Results from assays using a PCR-based test to detect a pyrethroid target site resistance-associated mutation in the tick sodium channel gene found only low levels of mutations in the Cz strain, while the SF strain had a high level of the mutated sodium channel alleles. A specific esterase, designated CzEst9, believed to be responsible for the esterase-mediated pyrethroid resistance in the Cz strain was purified, and the gene encoding CzEst9 cloned. This revised version was published online in July 2006 with corrections to the Cover Date.     

A generalized approach to detection of organophosphate resistance in mosquitoes

Insecticide bioassays and biochemical microtitre assays were compared for detection of resistance to the organophosphate insecticides malathion and fenitrothion, using inbred laboratory strains of malaria vectors Anopheles albimanus Wiedemann, An.arabiensis Patton and An.stephensi Liston. With susceptible mosquitoes, the LT100 values determined from bioassays corresponded closely with times taken to abolish the activity of acetylcholinesterase activity in biochemical assays: approximately 2 h for malathion and 3 h for fenitrothion. Resistant strains of all three anophelines showed longer survival correlated with prolonged acetylcholinesterase activity. An.albimanus strains with insensitive acetylcholinesterase survived bioassays with discriminating doses of 1 h exposure to 5% malathion or 1% fenitrothion and were judged as resistant. It is concluded that enzyme-specific microassays provide a reliable means of detecting resistant individuals, with practical advantages over bioassays which do not reveal the resistance mechanism and require large numbers of healthy mosquitoes.     

High expression of Cyp6g1, a cytochrome P450 gene, does not necessarily confer DDT resistance in Drosophila melanogaster

Cytochrome P450 monooxygenases, a family of detoxifying enzymes, are thought to confer resistance to various insecticides including DDT. Daborn et al. [Daborn, P., Yen, J.L., Bogwitz, M., Le Goff, G., Feil, et al. 2002. A single p450 allele associated with insecticide resistance in Drosophila. Science 297, 2253-2256.] suggested that the Accord transposable element causes overexpression of a Cyp6g1 allele, which has spread globally and is the basis of DDT resistance in Drosophila melanogaster populations. To determine whether the same phenomenon also operates in other Drosophila strains, we investigated 91-R, 91-C, ry(506), Wisconsin, Canton-SH and Hikone-RH strains. While the LC(50) values for the 91-R and Wisconsin strains are 8348 microg and 447 microg of DDT, respectively, values for the other four strains range between 0.74 to 20.9 microg. As expected, the susceptible ry(506) and 91-C strains have about 16-33-fold lower levels of CYP6G1 mRNA than the resistant 91-R and Wisconsin strains. Surprisingly, CYP6G1 mRNA and protein levels in the Canton-SH and Hikone-RH strains are as high as in the two resistant strains, yet they are as susceptible as the 91-C strain. The susceptible phenotype of the Canton-SH and Hikone-RH strains is not due to mutation in the Cyp6g1 gene; sequence analysis showed that Cyp6g1 alleles of resistant and susceptible strains are very similar and cannot be classified into resistant and susceptible alleles. As observed by others, we also found that only the 5'-upstream DNA of overexpressing alleles of Cyp6g1 has an insertional DNA, which is similar to Accord and Ninja elements. To examine the role of Cyp6g1 in DDT resistance, we substituted the Cyp6g1 allele of the 91-R strain with the allele from the susceptible 91-C strain via recombination and synthesized three recombinant lines. All three lines lacked Accord insertion and showed low expression of Cyp6g1 like the 91-C strain, yet they were as highly resistant as the 91-R strain. We conclude a strain may not have to have Accord insertion in the Cyp6g1 gene and the Cyp6g1 itself may not have to be overexpressed for DDT resistance to occur.     

Insecticide resistance genes confer a predation cost on mosquitoes, Culex pipiens

Newly occurring adaptive genes, such as those providing insecticide resistance, display a fitness cost which is poorly understood. In order to detect subtle behavioural changes induced by the presence of resistance genes, we used natural predators and compared their differential predation on susceptible and resistant Culex pipiens mosquitoes, using strains with a similar genetic background. Resistance genes were either coding an overproduced detoxifying esterase (locus Ester), or an insensitive target (locus ace-1). Differential predation was measured between susceptible and resistant individuals, as well as among resistant mosquitoes. A backswimmer, a water measurer, a water boatman and a predaceous diving beetle were used as larval predators, and a pholcid spider as adult predator. Overall, the presence of a resistance gene increased the probability of predation: all resistance genes displayed predation costs relative to susceptible ones, at either the larval or adult stage, or both. Interestingly, predation preferences among the susceptible and the resistance genes were not ranked uniformly. Possible explanations for these results are given, and we suggest that predators, which are designed by natural selection to detect specific behavioural phenotypes, are useful tools to explore non-obvious differences between two classes of individuals, for example when they differ by the presence or absence of one recent gene, such as insecticide resistance genes.     

Mutations in acetylcholinesterase genes of Rhopalosiphum padi resistant to organophosphate and carbamate insecticides.

Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.     

High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes

We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l⁻¹ and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1^R allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1^R and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1^V or the duplicated ace-1^D allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.     

Living in a jar: genetic variation and differentiation among laboratory strains of the red flour beetle

The red flour beetle, Tribolium castaneum, is a common pest, which has become an important model study organism, especially in genetic, ecological and evolutionary research. Although almost all studies on this species have been conducted using established laboratory strains, very little is known about the loss of genetic diversity within the strains and genetic divergence between different laboratory stocks. In this study, five long‐term laboratory strains and one wild strain were examined for genetic variation at 12 microsatellite loci, which were designed using publicly available sequences. One of the laboratory strains is resistant to phosphine and one to organophosphorous insecticides. All strains had significant amounts of molecular variation, but genetic diversity in the laboratory strains was lower than in the wild‐derived strain used as control. We observed significant molecular divergence among the strains, however, the relationship between them reflected resistance status rather than geographic origins. We found no evidence for recent bottlenecks, but the wild‐derived population showed signs of demographic expansion. A novel multivariate method, multiple co‐inertia analysis, revealed that the two loci contributing most to the divergence between the resistant strains were located on the eighth chromosome, near genes associated with insecticide resistance.     

The dominance of the herbicide resistance cost in several Arabidopsis thaliana mutant lines

Resistance evolution depends upon the balance between advantage and disadvantage (cost) conferred in treated and untreated areas. By analyzing morphological characters and simple fitness components, the cost associated with each of eight herbicide resistance alleles (acetolactate synthase, cellulose synthase, and auxin-induced target genes) was studied in the model plant Arabidopsis thaliana. The use of allele-specific PCR to discriminate between heterozygous and homozygous plants was used to provide insights into the dominance of the resistance cost, a parameter rarely described. Morphological characters appear more sensitive than fitness (seed production) because 6 vs. 4 differences between resistant and sensitive homozygous plants were detected, respectively. Dominance levels for the fitness cost ranged from recessivity (csr1-1, ixr1-2, and axr1-3) to dominance (axr2-1) to underdominance (aux1-7). Furthermore, the dominance level of the herbicide resistance trait did not predict the dominance level of the cost of resistance. The relationship of our results to theoretical predictions of dominance and the consequences of fitness cost and its dominance in resistance management are discussed.