Development and stability of insecticide resistance in the leafminer Liriomyza trifolii (Diptera: Agromyzidae) to cyromazine, abamectin, and spinosad

Three populations of the leafminer, Liriomyza trifolii (Burgess), were collected from commercial ornamental production greenhouses in the United States and tested for susceptibility to three commercial insecticides. A leaf dip bioassay of leaves containing young (1-2-d-old) larvae was used. Based on larval mortality and compared with a susceptible laboratory reference colony, the three strains varied in spectrum and level of resistance to the insecticides. CA-1, collected from Gerbera daisy, was moderately resistant to cyromazine (18.1-fold) and abamectin (22.0-fold), but highly resistant to spinosad (> 188-fold). CA-2, collected from chrysanthemums, was not resistant to abamectin, had a low level of resistance to cyromazine (8.2-fold), but was extremely resistant to spinosad (1,192-fold). GA-1, collected from chrysanthemums, had very low levels of resistance to cyromazine (5.4-fold) and spinosad (1.9-fold) but was moderately resistant to abamectin (30.6-fold). When reared in the absence of insecticide selection pressure, all three strains reverted to approximately the level of the reference strain. The CA-1 strain reverted in nine generations to cyromazine; however, the lowest levels of abamectin and spinosad resistance reverted to was 3.1-fold at F8 and 3.2 at the F10, respectively. The CA-2 strain reverted in five generations to both cyromazine and spinosad. GA-1 reverted in five generations to abamectin. Based on the results, resistance to these three insecticides was unstable. Additionally, there was no cross-resistance among these three insecticides.     

Genetics of organophosphate resistance in field populations of the house fly (Diptera: Muscidae)

The genetics of resistance to the organophosphate insecticide diazinon were investigated in four populations of the house fly, Musca domestica L., collected in the southern United States. Crosses were made between individual females of lines derived from each population and males of a susceptible strain with three recessive mutants on chromosome II. Individual F1 females were crossed to mutant males, and the progenies were scored for resistance to diazinon and for the presence of mutant phenotypes. A major chromosome II gene for resistance to diazinon was present in all populations at an overall frequency of 83%. Map distances between the resistance gene and the mutant aristapedia and between the mutants aristapedia and stubby wing were highly variable in all populations. Recombination among the visible mutants was usually reduced in resistant progenies relative to susceptible progenies. The data suggest that a single major gene for resistance to diazinon was present on chromosome II in all test populations at variable map positions and is usually associated with a chromosome rearrangement, probably an inversion. The results are similar to those obtained earlier with house fly populations selected for resistance to insecticides in the laboratory; therefore, they seem to be characteristic of field and laboratory populations of the house fly. Overall, the data offer an explanation for previous results suggesting the existence of multiple, closely linked genes for metabolic resistance to insecticides on house fly chromosome II.     

Larvicide resistance in Musca domestica (Diptera: Muscidae) populations in Denmark and establishment of resistant laboratory strains

We determined the toxicity of the two IGRs, diflubenzuron and cyromazine, in this survey of resistance in Danish field populations of Musca domestica (L.). We observed resistance toward diflubenzuron and for the first time in Denmark and we found field populations with some resistance to cyromazine. Eleven of the twenty-one field populations had larvae surviving a diagnostic dose of 1.6 times of susceptible LC95 of diflubenzuron and two of these populations had larvae surviving 6.1 times of LC95. Eight of the twenty-one field populations had larvae surviving 2.2 times of susceptible LC95 of cyromazine and one population had larvae surviving 4.4 times of LC95. A fivefold cyromazine resistant strain was established after selection with cyromazine. It was 3-, 5-, and 90-fold resistant to diflubenzuron, triflumuron, and methoprene, respectively. Two diflubenzuron resistant strains (120- and 86-fold, respectively) were established. They showed a high level of resistance to triflumuron (1000- and 200-fold, respectively), and moderate resistance to methoprene (73- and 50-fold, respectively). Both were susceptible to cyromazine. This study shows that by applying the recommendations of previous resistance risk assessments, severe control failures and detrimental development of a high level of resistance have been avoided. The development of resistance has not been completely avoided, but has not developed to a level of biological or economic importance.     

Insect growth regulator cross-resistance studies in field- and laboratory-selected strains of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae)

Abstract Resistance to diflubenzuron in the Australian sheep blowfly, Lucilia cuprina , has rendered this insecticide incapable of preventing flystrike in sheep from a few districts in eastern Australia. Wool producers affected by this situation must find suitable alternatives to protect their flocks. Results of laboratory bioassays against one population demonstrated that, despite extremely high diflubenzuron resistance (Resistance Factor >791), it had only very low (2x) tolerance of cyromazine and dicyclanil. It is unlikely that this level of tolerance would have any practical impact on field control with either insecticide. Consequently, wool producers in districts where diflubenzuron-resistant flies are common can rotate insecticide treatment to either of these compounds to prevent flystrike in their flocks. However, unlike the highly diflubenzuron-resistant field strain, a laboratory strain selected for resistance to diflubenzuron (Resistance Factor = 617) was 10 times more resistant to dicyclanil than a susceptible strain but, like the field strain, was only two times more tolerant of cyromazine. Conversely, a field-derived strain selected in the laboratory for cyromazine resistance was 20 times more resistant to dicyclanil and 362 times more resistant to diflubenzuron than the reference susceptible strain.     

Impact of an insecticide resistance strategy for house fly (Diptera: Muscidae) control in intensive animal units in the United Kingdom

A strategy for house fly (Musca domestica L.) control in intensive animal units in the United Kingdom was proposed by the Pesticide Safety Directorate (PSD) of the Ministry of Agriculture, Fisheries and Food in 1993. An advice leaflet was circulated to farmers, and label recommendations for insecticides used to control house flies were altered to prevent their long-term and frequent use. A study was carried out between 1996 and 1998 to gather data on insecticide use and resistance in house fly populations and compared with results from a study carried out in 1990-1992 to assess the impact of the 1993 label recommendations. As in the 1990-1992 study, resistance to methomyl, azamethiphos and pyrethrins + piperonyl butoxide was assessed. Larvicide tests with cyromazine, which had recently been released in the United Kingdom, were also included in this study. Most of the farmers claimed to have received and read the PSD insecticide advice leaflet, and half claimed to have altered insecticide treatments as a result. Comparing results for insecticides used before and after 1993, the proportion of farmers claiming to have used each of the insecticides had decreased. However, there had been no amelioration in resistance to synergised pyrethrins, and the number of house fly populations with reduced response to the insecticide baits had increased between 1990-1992 and 1996-1998. All the house fly populations tested were fully susceptible to cyromazine. There is an urgent need, therefore, to devise new strategies and particularly to minimize the risk of selecting for resistance to cyromazine.     

Cross-resistance to insecticides in a malathion-resistant strain of Ceratitis capitata (Diptera: Tephritidae)

Resistance to malathion has been reported in field populations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in areas of Spain where an intensive use of this insecticide was maintained for several years. The main goal of this study was to determine whether resistance to malathion confers cross-resistance to different types of insecticides. Susceptibility bioassays showed that the malathion-resistant W-4Km strain (176-fold more resistant to malathion than the susceptible C strain) has moderate levels of cross-resistance (three- to 16-fold) to other organophosphates (trichlorphon, diazinon, phosmet and methyl-chlorpyrifos), the carbamate carbaryl, the pyrethroid lambda-cyhalothrin, and the benzoylphenylurea derivative lufenuron, whereas cross-resistance to spinosad was below two-fold. The W-4Km strain was selected with lambda-cyhalothrin to establish the lambda-cyhalothrin-resistant W-1Klamda strain (35-fold resistant to lambda-cyhalothrin). The synergistic activity of the esterase inhibitor DEF with lambda-cyhalothrin and the increase in esterase activity in the W-1Klamda strain suggests that esterases may be involved in the development of resistance to this insecticide. Our results showed that resistance to malathion may confer some degree of cross-resistance to insecticides currently approved for the control of Mediterranean fruit fly in citrus crops (lambda-cyhalothrin, lufenuron, and methyl-chlorpyrifos). Especially relevant is the case of lambda-cyhalothrin, because we have shown that resistance to this insecticide can rapidly evolve to levels that may compromise its effectiveness in the field.     

The genetic bases of high-level resistance to diflubenzuron and low-level resistance to cyromazine in a field strain of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae)

Abstract  Several genes on chromosomes IV and VI have a significant influence on high-level resistance to diflubenzuron in a strain of the Australian sheep blowfly from Tara, Queensland. Low-level resistance to cyromazine in the same strain is due to genes on these chromosomes with a gene (gene complex) in the sv marker region of chromosome IV being particularly important. For both insecticides, genetic background influences resistance status. If the results of the Tara strain prove typical for those of other populations, resistance to diflubenzuron in the Australian sheep blowfly has potentially significant consequences for woolgrowers.     

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.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.     

Life-stage variation in insecticide resistance of the western flower thrips (Thysanoptera: Thripidae)

The life-stage variations in insecticide resistance of western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), to selective insecticides (acrinathrin, formetanate, and methiocarb) were studied using resistant laboratory strains. In each strain, the second-instar larva was less susceptible to the insecticides tested than the adults. The lower the resistance level of the adults, the higher the difference between larva and adult susceptibility: 32-fold to methiocarb, 15.4-fold to formetanate, and 180-fold to acrinathrin in the reference strain. In laboratory-selected resistant strains, these differences were much lower: 5.8-fold to methiocarb, 4.8-fold to formetanate, and 2.0-fold to acrinathrin. In selected strains, higher resistance levels for each insecticide were found, both for larvae and adults, compared with the reference strain. These results show that after insecticide resistance selection in adults, the resistance is carried over to the larvae, but at lower levels.     

A genetic analysis of cyromazine resistance in Drosophila melanogaster (Diptera: Drosophilidae)

Flies resistant to the insect growth regulator cyromazine were selected in the F1 generation from a cyromazine-susceptible strain of Drosophila melanogaster (Meigen) treated with ethyl methanesulfonate. Four resistant strains were isolated by screening with cyromazine at a concentration > LC100 of susceptibles. In each strain, resistance is conferred by a single gene mutation. Cyromazine resistance in two of the mutants (rst(1a)cyr1 and rst(1a)cyr2) localizes to map position 17 of the X chromosome. Evidence is presented that these mutations are alleles of the gene rst(1a)cyr. Cyromazine resistance in another of the mutants (Rst(1b)Cyr) is also X-linked, and localizes to map position 49 of the X chromosome. The location of the gene conferring cyromazine resistance in the other mutant (Rst(2b)Cyr) is map position 66 of chromosome II. This is possibly an allele of a previously characterized cyromazine resistance gene, Rst(2)Cyr. Dosage-mortality analyses demonstrate a low level of cyromazine resistance is conferred in all strains.     

Autosomal sex-associated pyrethroid resistance in a strain of house fly (Diptera: Muscidae) with a male-determining factor on chromosome three

Mechanisms and genetics of resistance to pyrethroid insecticides were investigated in a strain of house fly (ASPR) collected from a cattle ranch in Miyagi, Gunma Prefecture, Japan. Flies were selected in the laboratory with the pyrethroid insecticide permethrin. Both sexes were resistant to pyrethroids; however, females were 22- to 245-fold more resistant than males. Permethrin resistance could be partly suppressed by the monooxygenase inhibitor piperonyl butoxide in females, but not in males. In this strain, sex was determined by a male factor on the third autosome. The relationship of the autosomal male factor to the lower resistance levels observed in the males and the mechanisms of resistance expressed in each sex are discussed.     

Genetics and fitness costs of cyromazine resistance in the house fly (Diptera: Muscidae)

The genetic basis of cyromazine resistance was investigated in the house fly, Musca domestica L. The ED-R strain, which was collected in Mississippi and selected further in the laboratory, was 116.5-fold resistant compared with the laboratory susceptible strain, OR-S. The SEL strain, which was created by crossing ED-R with OR-S followed by three cycles of reselection and backcrossing to OR-S, was 84.7-fold resistant relative to the susceptible strain. Mortality data from reciprocal crosses of resistant and susceptible flies indicated that resistance was autosomal and not influenced by maternal effects. The relative position of probit lines from the parental strains and reciprocal crosses showed that resistance was expressed as an incompletely dominant trait with D = 0.30 and 0.32 for ED-R and SEL, respectively. To determine the number of genes involved, models of one, two, three, four, and five loci were used to compare observed and expected mortality of F1ED-R x susceptible backcross. Resistance was best described by a polygenic model of three loci when equal and additive effects of loci were assumed. Another approach, which was based on phenotypic variances, showed that nE, or the minimum number of freely segregating genetic factors for ED-R, equaled 3.07. ED-R showed greater reductions in fitness compared with SEL independent of the presence or absence of sublethal concentrations of cyromazine. These data suggested that reduced fitness was not due to deleterious pleiotropic effects of the resistance genes themselves but arose from other loci in the ED-R genotype.     

Acetylcholinesterase mutation in an insecticide-resistant population of the codling moth Cydia pomonella (L.)

Two strains of Cydia pomonella (L.) (Lepidoptera: Tortricidae) were selected in the lab by exposure to increasing concentrations of diflubenzuron (Rdfb strain) or azinphos-methyl (Raz strain). Insecticide bioassays showed that the adults of the Rdfb strain exhibited a 2.6-fold and a 7.7-fold resistance ratio to azinphos-methyl and carbaryl, respectively compared to a susceptible strain (S) whereas the adults of the Raz strain exhibited a 6.7-fold resistance ratio to azinphos-methyl and a 130-fold resistance ratio to carbaryl. In the Raz strain, a target site resistance mechanism was suggested by the inhibition of acetylcholinesterase (AChE) activity. In fact the ki values did not discriminate the S and Rdfb strains, while the Raz strain exhibited a 1.7-fold and a 14-fold increase in ki value compared to the S strain for azinphos-methyl oxon and carbaryl, respectively. To verify this hypothesis, two cloned AChE cDNAs sequences (named cydpom-ace2 e cydpom-ace1) were compared between the susceptible and the resistant strains. No difference in the deduced amino acid sequence was found in cydpom-ace2 (orthologous to the Drosophila melanogaster AChE). In the putative cydpom-ace1 (paralogous to the Drosophila AChE), a single amino acid substitution F399V was exclusively present in the Raz strain. The F399 lined the active site of the enzyme and the F399V substitution likely could influence the accessibility of different types of inhibitors to the catalytic site of the insensitive cydpom-ace1.     

Pleiotropy of insecticide resistance in the codling moth, Cydia pomonella

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), has developed resistance to various insecticides. Relative fitness of one susceptible strain (Sv) and two strains selected for resistance to diflubenzuron (Rt) and deltamethrin (Rv), respectively, was measured in the absence of insecticide selection pressure. Mating rate, fecundity, fertility, developmental time, fifth instar weight, and adult longevity were compared. Both resistant strains were less fecund and fertile, developed more slowly, weighed less, and had shorter life-spans than the susceptible strain. These results indicate that biological constraints are associated with insecticide resistance in the codling moth. We also found that fitness estimates of the Rv strain did not differ statistically from those of the Rt strain. Enhanced mixed-function oxidase and glutathione-S-transferase activities have been shown to be involved in insecticide resistance in both Rt and Rv strains. This suggests that the fitness cost described in both resistant strains was mainly associated to metabolic resistance. The impact of such deleterious pleiotropy of insecticide resistance in C. pomonella in terms of resistance management in the field is discussed.     

Differential expression of CYP6A5 and CYP6A5v2 in pyrethroid-resistant house flies, Musca domestica

Two cytochrome P450 alleles, CYP6A5 and CYP6A5v2, were isolated from a pyrethroid-resistant house fly stain, ALHF. The two alleles shared 98% similarity in amino acid sequence. To understand the importance of these two alleles in resistance and examine the expression profile of the two alleles between resistant and susceptible strains, quantitative real-time PCR (qRT-PCR) was performed and compared with the Northern blot analysis. We found that qRT-PCR was an efficient method to characterize the expression profiles between these two sequence-closely-related P450 genes between resistant and susceptible houses flies. One of them, CYP6A5v2, was constitutively overexpressed in ALHF house flies compared with susceptible house fly strains. Moreover, this gene was predominantly expressed in the abdominal tissues of ALHF, in which the primary detoxification organs of insects are located. However, there was no significant difference in the expression of CYP6A5 between ALHF and susceptible house flies. The genetic linkage analysis was conducted to determine the possible link between the constitutively overexpressed CYP6A5v2 and insecticide resistance. CYP6A5v2 was mapped on autosome 5, which is correlated with the linkage of resistance in ALHF. Taken together, the study suggests the importance of CYP6A5v2 in increasing metabolic detoxification of insecticides in ALHF. The distinct expression of CYP6A5 and CYP6A5v2 in resistant and susceptible house flies implies the functional difference of theses two genes in house flies and suggests that they are two recently diverged P450 genes presented in a single organism.     

Differential ovicidal and larvicidal resistance to benzoylureas in the codling moth, Cydia pomonella

French populations of the codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae), a major pest in apple and pear orchards, have developed resistance to different classes of insecticides including the benzoylurea diflubenzuron, a chitin synthesis inhibitor. Ovicidal tests performed on two susceptible strains and one strain selected for its resistance to diflubenzuron revealed the same order of magnitude in resistance ratios to this compound (30-fold) and two other benzoylureas teflubenzuron and flufenoxuron (22- and 11-fold, respectively). Field rates of these three compounds induced a 45–55% decrease in hatching in the resistant strain, compared to over 90% in the susceptible insects. Despite a 52-fold ovicidal resistance ratio to the juvenile hormone analog fenoxycarb, this compound induced a 85% decrease in hatching in the resistant strain. Conversely the newly hatched larvae of the resistant strain exhibited a 45 000-, 33- and 2.1-fold resistance ratio to diflubenzuron, teflubenzuron and flufenoxuron, respectively. The latter value was not significant, and the field rate of flufenoxuron killed over 97% of the resistant larvae while diflubenzuron had no effect. This lack of relationship between ovicidal and larvicidal resistance may be due to different transport properties together with differential enzymatic metabolization. Our results may limit the validity of substitution instars, which approach is frequently used for resistance monitoring. More importantly for resistance management, the resistance of different target instars to each compound has to be considered when establishing control strategies.     

二点叶螨对甲氰菊酯、氧乐果和四螨嗪抗药性的选育、衰退和恢复

以兰州吐鲁沟公园的二点叶螨Tetranychusurticae为敏感品系 ,分别用氧乐果、甲氰菊酯、四螨嗪喷雾处理 15次 ,其抗性水平分别为 38 5、 479 8和 6 7 3倍。将抗性品系分别与敏感品系进行杂交和回交的结果表明 :抗氧乐果品系的显性系数DRS (R♀×S♂ )为 0 470 0、DSR (S♀×R♂ )为 0 4749;抗甲氰菊酯品系的DRS (R♀×S♂ )为 0 5 15 5 ,DSR 为0 5 2 37;抗四螨嗪品系的DRS为 0 3134 ,DSR 为 0 2 46 6。表明二点叶螨对这 3种药剂的抗性均是由单个不完全显性基因所控制。在连续 10个月不接触药剂的情况下 ,3个抗性品系的抗药性都有下降 ,抗氧乐果品系的抗性下降最快 ,只有敏感品系的抗性倍数的 3 6倍 ;抗甲氰菊酯种群的抗性下降较慢 ,为敏感品系的 95 9倍。再经甲氰菊酯、氧乐果、四螨嗪分别连续15次喷雾处理后 ,3个抗性种群的抗性水平又再度回升 ,抗甲氰菊酯品系回升较快 ,抗性为敏感品系的 5 2 3 5倍 ,抗四螨嗪品系次之 ,抗氧乐果的品系抗性恢复最慢。     

DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid

Mutagenesis can be used as a means of predicting likely mechanisms of resistance to novel classes of insecticides. We used chemical mutagenesis in Drosophila to screen for mutants that had become resistant to imidacloprid, a neonicotinoid insecticide. Here we report the isolation of two new dominant imidacloprid-resistant mutants. By recombinational mapping we show that these map to the same location as Rst(2)DDT. Furthermore, we show that pre-existing Rst(2)DDT alleles in turn confer cross-resistance to imidacloprid. In order to localize the Rst(2)DDT gene more precisely, we mapped resistance to both DDT and imidacloprid with respect to P-element markers whose genomic location is known. By screening for recombinants between these P-elements and resistance we localized the gene between 48D5-6 and 48F3-6 on the polytene chromosome map. The genomic sequence in this interval shows a cluster of cytochrome P450 genes, one of which, Cyp6g1, is over-expressed in all resistant strains examined. We are now testing the hypothesis that resistance to both compounds is associated with over-expression of this P450 gene.     

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.