Resistant House Flies Research Articles

Resistance to Conventional and New Insecticides in House Flies (Diptera: Muscidae) From Poultry Facilities in Punjab, Pakistan.

House flies, Musca domestica L., are pests of poultry facilities and have the ability to develop resistance against different insecticides. This study was conducted to assess the resistance status of house flies to pyrethroid, organophosphate, and novel chemistry insecticides from poultry facilities in Punjab, Pakistan. Five adult house fly populations were studied for their resistance status to selected conventional and novel chemistry insecticides. For four pyrethroids, the range of resistance ratios was 14-55-fold for cypermethrin, 11-45-fold for bifenthrin, 0.84-4.06-fold for deltamethrin, and 4.42-24-fold for lambda-cyhalothrin when compared with a susceptible population. Very low levels of resistance were found to deltamethrin compared with the other pyrethroids. For the three organophosphate insecticides, the range of resistance ratios was 1.70-16-fold for profenofos, 7.50-60-fold for chlorpyrifos, and 4.37-53-fold for triazophos. Very low levels of resistance were found to profenofos compared with the other insecticides. For five novel chemistry insecticides, the range of resistance ratios was 1.20-16.00-fold for fipronil, 3.73-7.16-fold for spinosad, 3.06-23-fold for indoxacarb, 0.96-5.88-fold for abamectin, and 0.56-3.07-fold for emamectin benzoate. Rotation of insecticides with different modes of action showing no or very low resistance may prevent insecticide resistance in house flies. Regular insecticide resistance monitoring and integrated management plans on poultry farms are required to prevent resistance development, field control failures, and environmental pollution.

Does behaviour play a role in house fly resistance to imidacloprid-containing baits?

The objective of this research was to examine the role and type of behavioural mechanisms that function in house fly, Musca domestica L. (Diptera: Muscidae), resistance to an imidacloprid-containing commercial fly bait, QuickBayt(®) , using an insecticide-susceptible and an imidacloprid-resistant strain. Mortality and feeding behaviour were observed through choice bioassays of three post-imidacloprid selected house fly generations to determine whether flies would consume the bait in the presence of an alternative food source. Mortality rates in choice containers progressively decreased in post-selection flies as QuickBayt(®) no-choice selections proceeded. There were no differences between the proportions of flies observed contacting QuickBayt(®) and sugar, respectively, a finding that eliminates repellency as a mechanism of stimulus-dependent behavioural resistance. However, differences in QuickBayt(®) consumption and subsequent mortality between choice and no-choice containers provided strong support for the evolution of consumption irritancy- or taste aversion-related behavioural resistance. The results of this study support the responsible rotation of insecticide bait formulations for house fly control.

Resistance of the house fly Musca domestica (Diptera: Muscidae) to lambda-cyhalothrin: mode of inheritance, realized heritability, and cross-resistance to other insecticides

Lambda-cyhalothrin, a pyrethroid insecticide, has been used frequently for the control of house flies, Musca domestica L., worldwide including Pakistan. To assess the resistance risk and design a resistance management strategy, a house fly population was exposed to lambda-cyhalothrin in the laboratory to assess inheritance and heritability, and cross-resistance to other insecticides, including different chemical classes. After 11 generations of selection, the population developed 113.57-fold resistance to lambda-cyhalothrin compared to the susceptible population. There was no cross-resistance to bifenthrin and methomyl, but very low cross-resistance to abamectin and indoxacarb in the lambda-cyhalothrin selected population compared to the field population. Synergism bioassay with piperonyl butoxide and S,S,S-tributylphosphorotrithioate indicated that lambda-cyhalothrin resistance was associated with microsomal oxidases and esterases. The LC50 values of F1 (Lambda-SEL ♀ × Susceptible ♂) and F'1 (Lambda-SEL ♂ × Susceptible ♀) populations were not significantly different and dominance (DLC) values were 0.68 and 0.62. The resistance to lambda-cyhalothrin was completely recessive (DML = 0.00) at highest dose and completely dominant at lowest dose (DML = 0.95). The monogenic model of inheritance showed that lambda-cyhalothrin resistance was controlled by multiple factors. The heritability values were 0.20, 0.04, 0.003, 0.07 and 0.08 for lambda-cyhalothrin, bifenthrin, methomyl, indoxacarb and abamectin resistance, respectively. It was concluded that lambda-cyhalothrin resistance in house flies was autosomally inherited, incompletely dominant and controlled by multiple factors. These findings would be helpful to improve the management of house flies.

Genetics and mechanisms of imidacloprid resistance in house flies

Imidacloprid is the most widely used neonicotinoid insecticide against house flies, which are major pests at animal production facilities worldwide. However, cases of both physiological and behavior resistance have been reported. Recently, physiological resistance to imidacloprid was found in the United States (California and Florida). However, no studies have been undertaken to characterize this resistance in house flies from the United States.Three imidacloprid selections of a strain originally collected from Florida increased the level of resistance, ultimately resulting in a strain that had 2300-fold resistance in females and 130-fold in males. Imidacloprid resistance was not overcome with piperonyl butoxide (PBO) suggesting that resistance is not due to detoxification by cytochrome P450s. Resistance was mapped to autosomes 3 and 4. There was⩾100-fold cross-resistance to acetamiprid and dinotefuran, but no cross-resistance to spinosad. The resistance in this imidacloprid selected population was unstable and declined over a period of several months. The significance of these results to management of imidacloprid resistance in the field, and potential mechanisms of resistance involved, are discussed.

A whole transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies, Musca domestica

BackgroundStudies suggest that not only is insecticide resistance conferred via multiple gene up-regulation, but it is mediated through the interaction of regulatory factors. However, no regulatory factors in insecticide resistance have yet been identified, and there has been no examination of the regulatory interaction of resistance genes. Our current study generated the first reference transcriptome from the adult house fly and conducted a whole transcriptome analysis for the multiple insecticide resistant strain ALHF (wild-type) and two insecticide susceptible strains: aabys (with morphological recessive markers) and CS (wild type) to gain valuable insights into the gene interaction and complex regulation in insecticide resistance of house flies, Musca domestica.ResultsOver 56 million reads were used to assemble the adult female M. domestica transcriptome reference and 14488 contigs were generated from the de novo transcriptome assembly. A total of 6159 (43%) of the contigs contained coding regions, among which 1316 genes were identified as being co-up-regulated in ALHF in comparison to both aabys and CS. The majority of these up-regulated genes fell within the SCOP categories of metabolism, general, intra-cellular processes, and regulation, and covered three key detailed function categories: redox detailed function category in metabolism, signal transduction and kinases/phosphatases in regulation, and proteases in intra-cellular processes. The redox group contained detoxification gene superfamilies, including cytochrome P450s, glutathione S-transferases, and esterases. The signal transduction and kinases/phosphatases groups contained gene families of rhodopsin-like GPCRs, adenylate and guanylate cyclases, protein kinases and phosphatases. The proteases group contained genes with digestive, catalytic, and proteinase activities. Genetic linkage analysis with house fly lines comparing different autosomal combinations from ALHF revealed that the up-regulation of gene expression in the three key SCOP detailed function categories occurred mainly through the co-regulation of factors among multiple autosomes, especially between autosomes 2 and 5, suggesting that signaling transduction cascades controlled by GPCRs, protein kinase/phosphates and proteases may be involved in the regulation of resistance P450 gene regulation.ConclusionTaken together, our findings suggested that not only is insecticide resistance conferred via multi-resistance mechanisms or up-regulated genes, but it is mediated through the trans and/or cis co-regulations of resistance genes.

Insecticide resistance in house flies from the United States: Resistance levels and frequency of pyrethroid resistance alleles

Although insecticide resistance is a widespread problem for most insect pests, frequently the assessment of resistance occurs over a limited geographic range. Herein, we report the first widespread survey of insecticide resistance in the USA ever undertaken for the house fly, Musca domestica, a major pest in animal production facilities. The levels of resistance to six different insecticides were determined (using discriminating concentration bioassays) in 10 collections of house flies from dairies in nine different states. In addition, the frequencies of Vssc and CYP6D1 alleles that confer resistance to pyrethroid insecticides were determined for each fly population. Levels of resistance to the six insecticides varied among states and insecticides. Resistance to permethrin was highest overall and most consistent across the states. Resistance to methomyl was relatively consistent, with 65–91% survival in nine of the ten collections. In contrast, resistance to cyfluthrin and pyrethrins + piperonyl butoxide varied considerably (2.9–76% survival). Resistance to imidacloprid was overall modest and showed no signs of increasing relative to collections made in 2004, despite increasing use of this insecticide. The frequency of Vssc alleles that confer pyrethroid resistance was variable between locations. The highest frequencies of kdr, kdr-his and super-kdr were found in Minnesota, North Carolina and Kansas, respectively. In contrast, the New Mexico population had the highest frequency (0.67) of the susceptible allele. The implications of these results to resistance management and to the understanding of the evolution of insecticide resistance are discussed.

Resistance to conventional insecticides in Pakistani populations of Musca domestica L. (Diptera: Muscidae): a potential ectoparasite of dairy animals

The house fly, Musca domestica L., is an important hygienic pest of humans and dairy animals with the potential to develop resistance to most chemical classes of insecticides. Six adult house fly strains from dairy farms in Punjab, Pakistan were evaluated for resistance to selected insecticides from organochlorine, organophosphate, carbamate and pyrethroid classes. For a chlorocyclodiene and two organophosphates tested, the resistance ratios (RR) at LC50 were in the range of 5.60-22.02 fold for endosulfan, 7.66-23.24 fold for profenofos and 2.47-7.44 fold for chlorpyrifos. For two pyrethroids and one carbamate, the RR values at LC50 were 30.22-70.02 for cypermethrin, 5.73-18.31 for deltamethrin, and 4.39-15.50 for methomyl. This is the first report of resistance to different classes of insecticides in Pakistani dairy populations of house flies. Regular insecticide resistance monitoring programs on dairy farms are needed to prevent field control failures. Moreover, integrated approaches including the judicious use of insecticides are needed to delay the development of insecticide resistance in house flies.

Multiple cytochrome P450s overexpressed in pyrethroid resistant house flies (Musca domestica)

Cytochrome P450 monooxygenases-mediated detoxification is a major mechanism of pyrethroid resistance in house flies. Constitutive overexpression of P450 genes is the main cause for this type of insecticide resistance. In this study, three new P450 genes (CYP6A40, CYP6D8 and CYP6G4) over-expressed in a pyrethroid resistance strain (BJD) were identified. Developmental expression analysis showed that CYP6A40 and CYP6D8 were expressed in all life stages, while CYP6G4 was not transcribed in eggs or larvae. Permethrin resistance and expression level of cytochrome P450s in six house fly populations (the BJD strain and five field-derived house fly populations collected across China) were determined. Compared with the susceptible TJS strain, all the six populations exhibited different levels of resistance to permethrin. Piperonyl butoxide (PBO) reduced permethrin resistance level up to 109-fold, indicating that cytochrome P450-mediated detoxification is responsible for pyrethroid resistance in Chinese house flies. Significant overexpression of more than one of the eight P450 genes (CYP6A5v1, CYP6A5v2, CYP6A36, CYP6A40, CYP6D1, CYP6D3, CYP6D8 and CYP6G4) in the six pyrethroid resistant house fly populations was observed. Differential expression patterns of P450s over-represented in the resistance populations suggested evolutionary plasticity of P450-mediated pyrethroid resistance. CYP6G4 and CYP6D1, commonly over-expressed in resistant populations, were hypothesized to be the important P450s contributing to pyrethroid resistance of Chinese house flies.

Investigating the potential of selected natural compounds to increase the potency of pyrethrum against houseflies Muscadomestica (Diptera: Muscidae)

A study was undertaken to determine the efficacy of seven natural compounds compared with piperonyl butoxide (PBO) in synergising pyrethrum, with the intention of formulating an effective natural synergist with pyrethrum for use in the organic crop market. Discriminating dose bioassays showed PBO to be significantly more effective at synergising pyrethrum in houseflies than the seven natural compounds tested, causing 100% mortality in insecticide-susceptible WHO and resistant 381zb strains of housefly. The most effective natural synergists against WHO houseflies were dillapiole oil, grapefruit oil and parsley seed oil, with 59, 50 and 41% mortality respectively, compared with 18% mortality with unsynergised pyrethrum. Against 381zb houseflies, the most effective natural synergists were parsley seed oil and dillapiole oil. Esterase inhibition by the natural compounds and PBO in vitro showed no correlation with pyrethrum synergism in vivo, whereas the inhibition of oxidases in vitro more closely correlated with pyrethrum synergism in vivo. Dillapiole oil and parsley seed oil showed the greatest potential as pyrethrum synergists. PBO remained the most effective synergist, possibly owing to its surfactant properties, enhancing penetration of pyrethrins. The results suggest the involvement of oxidases in pyrethroid resistance in houseflies, with the efficacy of synergists showing a high correlation with inhibition of oxidases.

Micro-evolution of toxicant tolerance: from single genes to the genome’s tangled bank

Two case-studies published 55 years ago became textbook examples of evolution in action: DDT resistance in houseflies (Busvine) and the rise of melanic forms of the peppered moth (Kettlewell). Now, many years later, molecular studies have elucidated in detail the mechanisms conferring resistance. In this paper we focus on the case of metal tolerance in a soil-living arthropod, Orchesella cincta, and provide new evidence on the transcriptional regulation of a gene involved in stress tolerance, metallothionein. Evolution of resistance is often ascribed to cis-regulatory change of such stress-combatting genes. For example, DDT resistance in the housefly is due to insertion of a mobile element into the promoter of Cyp6g1, and overexpression of this gene allows rapid metabolism of DDT. The discovery of these mechanisms has promoted the idea that resistance to environmental toxicants can be brought about by relatively simple genetic changes, involving up-regulation, duplication or structural alteration of a single-gene. Similarly, the work on O. cincta shows that populations from metal-polluted mining sites have a higher constitutive expression of the cadmium-induced metallothionein (Mt) gene. Moreover, its promoter appears to include a large degree of polymorphism; Mt promoter alleles conferring high expression in cell-based bioreporter assays were shown to occur at higher frequency in populations living at polluted sites. The case is consistent with classical examples of micro-evolution through altered cis-regulation of a key gene. However, new data on qPCR analysis of gene expression in homozygous genotypes with both reference and metal-tolerant genetic backgrounds, show that Mt expression of the same pMt homozygotes depends on the origin of the population. This suggests that trans-acting factors are also important in the regulation of Mt expression and its evolution. So the idea that metal tolerance in Orchesella can be viewed as a single-gene adaptation must be abandoned. These data, added to a genome-wide gene expression profiling study reported earlier shows that evolution of tolerance takes place in a complicated molecular network, not unlike an internal tangled bank.

Autosomal Interactions and Mechanisms of Pyrethroid Resistance in House Flies, Musca domestica

Five BC₁ lines and 16 house fly mass-cross homozygous lines were generated from crosses of the pyrethroid resistant ALHF (wild-type) and susceptible aabys (bearing recessive morphological markers on each of five autosomes) strains. Each of the resulting homozygous lines had different combinations of autosomes from the resistant ALHF strain. Levels of resistance to permethrin were measured for each line to determine the autosomal linkage, interaction and, possibly, regulation in pyrethroid resistance of house flies. Results indicated that factors on autosome 4 are not involved in the development of resistance in house flies, while factors on autosomes 1, 2, 3 and 5 play important roles in pyrethroid resistance. The sodium channel gene has been mapped on autosome 3 and multiple cytochrome P450 genes overexpressed in resistant ALHF house flies have been genetically mapped on autosome 5, suggesting that P450 mediated detoxification and sodium channel-mediated target site insensitivity located on autosomes 3 and 5, respectively, are major factors related to resistance development in house flies. However, neither the factors on autosome 3 or 5 alone, nor the factors from both autosomes 3 and 5 combined could confer high levels of resistance to pyrethroid. In addition, strong synergistic effects on resistance was obtained when autosomes 1 and 2 interact with autosome 3 and/or 5, suggesting that the trans factors on autosomes 1 and 2 may interact with factors on autosomes 3 and 5, therefore, playing regulatory roles in the development of sodium channel insensitivity- and P450 detoxification-mediated resistance.

Mortality and knockdown effects of imidacloprid and methomyl in house fly (Musca domestica L., Diptera: Muscidae) populations

Mortality and knockdown effects of imidacloprid and methomyl in house fly (Musca domestica L., Diptera: Muscidae) populations

Survey of insect growth regulator (IGR) resistance in house flies (Musca domestica L.) from southwestern Turkey

Insect growth regulators (IGRs) are currently the fastest-growing class of insecticides, and in Turkey these products represent a new approach to pest control. In recent years, several IGRs were also registered for the control of the house fly, Musca domestica L. (Diptera: Muscidae), in Turkey. A field survey was conducted in the summers of 2006 and 2007 to evaluate resistance to some agriculturally and medically used IGRs on house flies from livestock farms and garbage dumps in the greenhouse production areas (Merkez, Kumluca, Manavgat, and Serik) of Antalya province (Southwestern Turkey). The results of larval feeding assay with technical diflubenzuron, methoprene, novaluron, pyripoxyfen, and triflumuron indicate that low levels (RF<10-fold) of resistance to the IGRs exist in the house fly populations from Antalya province. Exceptions, however, were two populations, Guzoren and Toptas, from the Kumluca area which showed moderate resistance to diflubenzuron with 11.8-fold in 2006 and 13.2-fold in 2007, respectively. We found substantial variation in susceptibility of field-collected house fly populations from year to year and from product to product. We generally observed an increase in resistance at many localities sampled from 2006 to 2007. The implications of these results to the future use of IGRs for house fly control are discussed. It will be critically important to continue monitoring efforts so that appropriate steps can be taken if resistance levels start to increase.

Nicotinoid and pyrethroid insecticide resistance in houseflies (Diptera: Muscidae) collected from Florida dairies

The housefly, Musca domestica L., continues to be a major pest of confined livestock operations. Houseflies have developed resistance to most chemical classes, and new chemistries for use in animal agriculture are increasingly slow to emerge. Five adult housefly strains from four Florida dairy farms were evaluated for resistance to four insecticides (beta-cyfluthrin, permethrin, imidacloprid and nithiazine). Significant levels of tolerance were found in most field strains to all insecticides, and in some cases substantial resistance was apparent (as deduced from comparison with prior published results). At the LC(90) level, greater than 20-fold resistance was found in two of the fly strains for permethrin and one fly strain for imidacloprid. Beta-cyfluthrin LC(90) resistance ratios exceeded tenfold resistance in three fly strains. The relatively underutilized insecticide nithiazine had the lowest resistance ratios; however, fourfold LC(90) resistance was observed in one southern Florida fly strain. Farm insecticide use and its impact on resistance selection in Florida housefly populations are discussed. Housefly resistance to pyrethroids is widespread in Florida. Imidacloprid resistance is emerging, and tolerance was observed to both imidacloprid and nithiazine. If these insecticides are to retain efficacy, producer use must be restrained.

High Performance Liquid Chromatography Assay for p-Nitroanisole o-Demethylation by Cytochrome P450 Enzymes in Musca domestica L.

Abstract A novel sensitive method was developed for measuring p -nitroanisole o -demethylation (PNOD) catalyzed by cytochrome P450 enzymes in crude homogenates of housefly, Musca domestica L., using high performance liquid chromatography (HPLC) with diode array detector. This method is based on the use of liquid-liquid extraction and HPLC with reversed-phase C 18 column and gradient ultraviolet detection at 316 nm. The detection limit ( S/N of 3/1) of p -nitrophenol ( p NP) was 0.1 ng. The relative standard deviation (RSD) of peak area response of the HPLC analysis system was 1.0%. The average recoveries of 0.351, 1.755 and 8.755 μg of p NP added to incubation mixtures ranged from 92.3% to 87.6%, and the RSD of recoveries of three added levels ranged 2.64%–5.90%. The relationships between peak areas and concentration were linear in the range of 9.72–486 ng with good coefficient of determination ( r 2 = 0.9995). This method is sensitive, precise, and reproducible and is also applicable to the assay of p -nitroanisole o -demethylases, which have low catalytic activities, present in other insects or organisms. With this method, the comparison of P450 o -demethylation activity of an imidacloprid-resistant strain with a susceptible strain of M. domestica (L.) was conducted to identify the roles of P450 enzymes involved in housefly resistance to imidacloprid. The results indicated that the resistance mechanism of housefly to imidacloprid was intensely assisted with elevated P450 activities.

Insecticide resistance of house fly, Musca domestica (L.) from Argentina

The status of resistance to cyromazine, 2,2-dichlorovinyl dimethyl phosphate (DDVP), and permethrin relative to field populations of the house fly, Musca domestica L. from Argentinean poultry farms was studied. All the three studied populations (SV, Q, and C) showed resistant ratios (RRs) to cyromazine of 3.9, 10.98, and 62.5, respectively. We observed high levels of resistance toward the organophosphate DDVP and permethrin. The RRs to DDVP ranged from 45.4 to 62.5. No significant differences were found among the studied populations. All the house fly populations were permethrin-resistant, in comparison with the susceptible strain. Two of the analyzed populations (SV and Q) differed significantly in toxicity to the population C. This is the first evidence that house flies from Argentina showed a multi-resistance pattern. The implementation of an insecticide monitoring program on poultry farms of Argentina is needed to prevent field control failures. Furthermore, integrated control strategies are needed to delay detrimental development of insecticide resistance.

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

BackgroundInsects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.ResultsThe expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.ConclusionOur study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.

50 years in insect toxicology: 1956–2006

50 years in insect toxicology: 1956–2006

Unraveling the mystery of spinosad resistance in insects

Spinosad is a new and highly promising insecticide, derived from the bacteria Saccharopolyspora spinosa, with efficacy against a wide range of insects. The mechanism of action of spinosad appears to be unique, with a primary site of attack being the nicotinic acetylcholine receptor (nAChR) and a secondary site of attack being γ-aminobutyric acid (GABA) receptors. Neural nAChRs are composed of five subunits, with a minimum of 2 αs. Each subunit possesses a large N-terminal extracellular domain that includes the acetylcholine (ACh) binding site and four transmembrane domains (M1-4) with M2 contributing most of the amino acids that line the ion channel. Spinosad resistance has been selected for and characterized in several insect species. Generally, resistance is monofactorial, recessive and cannot be overcome by insecticide synergists. Spinosad resistance in the house fly maps to chromosome 1 and three nAChR subunit genes (α5, α6, and β3) are predicted to exist on chromosome 1 based on Drosophila/Musca homology maps. However, cloning and sequencing of Mdα5, Mdα6, and Mdβ3 from susceptible and spinosad resistant strains of house fly found no differences that could be associated with resistance. Unraveling the mystery of spinosad resistance in house flies will require further study.

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.