House flies were collected from 16 different provinces in the Aegean and Mediterranean regions of Turkey, and the frequencies of pyrethroid resistance-associated mutations in Vssc1 and CYP6D1 in these field-collected populations were studied. Although there is no organized resistance management program for house fly control in Turkey, it is known that different groups of insecticides, including pyrethroids, are used. The frequencies of both Vssc1 and CYP6D1 alleles were weighted toward the susceptibles, with Vssc1-susceptible alleles having higher frequencies in both regions (0.75 in Aegean and 0.69 in Mediterranean populations) than CYP6D1-susceptible alleles (0.65 in Aegean and 0.56 in Mediterranean populations). The frequencies of kdr-his alleles were higher than the frequencies of kdr alleles in these populations. While the frequencies of kdr-his alleles were close to each other in the Aegean (0.23) and Mediterranean (0.17) populations, the frequencies of kdr alleles remarkably differed in these two regions, with values of 0.02 and 0.14, respectively. In contrast to Europe, Asia, and the U.S.A., no super-kdr allele was detected in the samples from both regions. We identified six and eight different Vssc1+CYP6D1 genotype classes in the Aegean and Mediterranean regions, respectively. The three most common genotype classes in the regions were susceptible Vssc1 with heterozygous CYP6D1v1 (29%), sus/kdr-his1 with heterozygous CYP6D1v1 (23%), and susceptible Vssc1 with CYP6D1 (22%). The total frequencies of these three most common genotype classes (approximately 75%) obtained in our study were very close to the value obtained in Florida in a previous study, which was related by the similarity of temperature patterns between Florida and the corresponding regions of Turkey. This may reflect the lack of overwintering fitness cost associated with resistance alleles in both climates.
The house fly (Musca domestica L.) is a serious pest of livestock and poultry facilities as well as a serious public health problem because it transmits human and animal pathogens. Among the different compounds used to control this organism, pyrethroid insecticides, which are the second most widely used class of insecticides, have been in use for more than 30 years and account for 25% of the worldwide insecticide market (Casida and Quistad 1998). They are effective, safe for the environment and less toxic to mammals, and therefore, are preferred insecticides for house fly control (Elliot and Janes 1978, Liu and Pridgeon 2002).
Although pyrethroids were initially highly effective against house flies, intensive use of these insecticides has resulted in the development of resistance (MacDonald et al. 1983, Scott et al. 1989, Shono et al. 2002). Resistance development is one of the major obstacles in the control of medically and agriculturally important pests. Increased frequency of application, increased rate of application, decreased yield, environmental damage, and outbreak of human and animal diseases due to uncontrolled spread of vectors are the major consequences of insecticide resistance. The World Health Organization has defined insecticide resistance as “the biggest single obstacle in the struggle against vector-borne diseases,” and it is estimated that the annual cost of resistance may be US$1.4 billion in the United States (Pimentel et al. 1992, Liu and Scott 1998).
Substantial attention has been devoted to study the mechanisms underlying resistance in house flies. Knowing the mechanisms of resistance is important to understand the pathways of resistance development and to design novel strategies to prevent or minimize the spread and evolution of resistance (Liu and Pridgeon 2002).
The primary target sites of pyrethroids are the voltage-gated sodium channels in nerve membranes. A mutation of leucine at position 1014 to phenylalanine residue (L1014F) in Vssc1 on autosome 3 in house flies results in insensitivity of the target site to pyrethroids and is termed the kdr mutation (knock-down resistant). In house fly strains with higher levels of resistance to pyrethroids than the kdr house flies, an additional mutation of methionine at position 918 to threonine (M918T) presents in combination with the kdr mutation and is termed super-kdr. Another mutation has been identified in Vssc1, that is, the substitution of leucine with histidine at position 1014 (L1014H), termed the kdr-his mutation. The kdr-his mutation confers lower levels of resistance than kdr (Williamson et al. 1996, Liu and Pridgeon 2002, Soderlund and Knipple 2003, Huang et al. 2004, Rinkevich et al. 2006).
To date, the target site insensitivity caused by kdr and super-kdr mutations in the house fly have also been identified in several additional pest species; Blatella germanica (Dong 1997, Dong et al. 1998), Haematobia irritans (Jamroz et al. 1998), Anopheles gambiae (Martinez-Torres et al. 1998, Ranson et al. 2000), Plutella xylostella (Schuler et al. 1998), Leptinotarsa decemlineata (Lee et al. 1999), and Myzus persicae (Martinez-Torres et al. 1999). However, 40 individual olive flies (Bactrocera oleae) collected from the olive orchards in Balıkesir Province that have been regularly treated with the pyrethroid α-cypermethrin for more than ten years were studied to investigate the possible occurrence of resistance-associated mutations within the IIS4–IIS6 region of the B. oleae para-type sodium channel gene. Comparison of the deduced amino acid sequences of the IIS4–IIS6 region of the samples with the corresponding region of susceptible LS strain (obtained from the NCBI database, GenBank EU253453) showed that all had identical sequences without any polymorphism of the amino acids previously associated with resistance (unpublished data).
The microsomal cytochrome P450 monooxgenases are one of the important metabolic systems found in all organisms. Increase in monooxygenase activity leading to enhanced detoxification of insecticides results in the development of insecticide resistance in insects (Kasai and Scott 2000). CYP6D1 is a house fly P450 known to be involved in pyrethroid resistance (Scott et al. 1998). A characteristic 15 bp insert in the 5´- flanking region, which defines the CYP6D1v1 allele, is responsible for CYP6D1 overexpression in resistant flies (Scott et al. 1999, Gao and Scott 2006, Rinkevich et al. 2006). It has been implicated that this 15 bp insert disrupts a putative repressor mdGfi-1 binding site in the CYP6D1v1 promoter, causing increased expression of CYP6D1 (Gao and Scott 2006). CYP6D1 was mapped to autosome 1, and the factors responsible for increased transcription of CYP6D1 were mapped to autosomes 1 and 2 (Liu et al. 1995, Liu and Scott 1996).
The early detection and characterization of field resistance in insects are critical to the development of strategies for resistance management. In Turkey, there is no resistance management program for house flies. In spite of the common and intensive use of insecticides for about 40 years, pyrethroids have been used for only about two decades for the control of house flies (Akiner and Caglar 2006). The pyrethroid resistance status of Turkish house fly populations collected from a limited number of locations was examined by a bioassay method (Akiner and Caglar 2006). The aim of the present study was to determine the frequencies of pyrethroid resistance-associated mutations in Vssc1 and CYP6D1 in house fly populations collected from 16 different provinces in the Aegean and Mediterranean regions of Turkey. These regions are the most important regions with respect to the agricultural and industrial sectors and are highly exposed to different groups of insecticides and other organic and inorganic xenobiotics. This study is important because it is the first to investigate the major mechanisms of pyrethroid resistance at the molecular level in natural M. domestica populations from this part of the world.
MATERIALS AND METHODS
House fly samples
The house fly samples were collected from farms and garbage disposal sites at three different locations from each of 16 different provinces in the Aegean and Mediterranean regions of Turkey during the summer of 2006 (mid-season 2006 collections). The provinces sampled were Muğla, Aydın, İzmir, Manisa, Kütahya, Uşak, Afyon, and Denizli in the Aegean region and Isparta, Burdur, Antalya, Mersin, Adana, Osmaniye, Kahramanmaraş, and Hatay in the Mediterranean region (Figure 1). From each location, 300 to 400 adult flies were collected. Males were stored at –80° C, and females were allowed to lay eggs. The samples were reared, as described by Taskin and Kence (2004). The first laboratory generation (F1) was frozen and stored at –80° C until use. The samples from each location were reared and kept separately.
Sequencing of Vssc1
To determine if kdr or super-kdr was present in the field-populations of house fly samples, a portion (a 335 bp fragment, including around 125 bp intron region, of Vssc1, putative transmembrane segments S4–S6 of domain II) of the sodium channel α-subunit was sequenced. From each province, four individual F1 generation house flies (64 house flies in total) were used for genetic analysis. Genomic DNA was isolated, according to the method of Bender et al. (1983), and PCR was performed, as described by Rinkevich et al. (2006). In this procedure, kdr fragments were amplified and sequenced using the primer pairs kdrDIGlongF and kdrDIGlongR. The M918 region was amplified and sequenced using the primer pairs SuperkdrLongF and SuperkdrLongR (Table 1).
Table 1. Primers used to amplify kdr fragments.
The PCR products were visualized and isolated from the 1.5% agarose gel by using Qiagen Qiaquick PCR purification kit by following the manufacturer's instructions. The PCR products were directly sequenced using Applied Biosytems Model A3100 Automated DNA Sequencer. Homozygous and heterozygous individuals were identified by manual inspection of the sequence electropherograms. Base sequence alignments and analysis were conducted using MEGA version 3.1 software (Kumar et al. 2004).
PCR-RFLP assay was performed to screen for CYP6D1 genotype in the house fly populations, by using the protocol described by Rinkevich et al. (2006). A 732 bp portion of CYP6D1 including the 5´ flanking sequences was amplified. Of the 160 individual flies (ten house flies from each province) used in this assay, 64 were also used for Vssc1 sequencing. In order to amplify the control region of CYP6D1, S35 (5´-AGCTGACGAAATTGATCAATCAGT-3´) and AS2 (5´-CATTGGATCATTTTTCTCATC-3´) primers were used. For the resistant (RR) and sensitive (SS) genotypes, 732 and 711 bp amplification products were detected, respectively. Hpy188III was the restriction enzyme used in the protocol. The presence of a 15 bp insert in the gene-flanking region in the resistant alleles disrupted the recognition sequence for this enzyme. Hpy188III treatment resulted in formation of 432 bp and 279 bp bands for the SS genotype; an uncut 732 bp band for the RR genotype; and 732 bp, 432 bp, and 279 bp bands for the heterozygous (RS) genotype.
Screening of field populations of house flies for pyrethroid resistance-associated mutations in Vssc1 and CYP6D1
Methionine was detected at position 918 of Vssc1 in all 64 flies screened, indicating the absence of super-kdr mutation in the populations. For Vssc1, the most common individuals in the populations were the susceptible homozygotes comprising 53.1% and 48% of the Aegean and Mediterranean populations, respectively. The second most common individuals were the heterozygous sus/kdr-his1, comprising 40.6% of the Aegean population and 28.1% of the Mediterranean population. The other heterozygotes detected in the populations were sus/kdr. In contrast to the other Vssc1 genotypes, the frequency of this genotype was higher in the Mediterranean population (15.6%) than in the Aegean population (3.1%), with an overall frequency of 9.3%. Homozygous kdr-his1 individuals were detected in both the Aegean and Mediterranean populations with the same frequency of 3.1%. While homozygous kdr individuals were not found in the Aegean population, the frequency of this genotype in the Mediterranean population was 6.25% (Figures 2A, B). Evaluation of Vssc1 allele frequencies revealed considerably higher frequencies for susceptible alleles (0.75 in Aegean and 0.69 in Mediterranean populations) than the frequencies of kdr and kdr-his alleles in both regions. Interestingly, the frequency of kdr-his alleles were higher than the frequency of kdr alleles in the house flies sampled. Although the frequencies of kdr-his alleles were close to each other in the Aegean (0.23) and Mediterranean (0.17) populations, the frequencies of kdr alleles were remarkably different in these two regions; the frequency in the Aegean population (0.02) was approximately seven-fold lower than that in the Mediterranean population (0.14) (Table 2).
Table 2. Frequency of pyrethroid resistance alleles in house flies from the Aegean and Mediterranean regions of Turkey.
Of 160 individuals screened, 70% of the Aegean population was heterozygous for CYP6D1v1, while the remaining 30% was CYP6D1 homozygous-susceptible. In the Mediterranean population, the frequency of heterozygous individuals was higher (85.6%) than in the Aegean population, and the ratio of susceptible CYP6D1 individuals was 13.8% in this region. Only one individual fly was homozygous for CYP6D1v1, belonging to the Mediterranean samples. Although there were no remarkable differences between them, the frequencies of CYP6D1 alleles were in favor of the susceptibles (Table 2).
Determination of the Vssc1+CYP6D1 genotype classes in the 64 individual flies that were common in the analysis of Vssc1 and CYP6D1 resulted in six different genotype classes in the Aegean population. Interestingly, it was determined that Vssc1 and CYP6D1 homozygous susceptible individuals formed 31% of the screened samples in this region. The second most common genotype class was composed of heterozygous sus/kdr-his1 and heterozygous CYP6D1v1 with a frequency of 25%. Vssc1 homozygous-susceptible with CYP6D1v1 heterozygous class was the third most common genotype class found in this region, constituting 22% of the flies studied (Figure 2A).
In the Mediterranean population, eight different genotype classes were detected. The susceptible Vssc1 with heterozygous CYP6D1v1 genotype, which was the third most common genotype in the Aegean population, was found to be the most common genotype in the Mediterranean population, with a frequency of 35%. The second most common genotype, sus/kdr-his1 and heterozygous CYP6D1v1, which was also the second most common genotype in the Aegean population, occupied the second order in the Mediterranean population, with a frequency of 22%. Vssc1 and CYP6D1 homozygous-susceptible individuals constituted the third most common genotype in this region with a frequency of 13%, while it was the most common genotype in the Aegean population. The two genotype classes, that is, sus/kdr with homozygous CYP6D1 and homozygous kdr with heterozygous CYP6D1v1, were found to be specific to the Mediterranean population with the same frequency of 6% (Figure 2B).
In the combined analysis of the individuals from both regions, the three most common genotype classes, susceptible Vssc1 with heterozygous CYP6D1v1, sus/kdr-his1 with heterozygous CYP6D1v1, and susceptible homozygous Vssc1 with CYP6D1, had frequencies of 29%, 23%, and 22%, respectively (Figure 2C).
Pest management has often relied heavily on insecticides, leading to resistance as an ever-growing, complicated, and global problem. The evolutionary routes arising between the pests (evolving resistance) and humans (developing novel strategies to control) are an interesting and attractive research area with many important implications for humans and the environment. Knowing the number of loci involved and the interactions between them is important in understanding resistance and for managing resistance in pest populations. Little is known about the frequencies of resistance alleles in the field populations of pests and the role of environmental differences in the evolution of these populations (Rinkevich et al. 2007).
Standardized bioassay methods and molecular assays can be used to evaluate the status and development of insecticide resistance in pest populations. Standardized bioassays are valuable for determining the phenotypes of populations to insecticides, but these assays are time-consuming and require a large number of samples compared to molecular assays. On the other hand, molecular assays are capable of successfully identifying heterozygotes. This is important in following populations that exhibit susceptible phenotypes while containing a large proportion of heterozygotes. However, molecular assays are effective in determining the frequencies of resistance alleles and changes in the allelic frequencies in response to management strategies only in cases in which resistance-associated mutations are already identified (Huang et al. 2004).
It has been suggested that the first pyrethroid resistance mechanism in house flies was kdr, likely due to the use of DDT for fly control starting in the 1940s (Shono 1985). Intensive use of pyrethroids since the 1980s has increased the frequency of kdr mutations (Kasai and Scott 2000, Rinkevich et al. 2006). As the M918T mutation has always been documented in flies that also have the L1014F mutation, it is suggested that super-kdr genotype arose from a kdr individual through a sequential accumulation of mutations (Rinkevich et al. 2006). The kdr-his mutation was identified for the first time in house flies from Alabama (Liu and Pridgeon 2002). Identification of the kdr allele in house fly populations from numerous locations worldwide made it unclear whether this was due to multiple evolutionary origins of the kdr mutation, or just a reflection of the high mobility rate of this organism. A recent phylogenetic analysis of Vssc1 alleles indicated multiple evolutionary origins for the kdr-his alleles but not for kdr alleles (Rinkevich et al. 2006).
For the first time in field populations, 14 populations from Denmark were screened by Huang et al. (2004) for kdr mutations, and the frequencies of this mutation varied from 0.46 to 0.99 in these populations. Subsequently, the frequencies of pyrethroid resistance alleles of Vssc1 were documented in flies collected from four dairies in Maine (ME), New York (NY), North Carolina (NC), and Florida (FL) in 2002 (Rinkevich et al. 2006). They reported that kdr and kdr-his1 alleles were present at high frequencies in each population sampled, but the relative frequencies of the two alleles differed between the dairies in the four states. More than 94% of all alleles detected at each location were composed of kdr and kdr-his1, while susceptible Vssc1 alleles comprised less than 6% of all alleles. In another study, Rinkevich et al. (2007) showed the dynamics of resistance alleles over different climatic conditions and time. They used house flies collected during the 2003–2004 season from dairies in New York and Florida before the first application of permethrin, during the middle of the field season, one to two weeks after the final application of permethrin, and again the following spring before insecticide use. They evaluated whether there was a difference in allele frequencies over time between the dissimilar climates of these two states. They found that there was a change in Vssc1 allele frequencies through the field season in NY, being consistent with selection by permethrin. They observed the presence of super-kdr allele, previously known to be restricted to Europe and Asia, at a low frequency in the collections from NY. This was the first report of super-kdr from any field population in the U.S.A. On the other hand, in the same study, the frequencies of Vssc1 alleles in FL did not follow the pattern observed in NY; susceptible alleles were found at high frequencies (≥ 0.73) during the study, and the frequencies of the resistance alleles were constant throughout the field season, showing no change in response to permethrin use at the dairies.
In our study, in the field populations of house flies collected in the mid-season of 2006, the frequencies of Vssc1-susceptible alleles were 0.75 and 0.69 in the Aegean and Mediterranean regions, respectively, with an overall frequency of 0.72. These are higher than the values obtained by Huang et al. (2004) for 14 populations from Denmark and remarkably higher than the values obtained by Rinkevich et al. (2006) for the populations from four states in the eastern U.S.A. When compared with the findings of Rinkevich et al. (2007), these values are still higher than the value (0.42) obtained for the samples collected from NY in mid-season 2003, but closer to the value (0.81) obtained for the samples collected from FL in the same season. It has been reported that target site insensitivity to pyrethroids has a fitness cost in horn flies; when the selection ended in autumn, an increase was observed in the frequency of Vssc1-susceptible alleles, resulting in a decrease in resistance in early-season populations (Scott et al. 1997, Guerrero et al. 2002). In addition, it has been shown by Rinkevich et al. (2007) that the frequency of resistance alleles at the NY dairy increased during the season and decreased over winter, suggesting an overwintering fitness cost associated with these alleles in the house fly. In the same study, no frequency changes were detected in the resistance alleles either during the spraying season or winter in FL, suggesting that no overwintering fitness cost was associated with resistance alleles in this climate, and that there was a substantial immigration of susceptible alleles to the FL dairy. Many other drivers should be considered that may also relate (or not) to environmental conditions. For example, the length of the activity season may drive the types of the chemical products used or the concentrations applied and will certainly affect the period of time during which insecticides are applied. House fly activity seasons differ between NY and FL, or Denmark and Turkey. In NY and Denmark, it is very short, while in FL, flies are active throughout the year. Furthermore, it was shown by Foster et al. (2003) that house fly genotypes possessing kdr mutation exhibited behavioral differences compared to the susceptible flies. In that study, resistant individuals had no positional preference along a temperature gradient, whereas susceptible genotypes showed a strong preference for warmer temperatures. Egg production and development were enhanced by warmer temperatures, and bacteria- and fungi-infected flies could cure themselves by behavioral fever after selecting a hot spot (Kalsbeek et al. 2001). The breeding success of susceptible individuals will increase with the preference for warmer temperatures, thereby lowering the frequencies of resistance alleles in the populations. Therefore, temperature preference is an important fitness parameter for house flies (Huang et al. 2004). These could be the possible explanations for the higher frequencies of Vssc1-susceptible alleles obtained in our study. The higher frequencies of kdr-his1 alleles compared to that of kdr alleles detected in both Aegean and Mediterranean populations are in accord with the findings of Rinkevich et al. (2006) for the NY and NC populations and the findings of Rinkevich et al. (2007) for the NY and FL collections belonging to all sampling times during the field season. Because a lower level of resistance is conferred by the kdr-his1 allele compared to the kdr allele, these findings were unexpected and we attempted to explain them by adaptation of the kdr-his1 mutation to the local environmental or insecticide pressures found in these regions. The super-kdr allele that was previously reported in Europe and Asia was not detected in the samples belonging to the transition region between these two continents used in this study.
Correlated with the rapid increase in pyrethroid use, CYP6D1-mediated monooxygenase resistance has begun to appear since 1985 as a second pyrethroid resistance mechanism in the house fly (Kasai and Scott 2000). For the first time in field populations, the frequency of CYP6D1v1 was documented across the eastern U.S. (ME, NY, NC, and FL) together with resistance alleles of Vssc1 by Rinkevich et al. (2006). In their study, a high frequency of CYP6D1v1 was observed, ranging from 0.63 (in FL) to 0.91 (in NC). In the study showing the dynamics of CYP6D1v1 over two different climates (NY and FL) and four different sampling times, Rinkevich et al. (2007) reported that, like kdr-type, the frequency of CYP6D1v1 in NY followed a pattern consistent with response to selection by permethrin, and in mid-season 2003 the frequency of CYP6D1v1 was 0.91. However, a limited change in the CYP6D1v1 allele frequencies across and between seasons was detected in FL, again in a manner similar to kdr-type in this state, and the 2003 mid-season frequency of CYP6D1v1 was 0.52. Compared to the findings of Rinkevich et al. (2006) and Rinkevich et al. (2007), the values obtained in our study from two regions (0.35 and 0.44 in Aegean and Mediterranean regions, respectively) related to the frequency of CYP6D1v1 are considerably lower than the values obtained in the three states (ME, NY, NC) for 2002 collections and the value obtained in NY for the mid-season 2003 collections, whereas they were relatively close to the values obtained from FL in both studies. The frequencies obtained in this study were due to excess heterozygotes because heterozygotes are reported to be resistant (Liu and Scott 1997), allowing susceptible alleles to be maintained in the population. Heterozygotes might have been driven by local environmental and selection pressures in the populations because of fitness advantages.
Susceptible Vssc1 with heterozygous CYP6D1v1 was the most common genotype class represented by 29% of the flies screened from both regions. This genotype was undetected in all four states by Rinkevich et al. (2006) and in NY in mid-season 2003 by Rinkevich et al. (2007). On the other hand, it was detected in pre-2003 and pre-2004 collections of NY with a relative frequency of approximately 10%, in mid-season 2003 collection in FL with a frequency of approximately 40%, and in FL collections sampled during the other times of the field season with a frequency of approximately 25% (Rinkevich et al. 2007). The second most common genotype class, sus/kdr-his1 and heterozygous CYP6D1v1, with 23% frequency in total flies screened in present study, was detected in all four states with less than 10% frequency, having the highest frequency in FL (Rinkevich et al. 2006), in all of the collections of NY, except the late-2003 season collection, with less than 15% frequency, and in all the FL collections, ranging between 20% (in late-2003 and pre-2004) and 27% (in pre-2003) frequencies (Rinkevich et al. 2007). Susceptible homozygous Vssc1 with CYP6D1, which was the third common genotype class with a frequency of 22% of the flies in our study, was undetected in all the four states by Rinkevich et al. (2006), detected with a very low frequency (<0.05) in NY collections by Rinkevich et al. (2007), but was detected in all the collections of FL, with the highest frequency in mid-season 2003 (approximately 15%) (Rinkevich et al. 2007). Interestingly, the total frequencies of these three most common genotype classes (approximately 75%) obtained in our study and in FL in mid-season 2003 were close to each other. This similarity observed between our findings and the results obtained from FL in terms of the frequencies of pyrethroid resistance alleles of Vssc1 and CYP6D1 in the house fly led us to compare the average temperatures recorded in FL and the Aegean and Mediterranean regions in Turkey for the related fly collection seasons. As indicated in Figure 3, a similar temperature pattern was obtained with FL and the corresponding regions in Turkey in this study. This might imply a lack of over-wintering fitness cost associated with resistance alleles in such climates or similar patterns of pesticide use in FL and in both regions of Turkey.
Although their orders and frequencies changed between the Aegean and Mediterranean regions, six genotype classes were found to be common in these regions. One possible explanation for this occurrence could be the gene flow between these two regions. Studies of house fly movement indicate that this species is highly mobile (Seifert and Scott 2002) and can move long distances. In addition, humans can play an important role in the transport of this species through cars, trains, planes, ships, and other means of transport. Intensive anthropogenic activities such as agriculture, trade, and tourism occur between the Aegean and Mediterranean regions, via which these flies can easily be transported. Also, flies may be dispersed between these regions by seasonal winds. The other possible explanation might be similar insecticide selection pressures encountered by the populations of the two regions in a wide geographical area. The detection of the two genotype classes specific to the Mediterranean region may be due to the adaptation of these genotype combinations to local environmental conditions, insecticide application rates, and/or exposure techniques, or perhaps due to chance selection.
In the bioassay performed by Akiner and Caglar (2006), the resistance factor (RF = LD50 of the test strain/LD50 of the reference strain) of Turkish house fly populations (collected from Antalya, İzmir, Adana, Ankara, İstanbul, and Şanlıurfa in 2002) for the pyrethroid group of insecticides ranged from 23.27 (permethrin-İstanbul fall strain) to 633.09 (cypermethrin-İzmir spring strain), which is very high. Although the samples originated from different collection seasons, the lack of correlation between the bioassay data of Akiner and Caglar (2006) and the genotype data obtained in our study may suggest the presence of detoxification by an additional P450 isoform, a glutathione-S-tranferase (GST) or an esterase. Previously, high levels of GST and esterase activities were reported in the same populations by Taskin et al. (2009).
Despite the higher frequencies of susceptible alleles, the detection of the resistance alleles in the populations indicates that these alleles can be swept through the populations if pressured by overuse of pyrethroid insecticides. For this reason, there is an urgent need to establish effective insecticide management programs to monitor all aspects of the resistance problem. Further, it should not be forgotten that, as well as the importance of keeping as many insecticides available as possible for future house fly management programs, a modern house fly management program should be a combination of sanitation, waste management, and insecticides against larvae and adult flies, and can also include traps and biological controls favoring natural enemies.