Frequencies of pyrethroid resistance-associated mutations of Vssc1 and CYP6D1 in field populations of Musca domestica L. in Turkey

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). There are three major mechanisms underlying resistance known to have evolved in house flies in response to intense pyrethroid use: (1) mutations in the voltage-sensitive sodium channel gene (Vssc1), (2) cytochrome P450 monooxygenase-mediated detoxifications, and (3) decreased cuticular penetration (Scott and Georghiou 1986, Williamson et al. 1993, Liu and Scott 1995, 1996, 1997, Tomita et al. 1995, Shono 1985, Shono et al. 2002, Liu and Pridgeon 2002, Rinkevich et al. 2007). 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. 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. Map showing the collection sites of M. domestica populations in Turkey. 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). 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 Homozygous individuals were detected in both the Aegean and Mediterranean populations with the frequency of While kdr individuals were found in the Aegean the frequency of this genotype in the Mediterranean population was of Vssc1 allele frequencies 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. the frequency of kdr-his alleles were higher than the frequency of kdr alleles in the house flies 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 the frequency in the Aegean population was lower than that in the Mediterranean population (Table of Vssc1 and CYP6D1 alleles in house flies collected from the Aegean region and Mediterranean region and for both regions. Vssc1 alleles are M918 and For CYP6D1 the 64 flies used for both Of 160 individuals screened, of the Aegean population was heterozygous for the was CYP6D1 In the Mediterranean the frequency of heterozygous individuals was higher than in the Aegean and the of susceptible CYP6D1 individuals was in this one individual fly was for to the Mediterranean Although there were no between the frequencies of CYP6D1 alleles were in of the (Table 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. it was that Vssc1 and CYP6D1 susceptible individuals of the samples in this The second most common genotype class was of heterozygous sus/kdr-his1 and heterozygous CYP6D1v1 with a frequency of Vssc1 with CYP6D1v1 heterozygous class was the most common genotype class found in this region, of the flies studied (Figure In the Mediterranean eight different genotype classes were The susceptible Vssc1 with heterozygous CYP6D1v1 which was the most common genotype in the Aegean was found to be the most common genotype in the Mediterranean with a frequency of The second most common sus/kdr-his1 and heterozygous which was also the second most common genotype in the Aegean the second order in the Mediterranean with a frequency of Vssc1 and CYP6D1 individuals the most common genotype in this region with a frequency of it was the most common genotype in the Aegean population. The two genotype that is, with CYP6D1 and kdr with heterozygous were found to be to the Mediterranean population with the frequency of (Figure In the analysis of the individuals from both regions, the three most common genotype susceptible Vssc1 with heterozygous sus/kdr-his1 with heterozygous and susceptible Vssc1 with CYP6D1, had frequencies of and (Figure management has on insecticides, leading to resistance as an and The between the and novel strategies to are an and with important for and the Knowing the number of involved and the between is important in resistance and for resistance in pest populations. is known about the frequencies of resistance alleles in the field populations of and the of environmental in the evolution of these populations et al. 2007). bioassay and molecular be used to the status and development of insecticide resistance in pest populations. are for the of populations to insecticides, these are and a number of samples to molecular the other molecular are of This is important in following populations that susceptible a of However, molecular are effective in the frequencies of resistance alleles and in the frequencies in response to management strategies only in in which resistance-associated mutations are identified et al. 2004). It has been that the first pyrethroid resistance in house flies was due to the use of for fly control in the use of pyrethroids the has increased the frequency of kdr mutations (Kasai and Scott Rinkevich et al. 2006). the mutation has been in flies that also have the it is that super-kdr genotype from a kdr individual a of mutations et al. 2006). The kdr-his mutation was identified for the first in house flies from (Liu and Pridgeon 2002). of the kdr allele in house fly populations from locations worldwide it this was due to of the kdr or a of the rate of this A analysis of Vssc1 alleles for the kdr-his alleles for kdr alleles et al. 2006). For the first in field populations, populations from were by Huang et al. for kdr and the frequencies of this mutation from to in these populations. the frequencies of pyrethroid resistance alleles of Vssc1 were in flies collected from four in and Florida in et al. 2006). They that kdr and alleles were present at frequencies in each population the frequencies of the two alleles differed between the in the four than of all alleles detected at each location were of kdr and susceptible Vssc1 alleles less than of all In study, Rinkevich et al. showed the of resistance alleles different and They used house flies collected during the from in and Florida the first of during the of the field one to two the of and the following insecticide use. They there was a in allele frequencies between the of these two They found that there was a in Vssc1 allele frequencies the field in with by They the presence of super-kdr allele, previously known to be to and Asia, at a frequency in the from This was the first of super-kdr from any field population in the the other in the study, the frequencies of Vssc1 alleles in the in susceptible alleles were found at frequencies during the study, and the frequencies of the resistance alleles were the field showing no in response to use at the In our study, in the field populations of house flies collected in the of 2006, the frequencies of Vssc1-susceptible alleles were and 0.69 in the Aegean and Mediterranean regions, with an overall frequency of These are higher than the values obtained by Huang et al. for populations from and remarkably higher than the values obtained by Rinkevich et al. for the populations from four in the with the of Rinkevich et al. these values are higher than the value obtained for the samples collected from in 2003, to the value obtained for the samples collected from in the It has been that target site insensitivity to pyrethroids has a fitness cost in the in an was in the frequency of Vssc1-susceptible in a in resistance in populations (Scott et al. 1997, et al. 2002). In it has been by Rinkevich et al. that the frequency of resistance alleles at the increased during the and decreased an overwintering fitness cost associated with these alleles in the house In the study, no frequency were detected in the resistance alleles during the or in that no overwintering fitness cost was associated with resistance alleles in this and that there was a of susceptible alleles to the other be that may also to environmental For the of the activity may the of the products used or the and the of during which insecticides are House fly activity between and or and Turkey. In and it is very in flies are the it was by et al. that house fly kdr mutation to the susceptible flies. In that study, resistant individuals had no a temperature susceptible showed a for and development were enhanced by and and flies by a et al. The of susceptible individuals with the for the frequencies of resistance alleles in the populations. temperature is an important fitness for house flies et al. 2004). These be the possible for the higher frequencies of Vssc1-susceptible alleles obtained in our The higher frequencies of alleles to that of kdr alleles detected in both Aegean and Mediterranean populations are in with the of Rinkevich et al. for the and populations and the of Rinkevich et al. for the and to all during the field a lower level of resistance is by the allele to the kdr allele, these were and to by of the mutation to the environmental or insecticide found in these regions. The super-kdr allele that was previously in and was detected in the samples to the region between these two used in this with the in pyrethroid monooxygenase resistance has to as a second pyrethroid resistance in the house fly (Kasai and Scott 2000). For the first in field populations, the frequency of CYP6D1v1 was the and with resistance alleles of Vssc1 by Rinkevich et al. (2006). In study, a frequency of CYP6D1v1 was from to In the study showing the of CYP6D1v1 two different and and four different Rinkevich et al. the frequency of CYP6D1v1 in a with response to by and in the frequency of CYP6D1v1 was However, a limited in the CYP6D1v1 allele frequencies and between was detected in in a to in this and the frequency of CYP6D1v1 was to the of Rinkevich et al. and Rinkevich et al. the values obtained in our study from two regions and in Aegean and Mediterranean regions, related to the frequency of CYP6D1v1 are lower than the values obtained in the three for and the value obtained in for the were close to the values obtained from in both The frequencies obtained in this study were due to heterozygotes because heterozygotes are to be resistant (Liu and Scott susceptible alleles to be in the population. have been by environmental and in the populations because of fitness Vssc1 with heterozygous CYP6D1v1 was the most common genotype class by of the flies from both regions. This genotype was in all four by Rinkevich et al. and in in by Rinkevich et al. the other it was detected in and of with a frequency of in collection in with a frequency of and in sampled during the other of the field with a frequency of 25% et al. 2007). The second most common genotype sus/kdr-his1 and heterozygous with frequency in total flies in present study, was detected in all four with less than having the frequency in et al. in all of the of the with less than and in all the between and and frequencies et al. 2007). Vssc1 with CYP6D1, which was the common genotype class with a frequency of of the flies in our study, was in all the four by Rinkevich et al. detected with a very frequency in by Rinkevich et al. was detected in all the of with the frequency in (approximately et al. 2007). the total frequencies of these three most common genotype classes (approximately 75%) obtained in our study and in in were close to each This similarity between our and the results obtained from in of the frequencies of pyrethroid resistance alleles of Vssc1 and CYP6D1 in the house fly to the in and the Aegean and Mediterranean regions in Turkey for the related fly collection in a temperature was obtained with and the corresponding regions in Turkey in this This a lack of fitness cost associated with resistance alleles in or patterns of use in and in both regions of Turkey. of the Aegean and Mediterranean regions of Turkey from to obtained from of and from to were obtained from Rinkevich et al. Although and frequencies between the Aegean and Mediterranean regions, six genotype classes were found to be common in these regions. possible for this occurrence be the gene between these two regions. of house fly that this is highly and Scott and In an important in the of this and other of as and between the Aegean and Mediterranean regions, which these flies be flies may be between these regions by The other possible be insecticide by the populations of the two regions in a The detection of the two genotype classes to the Mediterranean region may be due to the of these genotype to environmental insecticide or due to In the bioassay performed by and Caglar the resistance of the of the of Turkish house fly populations from Antalya, İzmir, Adana, and in for the pyrethroid of insecticides from to which is very Although the samples from different collection the lack of between the bioassay of and Caglar and the genotype obtained in our study may the presence of detoxification by an additional P450 a or an levels of and were in the populations by Taskin et al. the higher frequencies of susceptible the detection of the resistance alleles in the populations that these alleles be the populations if by of pyrethroid For this there is an to effective insecticide management to all of the resistance it be as well as the of as insecticides as possible for house fly management a house fly management program be a combination of and insecticides against and adult flies, and also and natural