Pyrethroid Insecticide Resistance kdr Gene in the House Fly , Musca domestica ( Diptera : Muscidae ) , in the United Arab Emirates

The house fly, Musca domestica L., is a very important insect because of its role as a vector of several human, livestock, and poultry diseases. Different groups of insecticides, including the pyrethroids, are used to control house flies. The objective of the current study was to conduct a survey of the kdr insecticide resistance gene in the house fly population in the United Arab Emirates (UAE). Flies were collected from five locations (Abu Dhabi, Dubai, Fujairah, Ajman, and Ras AlKhaymah). Genomic DNA was extracted and polymerase chain reaction (PCR) amplification of specific allele (PASA) was performed. The maximum percentage (70%) of homozygous genotype (kdr/kdr) occurred at Ajman, followed by Dubai (59.5%), and the minimum percentage occurred at Ras Al-Khaymah (14.3%). Resistant insects of the genotype kdr/kdr were detected in all of the tested house fly populations. Ajman and Fujairah house fly populations were in Hardy-Weinberg equilibrium. The resistance kdr allele was found at a high frequency (0.54 0.8) at all locations except at Ras Al-Khaymah (0.21). Together, this study demonstrated that the pyrethroid insecticide resistance kdr allele was found in UAE house fly populations, strongly suggesting that a countrywide pyrethroid insecticide resistance management program needs to be implemented.


Introduction
The house fly, Musca domestica L. (Diptera: Muscidae), is a very important insect that can have negative impacts on human, livestock, and poultry because of its role as a disease vector.It has been reported to be a poten-tial vector of metazoan parasites found in pig pens [1].House flies also can be mechanical vectors of Vibrio cholera (biotype El Tor), thus increasing its dissemination [2].In addition, a total of 497 fungal isolates of 15 genera were identified from the external surfaces of both female and male house flies, which showed that the house fly can be a vector for fungal spores [3].House flies are also effective vectors of Escherichia coli O157:H7 among cattle, as well as an effective vector from cattle to humans [4] [5].
Pyrethroid insecticides are used in the chemical control of house flies.The voltage-gated sodium channel (Vssc) is the main target of these insecticides, but target site insensitivity conferred by mutations in the Vssc has been a major mechanism of resistance to pyrethroids [6] [7].At amino acid residue 1014, a mutation of leucine to phenylalanine (L1014F), which is known as the kdr mutation in the para-type sodium channel gene, has been consistently associated with knockdown resistance in house flies [8] [9].Resistant flies have alleles such as CYP6D1 and Vssc1 that conferred resistance to permethrin and other pyrethroids [10].
In the United Arab Emirates (UAE), house flies can be disease vectors and can pose a risk to human and animal health.Although they are routinely controlled by chemical insecticides, there have been no reports on the prevalence of insecticide resistance, especially at the molecular level (detection of resistance alleles).Therefore, the objective of the current study was to conduct a survey on the presence of the kdr pyrethroid insecticide resistance gene in house fly populations at five locations in the UAE.

House Flies
A total of 122 adult house flies were collected from five field populations (Abu Dhabi, Dubai, Ras Al-Khaymah, Ajman, and Fujairah) in the UAE in 2013.

DNA Extraction
Genomic DNA was extracted from individual house flies and stored at −80˚C.Extractions were performed using an automated Maxwell 16 DNA extraction machine equipped with a tissue DNA purification kit (Promega, USA) using a procedure according to the manufacturer's protocol.

Presence of the kdr Resistance Gene in the House Fly Population Using PCR Amplification of Specific Alleles (PASA)
The PASA protocol described by Huang et al. [11] was followed.Briefly, it is an assay based on Williamson et al. [8] which genotypes each house fly for the presence of the L1014F replacement in the sodium channel's S6 transmembrane segment of domain II.Two outer allele-specific primers, kdr1, 5'-AAGGATCGCTTCAAGG-3' and kdr4, 5'-TTCACCCAGTTCTTAAAACGAG-3', were used.Also, two inner allele-specific primers, kdr2, 5'-TCGTGATCGGCAATT-3' and kdr3, 5'-GTCAACTTACCACAAG-3', were used.PASA was performed in thin walled microcentrifuge tubes using 25 μL reaction-volumes.The following are the optimized PCR conditions: 2 μL of genomic DNA (90 ng/μL) extracted from a single house fly, 10 pmol of each primer, 12.5 μL Taq PCR Master Mix (Qiagen, Germany), and 8.5 μL nuclease free water.Amplification was initiated by 95˚C for 2 minutes, followed by 40 cycles, 94˚C for 45 seconds, 54˚C for 30 seconds, 72˚C for 90 seconds, and a final extension step at 72˚C for 10 minutes.A control fragment of 480 bp was the result of amplification by using kdr1 and kdr4 primers.Amplification of the susceptible allele by kdr1 and kdr3 primers produced a 200 bp fragment.Instead of the 200 bp fragment, a 280 bp fragment was amplified by kdr2 and kdr4 primers from the kdr-type allele.Every PCR included a negative control (no-template DNA) to make sure there was no contamination.PCR amplifications were carried out in a Swift Max Pro thermocycler (ESCO, Singapore).The PCR amplified fragments were resolved by electrophoresis using 1.5% agarose gels that were stained with ethidium bromide and photographed under UV light using a UVDI gel documentation system (Major Science, Taiwan).Frequencies of the resistance kdr allele were tested for Hardy-Weinberg equilibrium for each house fly population at each location by using a chi-square X 2 test at P = 0.05 for one degree of freedom where X 2 = 3.84 [12].

Results
The 200 bp susceptible allelic fragment was amplified by kdr1 and kdr3 primers and the 280 bp kdr allelic fragment was amplified by kdr2 and kdr4 primers (Figure 1).The percentages of the three genotypes (kdr/kdr, kdr/sus, and sus/sus) varied among the five tested fly populations (Table 1).The maximum number of flies (70%) with the kdr/kdr genotype occurred at Ajman, followed by Dubai (59.5%), while the minimum number occurred at Ras Al-Khaymah (14.3%) (Figure 2).Moreover, insects with the kdr/kdr genotype were detected in all tested populations (Figure 3).Two of the house fly populations, namely Ajman and Fujairah, were in Hardy-    Weinberg equilibrium based on the chi-square X 2 test (1.41 and 1.61, respectively) (Table 1).The resistance allele kdr was found at a high frequency (0.54 -0.8) at all locations, except at Ras Al-Khaymah (0.21) (Table 1).

Discussion
Genotyping each house fly for the presence of the resistance kdr allele using PASA revealed that this allele was present in the five tested populations.This finding was confirmed by the amplification of a 280-bp kdr/kdr homozygous allelic fragment.Other flies were heterozygous (kdr/sus) as indicated by the amplification of two allelic fragments (280 bp for kdr and 200 bp for sus) or homozygous (sus/sus).These findings were in agreement with the results of Huang et al. [11].Because flies with the kdr/kdr genotype are resistant to pyrethroid insecticides, the results suggested that resistance levels could increase with time in fly populations of the UAE, which could lead to control failure.The screened house fly populations in the current study showed variabilty in the precentages of the three genotypes (kdr/kdr, kdr/sus, and sus/sus), indicating different levels of pyrethroid resistance.Similarly, Huang et al. [11] reported variabiliy in genotypes among house fly populations.Rinkevich et al. [10] reported that combinations of pyrethroid insecticide resistance alleles of Vssc1 and CYP6D1 were highly variable between the states from which the house flies were collected.Resistant insects of the genotype kdr/kdr were detected at Abu Dhabi, Dubai, Fujairah, Ajman, and Ras Al-Khaymah.However, only two of the examined house fly populations, namely Ajman and Fujairah, were in Hardy-Weinberg equilibrium based on the chi-square X 2 test in which the observed numbers of the kdr homo-and heterozygotes were compared with the expected numbers calculated from the kdr frequency.Deviation from Hardy-Weinberg equilibrium could indicate different levels of selection caused by the use of pyrethroid insecticides on house fly populations.The results of the current study are consistent with other studies, which indicated that insect populations are not always in Hardy-Weinberg equilibrium [11].Toloza et al. [13], in a study of the pyrethroid resistance allelic frequency in head lice (Pediculus humanus capitis), reported that tests for the Hardy-Weinberg equilibrium for each location showed that genotype frequencies differed significantly from expectations in four of the six studied sites.
The resistance kdr allele was found at a high frequency (0.54 -0.8) at all locations except at Ras Al-Khaymah (0.21).Similarly, Rinkevich et al. [10] reported that the resistance allele CYP6D1v1 was found at a high frequency (0.63 -0.91) in house fly populations at all sites.In the present study, the maximum frequency (0.8) of the kdr allele was found at Ajman.Thus, most of the fly population (70%) at that location was the resistant homozygous kdr/kdr genotype.Nonetheless, it should be mentioned that the tested number of flies in Ajman was small (n = 10) and may not reflect a precise representation of the kdr allelic frequency in the fly population.However, the minimum number (14.3%) of flies with kdr/kdr genotype was found at Ras Al-Khaymah.This finding indicated that the Ras Al-Khaymah house fly popualation was still dominated by susceptible flies and consequently could be managed by using pyrethroid insecticides.
Genomic DNA was extracted relatively from small numbers of individual house flies in the current study because of limited time and funding.Testing of larger numbers of flies is recommended in future studies, because larger fly numbers should provide more accurate estimations of the kdr allelic frequency and pyrethroid resistance levels.However, the current study provided sufficient evidence that the kdr allele was present in the tested house fly populations in the UAE.Furthermore, the current study is the first report on the kdr pyrethroid resistance allele in the UAE.Thus, it serves as a foundation for future studies on insecticide resistance.Once an insect population shows resistance to an insecticide, control programs using this insecticide are no longer effective.Accordingly, the pyrethroid insecticide list used for the control of house flies in the UAE should be reevaluated in order to reduce the frequency of the kdr resistance allele in the fly population.In addition, a country-wide operational implementation of a global plan for insecticide resistance management is also warranted [14].

Conclusion
This study demonstrates that the pyrethroid insecticide resistance kdr allele is found in UAE house fly populations.Future studies should include pyerethroid insecticide bioassays on house flies before performing screening for the kdr allele.This will examine the relationship between the kdr mutation and the toxicity of the pyrethroid insecticides and determine whether a countrywide pyrethroid insecticide resistance management program needs to be implemented.

Figure 1 .
Figure 1.PCR products from individual house flies after separation on a 1.5% agarose gel.The 200 bp susceptible allelic fragment was amplified by kdr1 and kdr3 primers.The 280 bp kdr allelic fragment was amplified by kdr2 and kdr4 primers.A control fragment (480 bp) was amplified by kdr1 and kdr4 primers.M = Marker 100-bp (Promega, USA); NC = Negative control.

Figure 2 .
Figure 2. Genotype percentages in house fly populations at different UAE locations.

Figure 3 .
Figure 3. UAE map showing the location of house fly populations containing the resistance kdr/kdr genotype.

Table 1 .
House fly genotypes and pyrethroid knockdown resistance kdr allelic frequency at different UAE locations.