Ace polymorphisms in field-collected populations
A total of 583 nucleotides, including the 85 bp intron region of the house fly Ace gene, was amplified by PCRs of genomic DNA from individual houseflies. Alignment of the sequences from all the 64 samples revealed many single nucleotide polymorphisms (SNPs); that is, some sites had a mixture of two bases because the individuals were heterozygous at those sites. Seven samples were polymorphic at only one site and six different alleles were identified from their sequences. Alleles 3 and 5 had L260-A342-Y407 and V260-A342-Y407 combinations, respectively, and corresponded to the alleles of v15 (GenBank Accession No. FJ174267) and v10 (GenBank Accession No. FJ174262), respectively (Kozaki et al. 2009). The other four alleles (allele 1, V260-V342-Y407; allele 2, L260-A342-Y407; allele 4, L260-A342-F407; allele 6, V260-G342-Y407) were specific to the Turkish house fly populations.
By examining the MdAce polymorphism, 13 different combinations at positions 260, 342, and 407 were detected in the studied populations. The L/V260-A/G342-F/Y407 combination had the highest frequency (21%). The second common phenotype was V260-A/G342-Y407 (19%). L/V260-A/V342-Y407 (12%), L/V260-A/G342-Y407 (12%), V260-A/V342-Y407 (9%), and L260-A342-Y407 (8%) were the other common phenotypes in the populations. The seven remaining combinations had a total frequency of 19% (Figure 2A).
Figure 2. Pie charts illustrating the frequencies of polymorphisms at the key 260, 342, and 407 residues in the Ace gene of the field-collected house fly samples. A: Genotypic combination patterns of the three resistance-associated mutations at positions 260, 342, and 407. B: Frequencies of mutations at position 260. C: Frequencies of mutations at position 342. D: Frequencies of mutations at position 407.
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The proportions of the three resistance-associated mutations in all the individuals are summarized in Figures 2B–D. At position 260, 20% and 32% of the individuals were homozygous for Leu and Val, respectively, whereas the remaining (48%) were heterozygous for these amino acids (Figure 2B). At position 342, five combinations were detected, suggesting the importance of this position for the enzyme. Walsh et al. (2001) reported that G342 is located close to the active-site triad at the base of the gorge, likely affecting the orientation of the catalytic serine, which is predicted to cause resistance by restricting the access and/or binding of bulky insecticide inhibitors within the active site; they identified G342A and G342V substitutions in OP and CB resistant house fly strains, respectively. Only 2% and 14% of the individuals were homozygous for Gly and Ala at this position, respectively; the remaining individuals (84%) were heterozygous at this position. Among them, 56% were heterozygous for Gly and Ala, 22% were heterozygous for Ala and Val, and 6% were heterozygous for Val and Gly (Figure 2C). V/V342 was not detected among the screened houseflies. At position 407, most of the individuals (67%) were homozygous for Tyr and the rest (33%) were heterozygous for Phe and Tyr (Figure 2D). The standard susceptible strain contained an allele coding a characteristic susceptible form of AChE (V260-A316-G342-F407). In addition, two samples from Aydın were found to be heterozygous for the amino acid position of 316; one allele contained Ala and the other contained Ser. This A316S change was recently reported as a new mutation appearing to be associated with resistance (Kozaki et al. 2009).
Region specific assessment of the data, given in Table 2, did not yield distinct differences between the Aegean and Mediterranean populations in terms of their compositions at the three resistance-associated amino acid positions. However, 11 samples of the Mediterranean populations were homozygous for Leu at position 260 compared with only two samples of the Aegean populations showing homozygosity at this position. Val residue in the homozygous condition was detected in higher frequency (75%) in the Izmir population of the Aegean region than in the other populations screened in this study. Among the Aegean populations, all the individuals from Denizli, 75% of the individuals from Kütahya, Uşak, and Afyon each, and 50% of the individuals from Manisa were heterozygous at position 260. On the other hand, among the Mediterranean populations, all the samples from Osmaniye and 50% of the samples from Kahramanmaraş, Burdur, and Isparta each were heterozygous at the same position. All of the three combinations at this position were detected in the Aydın and Muğla populations of the Aegean region and in the Antalya, Mersin, and Hatay populations of the Mediterranean region.
Table 2. The amino acid residues at positions 260, 342, and 407 inferred from the Ace sequence, alleles detected in the samples, and the percent remaining AChE activities of the field-collected house fly populations from the Aegean and Mediterranean regions.
Considering position 342, all the individuals from the Denizli and Osmaniye populations were heterozygous for Gly and Ala. The Kütahya, Aydın, and Afyon populations of the Aegean region and the Kahramanmaraş and Burdur populations of the Mediterranean region were also heterozygous for the same residues in high frequency (75% in each). The Uşak, Manisa, and Muğla populations of the Aegean region and the Antalya, Hatay, and Isparta populations of the Mediterranean region showed three different compositions at this position.
All the house flies screened from the Aydın, Uşak, Izmir, Manisa, and Burdur populations were homozygous for Tyr at position 407. On the other hand, only the Kahramanmaraş population was heterozygous for Tyr and Phe at this position, and 75% of the individuals from the Afyon and Osmaniye populations were heterozygous at the same position. No homozygous individuals for Phe were detected.
Evaluation of the populations in terms of the combinations at the three amino acid positions revealed a conspicuous situation in the Osmaniye population only, in which 75% of the individuals had the phenotype of L/V260-A/G342-F/Y407. The 12 remaining phenotypes were represented by only one or two samples in the other populations.