**4. Conclusions**

146 Gene Duplication

Three point mutations (Gly228Ser, Ala391Thr and Phe439W) have been identified using extensive sequence *c*omparisons of the AChE gene from the TSSM (*Tuace*) between OPresistant and susceptible strains. In addition, their functional roles have been assessed by analyzing the correlation between mutation frequencies and actual resistance levels of several field populations (Kwon et al., 2010b). The frequencies of the Gly228Ser and Phe439Trp mutations in resistant strains never reached 100% even after extensive selection with monocrotophos (Kwon et al., 2010b). To determine whether the lack of saturation for these mutation frequencies is due to the heterozygosity of the *Tuace* allele in individual mites, the frequencies of the three mutations in an individual diploid virgin female and her parthenogenetic haploid male progenies were have been determined using quantitative sequencing (Kwon et al., 2010a). The actual frequencies of the G228S, A391T and F439W mutations in a female mite and its haploid male progenies have been estimated as approximately 50%, 100% and 75%, respectively. These findings clearly suggested the presence of multiple copies of *Tuace*. Determination of *Tuace* copy number in three mite strains (highly resistant AD, moderately resistant PyriF and susceptible UD strains) using quantitative PCR has revealed that resistant strains have relatively more *Tuace* copies than the susceptible strain and that the levels of transcript were directly proportional to copy numbers (Kwon et al., 2010a). AChEs from the AD and PyriF strains have shown reduced catalytic efficiencies based on lower *kcat* values, suggesting that the resistant form of AChE is likely accompanied by fitness cost. Relative copy numbers of *Tuace* in field populations of TSSM ranged from 2.4 to 6.1 and are highly correlated with the respective resistance level,

**3.3 Extensive duplication of AChE gene in the two-spotted spider mite (TSSM)** 

suggesting that *Tuace* duplication itself contributes to resistance (Kwon et al., 2010a).

Western blot analysis using AChE-specific antibodies has been conducted to determine whether *Tuace* duplication results in TuAChE overproduction. The protein quantities of TuAChE in seven field-collected mite populations precisely correlated with the copy numbers (Lee and Kwon, unpublished data). To investigate the effects of each mutation on AChE insensitivity and possible fitness costs, eight variants of TuAChE were expressed *in vitro* using the baculovirus expression system. Kinetic analysis revealed that the Ala391Thr mutation did not alter the kinetic properties of AChE, whereas the Gly228Ser and Phe439Trp mutations significantly increased the insensitivity to monocrotophos. Moreover, when the Gly228Ser and Phe439Trp mutations were co-expressed, insensitivity increased over 1000-fold. These results show that both mutations confer resistance in a synergistic manner. However, the presence of the mutations considerably reduced the catalytic efficiency of AChE, suggesting an apparent fitness cost in monocrotophos-resistant mites. Reconstitution of the multiple copies of AChE with different compositions of the mutations revealed that the catalytic efficiencies of the six-copy and two-copy AChEs (resembling the AD and PyriF strains of mite, respectively) were lower but comparable to that of wildtype AChE. These finding clearly suggest that multiple rounds of *Tuace* duplication is needed to compensate the reduced catalytic activity of AChE caused by mutations. Whether mutation or gene duplication occurs first is unknown. However, the introduction of a single mutation (Gly228Ser or Phe439Trp) or a double mutation in a single copy of *Tuace* is unlikely because the fitness cost is severe based on the dramatic reductions in the catalytic efficiency. Therefore, at least a single event of *Tuace* duplication predates the introduction of mutations. A single Gly228Ser mutation likely occurs first in one of the duplicates as seen in the PyriF strain, in which the Gly228Ser mutation has been identified in one of the *Tuace* duplicates. Under continuous selection pressure by OPs, further duplication of mutations might have Duplications (or amplifications) of resistance-related genes are frequent mechanisms in insecticide resistance. Extensive forms of gene duplication (i.e., amplification) are commonly found in metabolic genes that are involved in insecticide detoxification, including esterase and Cyp450. In these cases, even with dramatically increased gene dosage, the apparent fitness cost is not severe. In other words, low fitness costs that are associated with high dosages of metabolic genes allow the amplification of genes. Gene duplication events have been found in insecticide target genes, including *ace* and *Rdl*, which play crucial functions in nerve impulse transmission. Unlike the amplification of metabolic genes, the level of duplication of these genes is precisely regulated due to the necessity to maintain the normal gene dosage. Mutations conferring target site insensitivity are always accompanied with duplication events. Because mutations in the insecticide target sites frequently alter catalytic or functional properties of target proteins, which usually increase fitness costs, duplication may act as a compensatory mechanism to restore the normal activity of the target protein, which is otherwise detrimental to maintaining the nervous system homeostasis. Conversely, if the increase of the target protein dose due to an incidental gene duplication event has different fitness consequences, the introduction and selection of any target site mutations conferring insecticide resistance is facilitated following duplication because the mutations that are associated with resistance usually reduce the normal function of target proteins. Taken together, it is difficult to determine whether duplication or mutation occurs first. However, these evolutionary events to acquire insecticide resistance may interact each other to balance the level of resistance and accompanied fitness costs. Genetic introgression between different populations plays a crucial role in spreading and formulating the degree of gene duplication and mutation. In *Anopheles gambiae*, for example, the genetic traits of the *ace1R* mutation and the *ace1* duplication are shared between populations through introgression (Djogbénou et al., 2008).
