Genetic variability in a given gene is the result of a complex interplay of evolutionary forces such as selection, mutation rate and recombination, and demographic history of the populations including changes in population size, migration and divergence
. The evolution of insecticide resistance in invasive pest species offers an opportunity to study these forces where the form, target and history of selection, as well as invasion routes are often well known
. Evolution due to a strong directional selection by insecticides can be a rapid process [
 and references therein], usually involving only a few genes with pronounced phenotypic influences
[4, 5]. Typically, mutations that confer resistance are considered to occur at very low frequency in the population due to fitness costs in non-insecticide environments
. Strong insecticide selection subsequently leads to an increase in the resistance allele frequency and a general change in the allele composition of populations (e.g.
). Furthermore, a strong reduction in genetic variation is often observed as the originally rare mutation increases in frequency
[7, 8]. This reduction may occur not only in the locus targeted by selection but also in the surrounding areas through linkage and genetic hitchhiking
[9, 10]. Alternatively, selection can act on standing genetic variation (that is, the beneficial mutation has relatively high frequency) leading to soft sweeps or even maintain variability
In addition to selection by insecticides, populations of invasive pest species are often affected by demographic events. For instance, genetic variation is often reduced by population bottlenecks and allele frequencies can be drastically changed by founder events
. Subsequent range expansions, typically observed in species invasions
, can contribute to the spread of an insecticide resistance allele across large regions without local insecticide selection
. However, demographic events can be difficult to disentangle from selection as the former can lead to patterns of genetic variation similar to those caused by selection
. These confounding effects can, in principle, be separated by examining multiple loci since demographic history modifies the genetic pattern of the whole genome whereas only certain loci are affected by selection
Studies of resistance genes have focused on revealing the nucleotide substitutions that are associated with resistance (e.g.
[17, 18]), while less attention has been paid to genetic variation in the resistance genes themselves. Yet the level and pattern of genetic variation at the gene and population level may give a more comprehensive picture of the evolutionary forces affecting populations
[7, 8, 19]. Comparing populations before and after selection (or populations differing in their exposure to pesticide selection pressure) can provide important insights on the origins of resistance and how selection modifies patterns of genetic variability
[10, 13]. For instance, the Colorado potato beetle (Leptinotarsa decemlineata), an invasive insect pest of potato, is well known for its ability to evolve resistance to insecticides
 and specific mutations at the target site of resistance to organophosphate and pyrethroids have been extensively investigated
[21–26]. However, few studies have examined the population genetic history of agricultural insect pest populations (but see e.g.
). By examining the population genetic history of agricultural insect pests, it will provide insight as to whether insect pests appear to be preadapted to insecticide resistance, or whether resistance arises from de novo mutations under intense selection.
Organophosphates are one of the most common classes of insecticides used worldwide. They were introduced as insecticides in the 1930s after which their use increased rapidly and peaked in the 1970s and 1980s
. Due to intense use of OP insecticides, resistance to OPs subsequently appeared in various insect pest species, including the Colorado potato beetle as early as 1964 in US
[3, 28, 29]. OP resistance in insects can be based on several mechanisms; the major ones are target-site insensitivity and enhanced detoxification of the toxicant. Also lowered availability of the toxicant may play a role in resistance (e.g.
, see also
). Target-site insensitivity is caused by point mutations in two AChE genes (AChE1 paralogous to and AChE2 orthologous to Drosophila melanogaster Ace gene)
 that encode for acetylcholinesterase enzymes that function in the synapses of the nervous system. These point mutations cause insensitivity at the target site of insecticides action (e.g.
). There are at least two point mutations in the AChE2 gene that have been associated with OP resistance in the Colorado potato beetle
[21, 22]. The major resistance-conferring mutation, an amino acid substitution from serine to glycine (S291G), seems to be unique to this species
. The second mutation (arginine to lysine, R30K), may enhance the resistance conferred by the S291G
In addition for being famous for its ability to evolve insecticide resistance, the invasion history of the Colorado potato beetle is relatively well known. The beetle is native to Mexico where it uses wild relatives of potato (Solanum rostratum Dunal, S. angustifolium Mill.) as host plants, and is not a pest of potato. It is thought that Spanish settlers that migrated northwards with cattle inadvertently carried S. rostratum, northwards. The beetle was first described feeding on S. rostratum in 1824 in Nebraska. The beetle became a serious pest after adapting to potato (S. tuberosum) in the American mid-west in the 1850s
. The first described outbreaks of the beetle occurred in 1859 in the Midwestern US
. Since, it has spread across the US and almost throughout Europe
[32, 33]. Although its establishment in Europe involved a founder event
 which reduced neutral genetic variability, the beetle has retained a substantial amount of genetic variability in life history traits
[33, 35]. Currently, the Colorado potato beetle is considered to be the major threat to potato crops worldwide. If left uncontrolled, it can cause serious damage to potato crops
. The beetle’s complicated and versatile life history has contributed to its success. For instance, the beetle has a very high reproductive potential, both larvae and adults feed on potato, and adults can survive harsh winter conditions by overwintering in diapause burrowed into the soil
We took advantage of the knowledge on one of the main mechanisms of OP resistance and combined it with the invasion history of the Colorado potato beetle to examine the origins of OP resistance-associated mutations, and the selective and demographic forces on the genetic variation in the AChE2 gene using a population genetic approach. In order to disentangle the confounding effects of demography from selection, we examined genetic variation at two other nuclear genes, the diapause protein 1 (DP1)
 and putative juvenile hormone esterase (JHE)
[39, 40]. If there was a clear sign of intense directional selection, we would expect lower genetic variability both at the AChE2 locus than at the other two loci and in the invasive agricultural (US and Europe) than in native Mexican beetle populations. We would also expect a higher frequency of the OP resistance-associated S291G mutation in the invasive than in the native populations. Finally, we searched for evidence of population genetic structure at the loci among geographic regions and investigated if nucleotide variability at the AChE2, DP1 and JHE genes are compatible with neutral expectations.