Vkorc1-sequencing of the house mouse (Mus musculus domesticus), Norway rat (Rattus norvegicus), and roof rat (R. rattus) from the USA enables new inferences regarding the evolution and spread of anticoagulant rodenticide resistance
Diaz, Juan C
Kohn, Michael H
Doctor of Philosophy
Resistance to anticoagulant rodenticides emerged within less than 10 years after their introduction in the 1950s in three species of commensal rodents; the house mouse (Mus musculus domesticus), the Norway rat (Rattus norvegicus), and the roof rat (Rattus rattus). The resistance phenomenon provides an example of evolution in action and how chemical pest control and the movement of animals around the globe can drive it. Resistance is of relevance to public health as resistance impairs rodent control. The study of resistance trait aids understanding of the physiological response of humans to the anticoagulant drug warfarin. Resistance has evolved to first-generation anticoagulants (FGARs), including warfarin, and then, to some second-generation anticoagulant rodenticides (SGARs), including SGARs++, which are recently developed SGARs that are more effective against rodents with resistance to FGARs and SGARs. The resistance phenomenon is a striking example of convergent evolution, as in the three rodent species resistance is at least partly, albeit strongly, mediated by mutations in the vitamin K epoxide reductase subcomponent 1 gene (Vkorc1). Some mutations are shared between the species, such as a Tyr139Cys resistance mutation seen in the house mouse and the Norway rat. In chapter 1 of this thesis, I provide background on rodenticide resistance and the rationale underlying my work. In chapter 2, I compile the published data on the distribution of rodenticide resistance. Available data mainly are in the form of physiological testing for resistance using feeding trials and blood coagulation tests, as well as in form of Vkorc1 nonsynonymous single nucleotide polymorphisms (nsSNPs) used to infer the resistance. Subsets of the nsSNPs are known to be associated with the resistance phenotype, but this association depends on the criteria used, such as to whether nsSNPs cause resistance of practical relevance in the field or merely affect vitamin K cycle kinetics. The survey reveals substantial research efforts were conducted in Europe in the past and present, but revealed the absence of any recent information on resistance in the USA, albeit a burst of effort testing for resistance occurred in the 1980s using feeding trials. The mapping of results point to Europe and North America as the regions where resistance has been studied most extensively. However, monitoring of resistance has generally not been conducted in a systematic fashion, that is, the distribution of resistance generally reflects a geographical bias in research efforts and a possible reporting bias towards the detection of resistance. Therefore, research is needed to systematically collect information on resistance in most other regions of the globe. Interesting, Vkorc1 polymorphism data is found to be nearly absent for North America. To fill this gap, in chapter 3, I conduct a DNA sequencing survey of Vkorc1 in house mice, Norway rats and roof rats from the USA. This geographical focus is justified because the USA is the place where anticoagulant rodenticides were invented and used first, yet no recent work comparable to the work done in Europe has been conducted in the USA since the 1980s. Thus, currently it is impossible to assess the role of ancient population structure, historic gene flow, and convergence by de novo mutations in the origin and spread of resistance at any scale, be it local, regional or global. Furthermore, this focus on the USA is of practical relevance as the Environmental Protection Agency (EPA) recently has passed legislation that only FGARs are made available as over-the-counter anticoagulant rodenticides. This legislation was passed in the absence of data on Vkorc1 in the USA. I report that numerous Vkorc1 nsSNPs in the house mouse (six nsSNPs found), in the Norway rat (two nsSNPs found) and the roof rat (one nsSNPs found) occur in the USA, out of those two (Leu128Ser and Tyr139Cys) are known to cause physiological and practical resistance (control problems in the field) in the house mouse. Arg35Pro and Tyr25Phe found in the Norway rat and roof rat, respectively, have physiological consequences and Arg35Pro appears to be responsible for practical resistance, such as reported from Chicago, USA. Other polymorphisms in Vkorc1 were also detected, but their role in mediating physiological responses or practical resistance causing rodent control problems in the field is unknown and requires further study. As inferred from the presence and absence of particular polymorphisms in the USA and Europe, resistance has evolved numerous times in geographically separated populations. In house mice, the Leu128Ser variant is rare in the USA but Tyr139Phe is common both in Europe and the USA, suggesting an independent geographic origin of both mutations. The occurrence of the Leu128Ser and Tyr139Cys variants in house mice from Europe and the USA also indicates a role for gene flow between populations within and between continents. However, at regional and local scales populations remain differentiated with several examples of adjoining populations carrying different resistance mutations. In the Norway rat the Arg35Pro variant is near exclusive to the USA. European Norway rats mainly carry Tyr139Phe/Cys/Ser variants, and Leu128Gln and Leu120Gln variants, and display considerable population differentiation at regional and local scales. Thus, in Norway rats, the signature of convergent molecular evolution to the trait is detectable still. Gene flow has not yet obscured this recent phenomenon. With only one widespread variant of uncertain effect on resistance, the roof rat may reflect a scenario of a single origin and subsequent widespread dispersal of a variant. To assess the role of ancient population structure and recent gene flow at global, regional and local scales, in chapter 4 I combined the previously published data and newly collected data on resistance and added published data and newly collected data on mitochondrial (mt) DNA haplotype variation. Analysis of mtDNA haplotype diversity and matrilineal gene flow estimates revealed the presence of widely (at a regional and global scale) distributed haplotypes that show association with resistance, and others that are locally restricted. Further analyses revealed the role of dispersal and resulting gene flow in the spatial occurrence of resistance. However, a prominent role for independent evolution at the Vkorc1 locus can be observed at the local, regional and global spatial scales. Thus, anticoagulant resistance provides an example of rapid convergent evolution of an adaptive trait at the population level when the selection regime is same or similar. Broader sampling of rodents needs to be done to fully understand the evolution of resistance and to provide more complete geographic coverage of the current distribution of resistance. My work, including mtDNA-based predictions, provides guidance as to where future sampling and testing should be conducted, and larger-scale analyses of gene flow in the nuclear genome, rather than the mtDNA genome, should be conducted.