Population genetics and vector biology of Amblyomma americanum

Amblyomma americanum has emerged as an important vector of human disease over the last decade, and the geographic range of this vector has expanded due to unknown factors. Genetic studies of arthropod vectors seek to describe the genetic variability between populations in order to predict the introduction of disease agents into new areas, the spread of resistant alleles, and potential epidemics. I describe the population structure of A. americanum and "R. amblyommii" from throughout the range of this tick. The prevalence in this tick of four bacteria that are potential or known pathogens of humans were analyzed. Microsatellites, amplified restriction length polymorphism analysis markers (AFLP), and denaturing high performance liquid chromatography (DHPLC) were evaluated to determine genetic variability within A. americanum. DHPLC was chosen to analyze single nucleotide polymorphisms and insertion deletion events (indels). I applied DHPLC to a 320 base pair fragment of the 16S mitochondrial gene and a 389 base pair fragment of the ITS2 ribosomal DNA gene of A. americanum to assess this method for the study of population genetics. Thirty-one haplotypes with twenty three variable positions and twenty-one genotypes with twenty-six variable positions were identified with DHPLC. DHPLC can be readily applied to other species for population genetic and evolutionary analyses. The data from DHPLC analysis was used to evaluate the genetic differentiation of A. americanum on a fine scale: ticks from nine populations in three physiogeographic regions in Georgia were analyzed for population differentiation using a single mitochondrial marker. The majority of the variation was among ticks within each population, indicating significant gene flow and little genetic differentiation between these regions. An overall FST value of 0.006 supported the lack of genetic structuring between collection sites in Georgia. Mantel regression analysis revealed no isolation by distance. Signatures of population expansion were detected in the shapes of the mismatch distribution and tests of neutrality. An absence of genetic differentiation combined with the rejection of the null model of isolation by distance might indicate recent range expansion in Georgia or insufficient time to reach an equilibrium where genetic drift might have affected allele frequencies. Alternatively, the high degree of panmixia found within A. americanum in Georgia might be due to bird dispersal of ticks increasing the genetic similarity between geographically separated populations. The data was expanded to assess the genetic structure of the tick across most of its geographic range using both genetic markers to differentiate between demographic and evolutionary events. A hierarchical population genetic study was conducted on 2158 individual ticks from 32 populations throughout its range. Thirty-two 16S haplotypes and twenty-one ITS2 genotypes were detected by DHPLC and characterized by DNA sequencing. The majority of the variation found was among ticks within each population, indicating high amounts of gene flow between regions. However, significant overall FST values indicated genetic differentiation existed between certain collection sites despite Mantel regression analysis that revealed no isolation by distance. Population expansion was indicated in several populations, and admixture events or population subdivision followed by expansion in others. I determined the prevalence of infection and confection with E. chaffeensis, E. ewingii, "Borrelia lonestari", and " R. amblyommii" in A. americanum. Overall infection prevalences were 4.7% for E. chaffeensis (range 0-27%), 3.5% for E. ewingii (range 0-18.6%), 2.5% for "B. lonestari " (range 0-12.2%) and 41.2% for "R. amblyommii" (range 0-84.0%). Eighty seven ticks (4.3%) were infected with two or more bacteria. I report new distribution records for E. ewingii, "B. lonestari", and "R. amblyommii" and underscore the importance of the nonhomogeneous distribution of pathogen foci of infection. Because A. americanum has a high prevalence of infection with "R. amblyommii", I assessed "R. amblyommii" for genetic variability and evaluated the implications of this data for coevolution of this agent with its host tick. Intergenic regions were used for analyzing genetic diversity of isolates of Rickettsia. I tested 51 primer pairs designed to amplify intergenic regions to assess levels of genetic diversity of "R. amblyommii" from 12 sources from eight states. Amplicons were obtained from 35 of the primer pairs; however, most intergenic regions had identical nucleotide sequences, and minor polymorphisms were found in only two markers. This suggests that little intraspecific variation exists in "R. amblyommii". I discuss the potential factors contributing to the homogenization of the species. Finally, I discuss the inferences and conclusions gained from the various studies as well as their limitations for understanding the evolution and population structure of A. americanum..