Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected during surveillance in Canada

The genetic diversity of Borrelia burgdorferi sensu stricto, the agent of Lyme disease in North America, has consequences for the performance of serological diagnostic tests and disease severity. To investigate B. burgdorferi diversity in Canada, where Lyme disease is emerging, bacterial DNA in 309 infected adult Ixodes scapularis ticks collected in surveillance was characterized by multilocus sequence typing (MLST) and analysis of outer surface protein C gene (ospC) alleles. Six ticks carried Borrelia miyamotoi, and one tick carried the novel species Borrelia kurtenbachii. 142 ticks carried B. burgdorferi sequence types (STs) previously described from the United States. Fifty-eight ticks carried B. burgdorferi of 1 of 19 novel or undescribed STs, which were single-, double-, or triple-locus variants of STs first described in the United States. Clonal complexes with founder STs from the United States were identified. Seventeen ospC alleles were identified in 309 B. burgdorferi-infected ticks. Positive and negative associations in the occurrence of different alleles in the same tick supported a hypothesis of multiple-niche polymorphism for B. burgdorferi in North America. Geographic analysis of STs and ospC alleles were consistent with south-to-north dispersion of infected ticks from U.S. sources on migratory birds. These observations suggest that the genetic diversity of B. burgdorferi in eastern and central Canada corresponds to that in the United States, but there was evidence for founder events skewing the diversity in emerging tick populations. Further studies are needed to investigate the significance of these observations for the performance of diagnostic tests and clinical presentation of Lyme disease in Canada.     

Pervasive recombination and sympatric genome diversification driven by frequency-dependent selection in Borrelia burgdorferi, the Lyme disease bacterium

How genomic diversity within bacterial populations originates and is maintained in the presence of frequent recombination is a central problem in understanding bacterial evolution. Natural populations of Borrelia burgdorferi, the bacterial agent of Lyme disease, consist of diverse genomic groups co-infecting single individual vertebrate hosts and tick vectors. To understand mechanisms of sympatric genome differentiation in B. burgdorferi, we sequenced and compared 23 genomes representing major genomic groups in North America and Europe. Linkage analysis of >13,500 single-nucleotide polymorphisms revealed pervasive horizontal DNA exchanges. Although three times more frequent than point mutation, recombination is localized and weakly affects genome-wide linkage disequilibrium. We show by computer simulations that, while enhancing population fitness, recombination constrains neutral and adaptive divergence among sympatric genomes through periodic selective sweeps. In contrast, simulations of frequency-dependent selection with recombination produced the observed pattern of a large number of sympatric genomic groups associated with major sequence variations at the selected locus. We conclude that negative frequency-dependent selection targeting a small number of surface-antigen loci (ospC in particular) sufficiently explains the maintenance of sympatric genome diversity in B. burgdorferi without adaptive divergence. We suggest that pervasive recombination makes it less likely for local B. burgdorferi genomic groups to achieve host specialization. B. burgdorferi genomic groups in the northeastern United States are thus best viewed as constituting a single bacterial species, whose generalist nature is a key to its rapid spread and human virulence.     

The effects of contemporary processes in maintaining the genetic structure of western song sparrows (Melospiza melodia)

Historic events and contemporary processes work in concert to create and maintain geographically partitioned variation and are instrumental in the generation of biodiversity. We sought to gain a better understanding of how contemporary processes such as movement and isolation influence the genetic structure of widely distributed vagile species such as birds. Song sparrows (Melospiza melodia) in western North America provide a natural system for examining the genetics of populations that have different patterns of geographic isolation and migratory behavior. We examined the population genetics of 576 song sparrows from 23 populations using seven microsatellite loci to assess genetic differentiation among populations and to estimate the effects of drift and immigration (gene flow) on each population. Sedentary, isolated populations were characterized by low levels of immigration and high levels of genetic drift, whereas those populations less isolated displayed signals of high gene flow and little differentiation from other populations. Contemporary dispersal rates from migratory populations, estimated by assignment test, were higher and occurred over larger distances than dispersal from sedentary populations but were also probably too low to counter the effects of drift in most populations. We suggest that geographic isolation and limited gene flow facilitated by migratory behavior are responsible for maintaining observed levels of differentiation among Pacific coastal song sparrow populations.     

Whole-genome sequences of thirteen isolates of Borrelia burgdorferi

Borrelia burgdorferi is a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence of B. burgdorferi strain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease. Because a single genome sequence is not sufficient to understand the relationship between genotypic and geographic variation and disease phenotype, we determined the whole-genome sequences of 13 additional B. burgdorferi isolates that span the range of natural variation. These sequences should allow improved understanding of pathogenesis and provide a foundation for novel detection, diagnosis, and prevention strategies.     

Range-wide comparisons of northern leatherside chub populations reveal historical and contemporary patterns of genetic variation

Patchily distributed species are those taxa whose populations occupy geographically insular habitats and their conservation often depends on an understanding of the relationship among disjunct populations. The objective of our study was to use molecular data and analytical techniques to separate the effects of historical and contemporary processes influencing the distribution of a high-desert minnow, the northern leatherside chub (Lepidomeda copei). Individuals from 23 populations were sequenced for 1,140 base pairs of the cytochrome B gene of the mitochondrial genome and genotyped at seven nuclear microsatellite loci. We estimated gene flow and examined population structure using both microsatellite and mtDNA data. Low sequence divergence and the distribution of shared haplotypes in multiple watersheds suggest historical connectivity between populations over a large geographic area. In contrast, patterns of microsatellite diversity indicate that populations of leatherside chub are isolated from one another with low levels of contemporary gene flow between populations. Our results suggest that populations of leatherside chub were historically more widely inter-connected and have recently been isolated, likely through a combination of natural and anthropogenic habitat fragmentation. As populations become increasingly isolated, they are more vulnerable to extirpation as a result of stochastic events. For northern leatherside chub, recent isolation and lack of gene flow among populations may affect their long-term survival in the arid landscapes of the Great Basin and surrounding watersheds because of widespread and increasing habitat alteration and fragmentation.     

UNCOORDINATED PHYLOGEOGRAPHY OF BORRELIA BURGDORFERI AND ITS TICK VECTOR,IXODES SCAPULARIS

Vector‐borne microbes necessarily co‐occur with their hosts and vectors, but the degree to which they share common evolutionary or biogeographic histories remains unexplored. We examine the congruity of the evolutionary and biogeographic histories of the bacterium and vector of the Lyme disease system, the most prevalent vector‐borne disease in North America. In the eastern and midwestern US, Ixodes scapularis ticks are the primary vectors of Borrelia burgdorferi, the bacterium that causes Lyme disease. Our phylogeographic and demographic analyses of the 16S mitochondrial rDNA suggest that northern I. scapularis populations originated from very few migrants from the southeastern US that expanded rapidly in the Northeast and subsequently in the Midwest after the recession of the Pleistocene ice sheets. Despite this historical gene flow, current tick migration is restricted even between proximal sites within regions. In contrast, B. burgdorferi suffers no barriers to gene flow within the northeastern and midwestern regions but shows clear interregional migration barriers. Despite the intimate association of B. burgdorferi and I. scapularis, the population structure, evolutionary history, and historical biogeography of the pathogen are all contrary to its arthropod vector. In the case of Lyme disease, movements of infected vertebrate hosts may play a larger role in the contemporary expansion and homogenization of the pathogen than the movement of tick vectors whose populations continue to bear the historical signature of climate‐induced range shifts.     

The population genetic structure of a large temperate pollinator species, Bombus pascuorum (Scopoli) (Hymenoptera: Apidae)

The genetic population structure of the bumble bee Bombus pascuorum was studied using six microsatellite loci and a partial sequence of the mitochondrial gene cytochrome b . Eighteen populations from central and northern Europe were included in the analysis. Observed levels of genetic variability and heterozygosity were high. Estimates of population differentiation based on F - and Φ-statistics revealed significant genetic differentiation among B. pascuorum populations and suggest that two partially isolated gene pools, separated by the Alps, do exist. The distribution of mtDNA haplotypes supports this view and presents direct evidence for gene flow across the Alps. Estimates of the number of migrants exchanged among populations north of the Alps suggest that historical events may have left a strong imprint on population structure.     

Molecular interactions that enable movement of the Lyme disease agent from the tick gut into the hemolymph

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to humans by bite of Ixodes scapularis ticks. The mechanisms by which the bacterium is transmitted from vector to host are poorly understood. In this study, we show that the F(ab)(2) fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the migration of the spirochete from tick gut into the hemolymph during tick feeding. The decreased hemolymph infection results in lower salivary glands infection, and consequently attenuates mouse infection by tick-transmitted B. burgdorferi. Using a yeast surface display approach, a tick gut protein named TRE31 was identified to interact with BBE31. Silencing tre31 also decreased the B. burgdorferi burden in the tick hemolymph. Delineating the specific spirochete and arthropod ligands required for B. burgdorferi movement in the tick may lead to new strategies to interrupt the life cycle of the Lyme disease agent.     

Lyme borreliosis situation in North Africa

Many epidemiological studies were conducted for studying Lyme borreliosis (LB) which represents a new global public health problem. It is now the most common vector-borne disease in Europe and North America. The causative agent Borrelia burgdorferi sl is a bacterial species complex comprising 12 delineated and named species. In North Africa, few studies based on clinical and serological features, have suggested that LB could occur. Indeed, recent studies conducted in Tunisia, Algeria and Morocco have showm that Ixodes ricinus is present in cooler and humid area of these regions. These studies also revealed that this species is a vector of B. burgdorferi sl with high prevalence of infection. Using IFI and PCR tests, the mean rate of Borrelia-infection ranged from 50 to 60% in I. ricinus adult collected in Tunisia and Morocco and from 30 to 40% in nymphs; in contrast, the prevalence in larvae is less than 2.5%. Several strains of B. burgdorfer were isolated from adult and nymph I ricinus collected in Tunisia and Morocco. The identification of these strains and DNAs directly extracted from Ixodes was done by PCR-RFLP and sequence analysis. The results showed that B. lusitaniae (genotypes Poti B2 and Poti B3) is the predominant species circulating in I. ricinus in Tunisia and Morocco, B. garinii and B. burgdorferi ss and B lusitaniae were also present but very rare. These results provide the evidence for the existence of B. burgdorferi sl in North Africa; however, the impact of LB in the human population seem to be negligible and the seroprevalence of Borrelia in forest workers (considered as population at high risk) in Tunisia is less than 4%.     

Genomic diversity,population structure,and migration following rapid range expansion in the Balsam Poplar,Populus balsamifera

Rapid range expansions can cause pervasive changes in the genetic diversity and structure of populations. The postglacial history of the Balsam Poplar, Populus balsamifera, involved the colonization of most of northern North America, an area largely covered by continental ice sheets during the last glacial maximum. To characterize how this expansion shaped genomic diversity within and among populations, we developed 412 SNP markers that we assayed for a range‐wide sample of 474 individuals sampled from 34 populations. We complemented the SNP data set with DNA sequence data from 11 nuclear loci from 94 individuals, and used coalescent analyses to estimate historical population size, demographic growth, and patterns of migration. Bayesian clustering identified three geographically separated demes found in the Northern, Central, and Eastern portions of the species’ range. These demes varied significantly in nucleotide diversity, the abundance of private polymorphisms, and population substructure. Most measures supported the Central deme as descended from the primary refuge of diversity. Both SNPs and sequence data suggested recent population growth, and coalescent analyses of historical migration suggested a massive expansion from the Centre to the North and East. Collectively, these data demonstrate the strong influence that range expansions exert on genomic diversity, both within local populations and across the range. Our results suggest that an in‐depth knowledge of nucleotide diversity following expansion requires sampling within multiple populations, and highlight the utility of combining insights from different data types in population genomic studies.     

A novel fold for the factor H-binding protein BbCRASP-1 of Borrelia burgdorferi

Borrelia burgdorferi, a spirochete transmitted to human hosts during feeding of infected Ixodes ticks, is the causative agent of Lyme disease. Serum-resistant B. burgdorferi strains cause a chronic, multisystemic form of the disease and bind complement factor H (FH) and FH-like protein 1 (FHL-1) on the spirochete surface. Here we report the atomic structure for the key FHL-1- and FH-binding protein BbCRASP-1 and reveal a homodimer that presents a novel target for drug design.     

Spatial and temporal stability of mtDNA haplogroup frequencies in native North America

Mitochondrial DNA lineage frequencies in prehistoric Aleut, eastern Utah Fremont, Southwestern Anasazi, Pyramid Lake, and Stillwater Marsh skeletal samples from northwest Nevada and the Oneota of western Illinois are compared with those in 41 contemporary aboriginal populations of North America. The ancient samples range in age from 300 years to over 6,000 years. The results indicate that the prehistoric inhabitants of North America exhibit the same level of mtDNA variability as contemporary populations of the continent. Variation in modern mtDNA haplogroup frequencies is highly geographically structured, and the prehistoric samples exhibit the same geographic pattern of variation. This indicates that differentiation of regional patterns of mtDNA lineage variation occurred early in North American prehistory (much more than 2,000 years B.P.), has remained relatively stable since its origin, and was little influenced by the disruptions hypothesized for other genetic systems as a result of population declines and relocations at contact.     

Genetic structure of muskrat (Ondatra zibethicus) and its concordance with taxonomy in North America

Extrinsic factors such as physical barriers play an important role in shaping population genetic structure. A reduction in gene flow leading to population structuring may ultimately lead to population divergence. These divergent populations are often considered subspecies. Because genetic differentiation may represent differences between subspecies, patterns of genetic structure should reflect subspecies groupings. In this study, we examine the contemporary population genetic structure of muskrat (n = 331) and assess the relevance of 4 geographically distinct subspecies designations across northern North America using 9 microsatellite loci. We predicted that patterns of gene flow and genetic structure would reflect the described subspecies. We found evidence of genetic differentiation between western and eastern regions, and muskrats from Newfoundland (NF) showed significantly lower genetic diversity than central regions. A strong isolation by distance pattern was also detected within the eastern cluster. Our results did not differentiate Ondatra zibethicus spatulus (northwest) from O. z. albus (central), but they suggest a distinction between O. z. obscurus (NF) and O. z. zibethicus (east). This study highlights the need for more phylogenetic studies in order to better understand intraspecific divergence and the genetic characterization of subspecies.     

Characterization of Lyme disease spirochetes isolated from ticks and vertebrates in North Carolina

Borrelia burgdorferi isolates obtained from numerous locations and from different hosts in North Carolina, were compared to previously characterized strains of the Lyme disease spirochete and other Borrelia spp. The spirochete isolates were confirmed to be B. burgdorferi sensu stricto based on immunofluorescence (IFA) using a monoclonal antibody to outer surface protein A (Osp A [H5332]) and polymerase chain reaction (PCR) using a species-specific nested primer for a conserved region of the gene that encodes for flagellin. In addition, the isolates tested positive in Western blots with species-specific monoclonal antibodies for outer surface protein A and OspB (84c), and the genus-specific, monoclonal antibody to flagellin (H9724). Infectivity studies with several of these isolates were conducted using Mus musculus and Oryzomys palustris and the isolates exhibited markedly different levels of infectivity. This study demonstrates that B. burgdorferi sensu stricto is present and naturally transmitted on the Outer Banks and in the Coastal Plain and Piedmont regions of North Carolina.     

Genetic structure in the European endemic seabird,Phalacrocorax aristotelis,shaped by a complex interaction of historical and contemporary,physical and nonphysical drivers

Geographically separated populations tend to be less connected by gene flow, as a result of physical or nonphysical barriers preventing dispersal, and this can lead to genetic structure. In this context, highly mobile organisms such as seabirds are interesting because the small effect of physical barriers means nonphysical ones may be relatively more important. Here, we use microsatellite and mitochondrial data to explore the genetic structure and phylogeography of Atlantic and Mediterranean populations of a European endemic seabird, the European shag, Phalacrocorax aristotelis, and identify the primary drivers of their diversification. Analyses of mitochondrial markers revealed three phylogenetic lineages grouping the North Atlantic, Spanish/Corsican and eastern Mediterranean populations, apparently arising from fragmentation during the Pleistocene followed by range expansion. These traces of historical fragmentation were also evident in the genetic structure estimated by microsatellite markers, despite significant contemporary gene flow among adjacent populations. Stronger genetic structure, probably promoted by landscape, philopatry and local adaptation, was found among distant populations and those separated by physical and ecological barriers. This study highlights the enduring effect of Pleistocene climatic changes on shag populations, especially within the Mediterranean Basin, and suggests a role for cryptic northern refugia, as well as known southern refugia, on the genetic structure of European seabirds. Finally, it outlines how contemporary ecological barriers and behavioural traits may maintain population divergence, despite long‐distance dispersal triggered by extreme environmental conditions (e.g. population crashes).     

Mate recognition in a freshwater fish: geographical distance, genetic differentiation, and variation in female preference for local over foreign males

It often is assumed that more distant allopatry should reflect reduced rates of contemporary gene flow and/or greater divergence in mate recognition systems. This assumption, however, is rarely tested and may not always be appropriate. Here we investigated female preference for local and foreign males in a morphologically variable Australian freshwater fish, the Pacific blue-eye Pseudomugil signifer. Using a multidisciplinary approach that combined molecular phylogeography with conventional mate choice experiments, we found female blue-eyes spent more time in association with local males only when the alternative was a foreigner from a geographically and genetically more distant population. When offered the choice between two foreign males, females associated more with males from the population that was more closely adjacent to their own. Our results suggest that female preference for local over foreign males in blue-eyes may depend on how genetically and geographically separated populations are from one another.     

Comparative genetic analyses of historical and contemporary collections highlight contrasting demographic histories for the bumble bees Bombus pensylvanicus and B. impatiens in Illinois

Direct comparison of genetic patterns between museum specimens and contemporary collections can be a powerful approach for detecting recent demographic changes. Using microsatellite markers, we examined historical and contemporary genetic variation from an apparently declining bumble bee species, Bombus pensylvanicus , and from a stable species, Bombus impatiens , in central Illinois. For each species, we genotyped specimens from the Illinois Natural History Survey collected from three populations between 1969–1972 and from a resurvey of the same areas conducted in 2008. Population structure in B . pensylvanicus increased markedly over the last four decades (from θ_ST = 0.001 to 0.027) while no structure was detected in B . impatiens for either time period (θ_ST = –0.006 to –0.003). Changes in genetic diversity were not significant for either species, although small reductions were observed for B . pensylvanicus in all three populations. Coalescent simulations incorporating both contemporary and historical samples suggest that this small change is not surprising for recent population declines, as large reductions in genetic diversity were only apparent under the most severe bottleneck scenarios. These results demonstrate how comparisons of genetic patterns between temporal periods and species can help elucidate potential threats to population health and suggest several strategies that might be useful in the conservation of B . pensylvanicus in the Midwestern USA.     

Population genetic structure of a common host predicts the spread of white‐nose syndrome,an emerging infectious disease in bats

Landscape complexity influences patterns of animal dispersal, which in turn may affect both gene flow and the spread of pathogens. White‐nose syndrome (WNS) is an introduced fungal disease that has spread rapidly throughout eastern North America, causing massive mortality in bat populations. We tested for a relationship between the population genetic structure of the most common host, the little brown myotis (Myotis lucifugus), and the geographic spread of WNS to date by evaluating logistic regression models of WNS risk among hibernating colonies in eastern North America. We hypothesized that risk of WNS to susceptible host colonies should increase with both geographic proximity and genetic similarity, reflecting historical connectivity, to infected colonies. Consistent with this hypothesis, inclusion of genetic distance between infected and susceptible colonies significantly improved models of disease spread, capturing heterogeneity in the spatial expansion of WNS despite low levels of genetic differentiation among eastern populations. Expanding our genetic analysis to the continental range of little brown myotis reveals strongly contrasting patterns of population structure between eastern and western North America. Genetic structure increases markedly moving westward into the northern Great Plains, beyond the current distribution of WNS. In western North America, genetic differentiation of geographically proximate populations often exceeds levels observed across the entire eastern region, suggesting infrequent and/or locally restricted dispersal, and thus relatively limited opportunities for pathogen introduction in western North America. Taken together, our analyses suggest a possibly slower future rate of spread of the WNS pathogen, at least as mediated by little brown myotis.     

Borrelia burgdorferi sensu stricto invasiveness is correlated with OspC-plasminogen affinity

Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is transmitted through tick bite. Lyme borreliosis evolves in two stages: a primary red skin lesion called erythema migrans; later on, invasive bacteria disseminate to distant sites inducing secondary manifestations (neuropathies, arthritis, carditis, late skin disorders). It has been previously suggested that the ospC gene could be associated with invasiveness in humans depending on its sequence. Here, we confirm the pattern of invasiveness, according to B. burgdorferi sensu stricto (B. b. ss) ospC group, using the mouse as an experimental host of B. b. ss. As it has been shown that the host plasminogen activation system is used by B. burgdorferi to disseminate throughout the host, we studied the interaction of plasminogen with OspC proteins from invasive and non-invasive groups of B. b. ss. Using two methods, ELISA and surface plasmon resonance, we demonstrate that indeed OspC is a plasminogen-binding protein. Moreover, significant differences in binding affinity for plasminogen are correlated with different invasiveness patterns in mice. These results suggest that the correlation between ospC polymorphism and Borrelia invasiveness in humans is linked, at least in part, to differences in OspC affinity for plasminogen.