Genetic variation in the mitochondrial 16S rRNA gene of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

Genetic variation in the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene was examined for the American dog tick, Dermacentor variabilis (Say, 1821). Nine different haplotypes were detected among 369 adult D. variabilis collected from four localities in Canada. There were eight variable nucleotide positions in the 404 bp sequence alignment. Individuals of haplotype 1 occurred at frequency of >75% at all localities. Five haplotypes were detected at only one of the four localities. High haplotype diversity and low nucleotide diversity, combined with significantly negative Fs values for ticks at three localities, suggest a recent population expansion. Genetic differences were found between populations at different localities, but a Mantel regression analysis revealed no association between genetic differences and geographical distances. There was also no association between tick haplotype and the prevalence of the bacterium, Rickettsia montanensis Weiss and Moulder, 1984, in D. variabilis among localities or on opposite sides of Blackstrap Lake (Saskatchewan). The 16S rDNA haplotypes from Canadian populations of D. variabilis formed a clade with those from the eastern and central U.S.A., to the exclusion of D. variabilis from geographically isolated populations in the western U.S.A. Although sample sizes for D. variabilis in the eastern U.S.A. are small, there may be genetic divergence between populations in Canada and those in the eastern U.S.A., which may have implications for studies on the pathogenic agents transmitted by D. variabilis to its hosts. The American dog tick, Dermacentor variabilis (Say, 1821), and the Rocky Mountain wood tick, Dermacentor andersoni Stiles, 1908, are important vectors of pathogenic bacteria to humans, domestic animals, and wildlife. These pathogens include Rickettsia rickettsii (Wolbach, 1919), the causative agent of Rocky Mountain spotted fever, Anaplasma marginale Theiler, 1910, the agent responsible for bovine anaplasmosis, and Francisella tularensis (McCoy and Chapin, 1912), the causative agent of tularemia (Azad and Beard 1998, Treadwell et al. 2000, de la Fuente et al. 2001, Goethert et al. 2004, Farlow et al. 2005, Chapman et al. 2006, Clay et al. 2006, Goethert and Telford 2009). Several species of endosymbiotic bacteria have also been detected in D. variabilis and D. andersoni (see Bell et al. 1963, Feng et al. 1980, Burgdorfer et al. 1981, Clay et al. 2006, Dergousoff et al. 2009). The presence of some non-pathogenic bacteria in ticks can interfere with the acquisition and/or transmission of pathogenic bacteria (Burgdorfer et al. 1981, Clay et al. 2006). The prevalence of pathogenic and endosymbiotic bacteria vary considerably throughout the distributional ranges of their vectors, even between tick populations separated by relatively short distances. For example, reported prevalences of R. rickettsii in D. variabilis range from 0.1% in Ohio (Pretzman et al. 1990) to 8.6% in Maryland, U.S.A. (Schriefer and Azad 1994), whereas this pathogenic bacterium was not detected in D. variabilis from 13 localities in southern Canada (Dergousoff et al. 2009). The prevalence of the endosymbiont Rickettsia montanensis Weiss and Moulder, 1984 also varies considerably (0–33%) in D. variabilis populations from the northeastern U.S.A. and southern Canada (Feng et al. 1980, Pretzman et al. 1990, Ammerman et al. 2004, Dergousoff et al. 2009). At Blackstrap Lake, near the northwestern distributional limit of D. variabilis in Saskatchewan (Canada), the prevalence of R. montanensis in ticks on the western shore was markedly higher (39% vs 4%) than on the eastern shore (Dergousoff et al. 2009). The American dog tick occurs throughout the eastern and central U.S.A. and in parts of California, Idaho, Oregon, Mexico, and Canada (Bishopp and Trembley 1945, Gregson 1956, Wilkinson 1967, Stout et al. 1971, Sonenshine 1979, Rand et al. 2007). Its distributional range in Canada extends from central Saskatchewan eastward to southern Manitoba and Ontario, with disjunct populations in Nova Scotia (Gregson 1956, Wilkinson 1967, Dodds et al. 1969, Garvie et al. 1978, Campbell and MacKay 1979, Burachynsky and Galloway 1985). D. variabilis was introduced into Nova Scotia from the U.S.A. on imported dogs around 1900 (McEnroe 1985), and subsequently expanded its range in the province during the 1940s (Dodds et al. 1969). The distributional limits of D. variabilis are related to climatic factors (i.e., temperature) and the physiological limitations of the different life cycle stages (Wilkinson 1967, Sonenshine 1979). It was suggested by McEnroe (1978) that D. variabilis populations in marginal areas for survival and development (i.e., near the species distributional limits) would be localized into discrete populations and that there would be no gene flow between these populations. It was further hypothesized that these isolated populations could respond to climatic selection and that they would genetically diverge from populations in more central parts of the species' range (McEnroe 1978). Other factors, such as genetic drift, can also reduce genetic variation in marginal populations (Eckert et al. 2008, Kawecki 2008). Unfortunately, there is little information on the genetic variation of D. variabilis from different parts of its distributional range. The mitochondrial (mt) 16S ribosomal RNA (rRNA) gene has been used as the target for most population genetic studies of ixodid ticks (Rich et al. 1995, Norris et al. 1996, Norris et al. 1997, Crosbie et al. 1998, Qiu et al. 2002, de la Fuente et al. 2005, Mixson et al. 2006, Patterson et al. 2009, Trout et al. 2009). It has been demonstrated that the black-legged tick, Ixodes scapularis Say, 1821, comprises two distinct lineages (i.e., clades) based on analyses of the 16S rRNA gene (Rich et al. 1995, Norris et al. 1996, Qiu et al. 2002). One lineage is restricted to the southeastern U.S.A., whereas the other occurs both in southeastern U.S.A. and to the north (Rich et al. 1995, Norris et al. 1996, Qiu et al. 2002). There are differences between ticks of these two lineages in their physiology and ecology (Rich et al. 1995) and in the prevalence of infection with Borrelia burgdorferi Johnson et al., 1984, the agent of Lyme borreliosis (Qiu et al. 2002). It has been proposed that differences in prevalence of B. burgdorferi infection among geographical areas are associated with genetic and ecological factors involving the ticks (Qiu et al. 2002). Other studies have also examined if different populations or “strains” of other tick species differ in their infection levels or vector competence in the transmission of pathogenic bacteria to hosts (de la Fuente et al. 2005, Scoles et al. 2005). Published sequences of the mt 16S rRNA gene for D. variabilis are limited to a few individuals in the United States (Caporale et al. 1995, Crosbie et al. 1998, de la Fuente et al. 2001, Scoles 2004). The aim of this study was to determine the extent of genetic variation in this gene for D. variabilis from different localities. In addition, we examined whether there was an association between infection with R. montanensis and the 16S haplotype of individual ticks from different localities, and from the eastern and western shores of Blackstrap Lake. Adult D. variabilis were collected at four localities in Canada (Figure 1): Saskatchewan Landing Provincial Park, Saskatchewan (50°38′N, 107°57′W), near the shores of Blackstrap Lake, Saskatchewan (51°47′N, 106°25′W), Minnedosa, Manitoba (50°14′N, 99°50′W), and Kenora, Ontario (49°45′N, 94°29′W). Saskatchewan Landing Provincial Park is an area of mixed prairie grassland. Minnedosa is located in aspen parkland, while Kenora is situated within the boreal shield ecozone consisting of coniferous forest. Blackstrap Lake (14.5 km long and 0.8 km wide) is situated near a transition area between mixed prairie grassland and aspen parkland. Mixed grass prairie covers the western slope of the lake, with woody vegetation primarily associated with drainage flows down the valley slope, while the eastern slope has more continuous wooded areas. The trees and shrubs (Salix, Prunus, Populus, Cornus, Alnus, and Rosa spp.) in these wooded areas are similar for both sides of the lake. A causeway (500 m long) connects the two sides of the lake. Ticks collected from Blackstrap were divided into those from the eastern and western sides of the lake based on differences in the prevalence of R. montanensis infection (Dergousoff et al. 2009). The species identity of each tick collected was determined using the morphological criteria of Gregson (1956). Genomic DNA (gDNA) was extracted and purified from individual ticks using the methods described by Dergousoff and Chilton (2007). Given that D. andersoni also occurs in Saskatchewan Landing Provincial Park (Dergousoff and Chilton 2007, Dergousoff et al. 2009), the identity of all adult D. variabilis from this locality was confirmed using the molecular assay of Dergousoff and Chilton (2007). Approximate distributional range of D. variabilis in the USA and Canada based on Bishopp and Trembley (1945), Wilkinson (1967), Stout et al. (1971), Sonenshine (1979), and Rand et al. (2007) and the four localities in Canada (A = Saskatchewan Landing, B = Blackstrap, C = Minnedosa, and D = Kenora) from where adult ticks were collected for this study. The distribution of D. variabilis in Saskatchewan (SK) has expanded further to the west since the study of Wilkinson (1967) and includes several breeding populations (Dergousoff & Chilton, unpublished data). Also shown are localities in the U.S.A. (solid circles) from where mt 16S rDNA sequence data are available on Genbank for D. variabilis. These localities are Portland, Maine (Caporale et al. 1995), Payne County, Oklahoma, Gainsville, Florida (de la Fuente et al. 2001), Placer County and Monterey, California (Crosbie et al. 1998, Scoles 2004), Nez Perce County and near Lewiston, Idaho (Scoles 2004, de la Fuente et al. 2001), Pullman and Dusty, Washington (Scoles 2004), and unspecified localities in Virginia (de la Fuente et al. 2001) and Kansas (Black and Piesman 1995). For each adult D. variabilis, ≈450 bp of the mt 16S rRNA gene was amplified by PCR from gDNA using primers 16S-1 (5′-CCACAGCAATTTAAAAAATCATTGAGCAG-3′) and 16S+1 (5′-CCGGTCTGAACTCAGATCAAGT-3′) (Norris et al. 1996). PCRs were performed in 50 μl volumes containing 200 μM of each dNTP, 1.8 mM MgCl2, 50 pmol of each primer, and of DNA using a with the C for of C for C for and C for by C for gDNA (i.e., were in each PCR were to on mM mM mM were to analyses et al. 2006). was used to DNA sequences that differ by one or more was mixed with and μl of The were at C for in for sample was into the of and to for at and C in a were for with in and using a of the different were PCR and to DNA using primers 16S-1 and 16S+1 in sequence data have been in the The et al. was used to the haplotype diversity and nucleotide diversity of D. variabilis from each locality and the data where is the that two individuals not have the and is the of nucleotide differences between two sequences and The D of and the Fs of were using a of to for D is by the of in to the of nucleotide differences between DNA sequences the D be D values are by population whereas significantly negative values are to population and 1996). Fs values are by if there is an of recent (i.e., of The Fs of are more to from to population et al. 2005). Fs values are if 1997, et al. 2005). A 1990) was also performed using to determine if there were significantly more haplotypes in each population than was also used to a of genetic between each of localities and to the of from using A Mantel was using to determine if there was a between geographical and genetic among localities. also examined if there was an association between tick haplotype and infection with R. montanensis among localities and on opposite sides of Blackstrap Lake. The data for R. montanensis infection in individual ticks were from the PCR analyses of the gene by Dergousoff et al. The et al. was used to a to among the different D. variabilis mt 16S rDNA Also in this analysis were 13 mt 16S rDNA sequences of the D. variabilis from the U.S.A. and (Black and Piesman et al. 1995, Crosbie et al. 1998, de la Fuente et al. 2001, Scoles 2004). A analysis was also on the sequence data using the in The 16S sequence of D. andersoni Patterson et al. was used to the A analysis was used to determine the of the different in the A was detected on for the mt 16S rDNA of individual D. variabilis whereas no were detected for negative (i.e., no sample not there were differences among in their (i.e., on (Figure The of from two to and there were differences in the of on an The of were on different A of different were detected among the 369 adult D. variabilis the were to Individuals with the an 16S whereas those with a different each a different sequence (i.e., There were eight in the sequence and these two and an of the sequences of the haplotypes revealed differences at one to three nucleotide positions The of differences between haplotypes was of the mt 16S rDNA of eight adult D. variabilis. The of four of the haplotypes and are shown on this The of the mt 16S rDNA haplotypes at the four localities are shown in The of haplotypes locality from two Landing Provincial to one haplotype was detected at all four localities, and occurred at a frequency of Five haplotypes and were detected at only one of the four localities, and each of the at that The haplotype diversity was to be at each while the nucleotide diversity of D. variabilis was low The of the D for for three localities Minnedosa and there were negative D the of an of recent each population was not significantly different from In the D for the data in a negative D The on the data for Blackstrap, Minnedosa, and Kenora each in negative Fs values that significantly from an of recent (i.e., and population expansion. The Fs for Saskatchewan Landing a population but there was no from The of the also that there were significantly more haplotypes than for the populations at Blackstrap and There were as haplotypes than for the population at Kenora, but this was not There was also no between the and of haplotypes for the population at Saskatchewan of genetic revealed differences among ticks from the four localities, between those of Kenora and Minnedosa the of the Mantel values between localities revealed no association between genetic and geographical was also of the population genetic for D. variabilis from the western and eastern sides of Blackstrap Lake where there was a in the of ticks with R. the adult ticks collected on the eastern of the lake, were of haplotype 1 and two were of haplotype whereas of the individuals collected on the western were of haplotype were of haplotype and three were of haplotype Although the haplotype and nucleotide were there were genetic differences in the population of ticks on the western and eastern sides of Blackstrap Lake. There were no D. variabilis individuals of haplotypes and that R. some ticks of haplotype 1 from the west and one from the were with R. the of D. variabilis individuals of each haplotype at each locality that were by R. the D. variabilis with R. most were of haplotype the was an individual from Kenora that was of haplotype A the of the D. variabilis haplotypes detected within Canada and the different sequence from the U.S.A. (Caporale et al. 1995, Crosbie et al. 1998, de la Fuente et al. 2001, Scoles is shown in The different D. variabilis haplotypes can be divided into two haplotypes from the four Canadian localities to the clade as those from the central and eastern U.S.A. haplotype in this clade by one to four whereas they all from individuals in the western U.S.A. by at (Figure The by the analysis of the 16S sequence data also in the of the haplotypes into the two with values of as for the A of the 16S haplotypes of D. variabilis detected in Canada. Also are sequence of individual ticks from Florida and and Kansas Virginia and and Maine California and and Washington and and and Idaho and and (Black and Piesman et al. 1995, Crosbie et al. 1998, de la Fuente et al. 2001, Scoles 2004). 1 is in sequence to the two individual ticks from and The areas of the each haplotypes for haplotype 1 which comprises of all ticks is to the of individuals with that between two haplotypes the of nucleotide differences in DNA have been divided into two based on the extent of the sequence differences between ticks from different geographical of the ecology of a of the population of the vector et al. 2006). the geographical distribution of D. variabilis in (Wilkinson 1967, Sonenshine and the of this tick species as a vector of pathogenic bacteria to and domestic (de la Fuente et al. 2001, Farlow et al. 2005, Chapman et al. little or no information is available on the extent of genetic variation in D. variabilis throughout parts of its range. In the different haplotypes of the mt 16S rRNA gene were detected among 369 adult D. variabilis collected from Saskatchewan Landing Provincial Park and near Blackstrap Lake Minnedosa and Kenora localities in southern Canada where the American dog tick the limit of its range (Wilkinson These localities are situated in different (i.e., mixed prairie aspen parkland, and the boreal and are separated by of to There are also differences between these localities in of adult D. variabilis with R. montanensis Ticks near Blackstrap Lake have the prevalence of R. montanensis infection in southern Canada the frequency of R. montanensis in D. variabilis on the western shore is than on the eastern shore (Dergousoff et al. 2009). genetic differences were detected between ticks on of the lake, which can be to the presence of haplotypes of low frequency one on the eastern and two and on the western there was no association found between the of R. montanensis in individual ticks at Blackstrap and their mt 16S DNA haplotype all ticks were of haplotype and this haplotype of the ticks on both sides of the lake. data from Blackstrap, Saskatchewan Landing, Minnedosa, and Kenora were all but one individual of the D. variabilis with R. montanensis were of haplotype 1 was the most D. variabilis haplotype at all four Canadian localities, at a frequency of to 1 has also been in the U.S.A. from one adult D. variabilis in Kansas (Black and Piesman 1994), and in (de la Fuente et al. The other eight haplotypes detected in the Canadian populations of D. variabilis have not been reported in the U.S.A. Five of these haplotypes were detected at only a locality and occurred at a low There were genetic differences on analyses of the between localities, between Minnedosa and Kenora, which are separated by a of the of the Mantel that there was no association between the genetic differences and geographical between populations. and gene flow between populations at Minnedosa and Kenora is an for their genetic D. variabilis are relatively short distances. D. variabilis have been found to than m et al. while and have more restricted of their of to as a of their et al. 1979). of ticks be more on the and ecology of the hosts they D. variabilis and on a of and (Gregson 1956, Dodds et al. 1969, Campbell and MacKay 1979, Burachynsky and Galloway et al. These hosts would also ticks relatively distances. The and of other tick species can be relatively long (i.e., of by et al. 2008). There are a few isolated where D. variabilis have been collected from for example, in of ticks from et al. and of ticks from et al. in the study by et al. there was no of D. variabilis on of the collected from in southern Canada. is that the of D. variabilis of of ticks on or hosts. Adult D. variabilis a of to and (Gregson 1956, Dodds et al. 1969, et al. some of which have ranges and limited to ticks distances. The limited of D. variabilis may an as to R. montanensis infection in adult ticks may be localized to localities (Dergousoff et al. 2009). is by the of dogs and It has been suggested that dogs have been responsible for the of D. variabilis into 1979, McEnroe Farlow et al. 2005). There was haplotype diversity but low nucleotide diversity for D. variabilis at all four localities is for species that have a range where the population is an of haplotypes sequence differences and A for species with populations is the presence of a few haplotypes with low frequency (i.e., haplotypes localized to populations and 2006). a was detected for D. variabilis populations in southern Canada. There was no of selection haplotype based on analyses using negative D values were for three localities Minnedosa, and In the significantly negative Fs values at these three localities are of population and 1996). The of haplotypes for D. variabilis at Blackstrap Lake and Minnedosa were also significantly than The D values to and Fs values to for D. variabilis from Blackstrap, Minnedosa, and Kenora within the ranges of the values = to and to for populations of the tick, in that are to a recent range et al. 2006). population for D. variabilis, a is a for those populations near Blackstrap, D. variabilis was not from this area of Saskatchewan in studies to the (Gregson 1956, Wilkinson the of breeding populations of D. variabilis further west of Blackstrap, such as those to Saskatchewan Landing Provincial Park (Dergousoff and Chilton 2007, Dergousoff et al. 2009), are also The of haplotypes was detected in D. variabilis from Saskatchewan Landing Provincial Park, a locality near its northwestern distributional Although the D and Fs values for Saskatchewan Landing were a population they were not significantly different from selection 1997, The two haplotypes detected at Saskatchewan Landing were also found at Blackstrap Lake, D. variabilis of haplotype a of the ticks at Saskatchewan Landing but occurred at a low frequency at The Rocky Mountain wood tick, D. also occurs in Saskatchewan Landing Provincial Park (Dergousoff et al. 2009, Patterson et al. 2009), where is its northeastern distributional limit in and nucleotide for D. andersoni at Saskatchewan Landing Provincial Park and et al. were than for D. variabilis and The and of the genetic diversity in D. variabilis in populations near the of the species range to be further using other genetic A analysis and a analysis of the D. variabilis haplotypes detected in Canada and the 16S rDNA sequences of 13 D. variabilis from the U.S.A. (Black and Piesman et al. 1995, Crosbie et al. 1998, de la Fuente et al. 2001, Scoles both in of the haplotypes into two which from one by at of D. variabilis into two is with the of de la Fuente et al. One clade D. variabilis haplotypes from localities in Ontario, Oklahoma, and all of which within the distributional range of this tick species in the eastern of 1979). 1 the of the for clade A with all other haplotypes from by one or two The other clade the two D. variabilis haplotypes from the western U.S.A. and populations that are geographically isolated from those in central and eastern 1979). It has been suggested by Farlow et al. that D. variabilis may have been introduced into in California on dogs during the of the or Genetic divergence of the geographically isolated D. variabilis populations in the western U.S.A. from those in central and eastern may in the of genetic divergence between these two mitochondrial lineages of D. variabilis is similar to that between two mitochondrial lineages of differences (Qiu et al. which a based on sequence of ribosomal DNA et al. The of the study by Crosbie et al. on the mt 16S rRNA gene of eight Dermacentor species also revealed that D. variabilis one from Kansas and a California, from A and formed a clade to the exclusion of the other species in the The of the two mitochondrial lineages of D. variabilis to be further using There may also be genetic divergence between populations in Canada and the central U.S.A., and those on the (i.e., within clade Although sizes are for the eastern and central U.S.A., the most haplotype in populations from southern Canada has not been reported in the of and Florida (Caporale et al. 1995, de la Fuente et al. the sequence of two individuals from and Kansas in the central U.S.A. were haplotype 1 (Black and Piesman de la Fuente et al. The haplotypes reported from the eastern U.S.A. and were not detected in population from southern Canada. sequences of the mt 16S rRNA gene are for significantly more individual ticks from the eastern populations to if there is genetic divergence between populations from different in central and eastern have been for D. variabilis (see et al. 2009). These genetic in with the mt 16S may important into the genetic divergence and of D. variabilis populations throughout its distributional range. have important implications for studies on the different of pathogenic agents transmitted by the American dog tick to humans, domestic animals, and wildlife. are to and some of the ticks used in this study. also the for their on the for this was to from the and of Canada and the Canadian for also to and and

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