Phylogenetic and phylogeographic relationships in Ixodes holocyclus and Ixodes cornuatus (Acari: Ixodidae) inferred from COX1 and ITS2 sequences

We inferred the phylogenetic and phylogeographic relationships in ticks, which were identified morphologically as Ixodes holocyclus and Ixodes cornuatus, from mitochondrial cytochrome oxidase subunit 1 (COX1) and nuclear internal transcribed spacer 2 (ITS2) sequences. We obtained COX1 (640bp) and ITS2 (527-568bp) sequences from 429 ticks from 49 localities in Tasmania, Victoria, New South Wales and Queensland, Australia. Our analyses show that there are two species of Ixodes in eastern Australia that cause paralysis in dogs and other mammals: I. holocyclus and I. cornuatus. Further, we conclude that the morphological criteria used to differentiate female I. holocyclus and I. cornuatus are equivocal but I. holocyclus can be distinguished from I. cornuatus using COX1 and/or ITS2 sequences. Intraspecific genetic variation in I. holocyclus and I. cornuatus was less than 0.86% and 0.19% for COX1 and ITS2, respectively. Ixodes holocyclus could be genetically distinguished between different geographic ranges. There were no significant genetic differences between I.cornuatus from Tasmania and mainland Australia, but there are some COX1 haplotypes of I. cornuatus from the mainland that were not detected in Tasmanian and vice versa.     

Migratory songbirds disperse ticks across Canada, and first isolation of the Lyme disease spirochete, Borrelia burgdorferi, from the avian tick, Ixodes auritulus

During a 3-yr comprehensive study, 196 ixodid ticks (9 species) were collected from 89 passerine birds (32 species) from 25 localities across Canada to determine the distribution of avian-associated tick species and endogenous Lyme disease spirochetes, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, and Brenner. We report the following first records of tick parasitism on avian hosts: the rabbit-associated tick, Ixodes dentatus Marx, from Manitoba and Ontario; the mouse tick, Ixodes muris Bishopp and Smith, from British Columbia; and the blacklegged tick, Ixodes scapularis Say, from New Brunswick. Moreover, we provide the first record of the Neotropical tick, Amblyomma humerale Koch (1 nymph), in Canada and its parasitism of any bird. This tick was compared morphologically with nymphs of other Neotropical Amblyomma spp., and genetically, using a 344-bp fragment of the 12S rDNA sequence of 41 New World Amblyomma species. The first collections of the western blacklegged tick, Ixodes pacificus Cooley and Kohls, from passerine species in Alberta and British Columbia, are also reported. Notably, we further report the first isolation of B. burgdorferi from the bird tick, Ixodes auritulus Neumann, collected from an American robin, Turdus migratorius L., on Vancouver Island. Furthermore, B. burgdorferi-positive I. auritulus larvae were collected from a reservoir-competent fox sparrow, Passerella iliaca (Merrem). Our findings indicate that ground-dwelling passerines, in particular, are parasitized by certain ixodid ticks and play an important role across Canada in the wide dispersal of B. burgdorferi-infected ticks and increased risk of Lyme disease exposure.     

Seasonal patterns of abundance and host relationships of the Australian paralysis tick,Ixodes holocyclus Neumann (Acarina: Ixodidae), in southeastern Queensland

Estimates of seasonal abundance of larvae, nymphs and females of the paralysis tick, Ixodes holocyclus, were obtained by collecting ticks that engorged on small mammals and birds trapped in two localities in southeastern Queensland: Brisbane (wet sclerophyll forest) and Tamborine Mountain (cleared rain forest). The long-tailed short-nosed bandicoot, Isoodon macrourus, was the most common mammal trapped but small numbers of other marsupials, rodents and ground frequenting birds were also captured. Small numbers of five other tick species were also collected. In both habitats there was clearly one dominant generation of I. holocyclus;7er year, although the presence of all stages at most times of the year indicated overlapping of smaller cohorts. Females were most abundant in spring and early summer, larvae in summer-autumn, and nymphs in autumn-winter. I. holocyclus was abundant on I. macrourus and rare on most other mammals and birds captured. At the peak of abundance of each instar, each bandicoot dropped from 500 to 2000 engorged larvae, 100 to 200 engorged nymphs, and four to six engorged females. Life tables were compiled for the tick in both habitats and these indicate that there was relatively high survival from engorged larva to engorged nymph and thence to engorged female and that most mortality occurred between detaching of the engorged female and the detaching of the engorged larva. The tick was more abundant on bandicoots from cleared rain forest and rain forest edge, than on those from sclerophyll woodland. The survival of engorged larvae and nymphs of I. holocyclus and the larval productivity of engorged females were examined in a warm moist climate where the tick was abundant (Tamborine Mountain) and in a hotter dryer climate where the tick was rare or absent (Amberley). In both localities engorged larvae and nymphs survived to the next instar in all seasons of the year. On most occasions engorged females produced eggs which hatched. Mature bandicoots from tick infested areas showed little or no resistance to infestation with larvae or nymphs of I. holocyclus, whereas other small mammals from the same area showed an appreciable degree of resistance to the immature stages of the tick. Feeding larvae and nymphs exposed to normal light-dark cycles in the laboratory detached during the afternoon and early evening. This behaviour and host resistance are discussed in relation to the daily activity cycles of host species, their habitat preferences, and their role as hosts for I. holocyclus.     

The isolation of Saumarez Reef virus, a new flavivirus, from bird ticks Ornithodoros capensis and Ixodes eudyptidis in Australia.

Strains of a new flavivirus, for which the name Saumarez Reef Virus is proposed, were isolated from seabird ticks collected from four localities. Two strains were isolated from ticks of the species Ornithodoros capensis Neumann 1901 collected from the nests of Sooty Terns, Sterna fuscata Linnaeus 1766 on coral cays off the east coast of Queensland, Australia. The other three strains were isolated from ticks of the species Ixodes eudyptidis Maskell 1885 taken from two dead Silver Gulls Larus novaehollandiae Stephens 1826 in northern Tasmania. The new virus was compared serologically with 50 other flaviviruses at the Yale Arbovirus Research Unit and was found to be most closely related to Tyuleniy virus.     

Body size and shape evolution in host races of the tick Ixodes uriae

The tick Ixodes uriae is a common ectoparasite of seabirds, and is widely distributed across the circumpolar regions of both hemispheres. Previous work demonstrated the existence of genetically distinct host races of this ectoparasite, occurring across its current range. The objective of the present study was to examine whether these host races have evolved measurable morphological differences. We measured a set of morphological variables on 255 non‐engorged ticks (nymphs and adults) collected from three sympatrically occurring host species in the North Atlantic. Genotyping at eight microsatellite markers enabled us to analyse the relationship between patterns of morphological and neutral genetic variation. Multivariate analyses showed that most morphological variation was associated with size differences among tick individuals. Body size differed among races, but only in adult life stages. A linear discriminant analysis based on shape variation revealed three distinct morphological clusters corresponding to the three tick host races. These results, along with correlated patterns of host‐related genetic variation, suggest that differences among host‐related groups are not simply the result of phenotypic plasticity or drift, but rather reflect host‐associated adaptations. Experimental work and observations across the range of I. uriae will now be required to test the genetic basis and adaptive nature of morphological differences.     

Species identification of <Emphasis Type="Italic">Ixodes granulatus</Emphasis> (Acari: Ixodidae) based on internal transcribed spacer 2 (ITS2) sequences

To investigate the genetic specificity of Ixodes granulatus ticks collected from Taiwan, the genetic identities and phylogenetic relationships were analyzed by comparing the sequences of the internal transcribed spacer 2 (ITS2) region obtained from 27 strains of ticks representing twelve species of Ixodes. Five major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks collected from Taiwan and Japan were genetically affiliated to a monophyletic group with highly homogeneous sequences (95.8–99.5% similarity), and can be discriminated from other species and subgenera of Ixodes ticks with a sequence divergence ranging from 13.6% to 62.9%. Moreover, interspecific analysis revealed that four distinct lineages are evident between Ixodes ticks, and all these I. granulatus ticks collected from Taiwan and Japan belong to the same lineage. Our results provide the first investigation on the genetic specificity of I. granulatus ticks, and demonstrate that all these I. granulatus ticks represent a unique lineage distinct from other species and subgenera of Ixodes ticks. The feasibility of ITS2-based genetic analysis for species-specific identification of I. granulatus ticks around East Asia was highly anticipated.     

Molecular analysis of <Emphasis Type="Italic">Ixodes granulatus</Emphasis>, a possible vector tick for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in Taiwan

The genetic identity of Ixodes granulatus ticks was determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 19 strains of ticks representing seven species of Ixodes and two outgroup species (Rhipicephalus sanguineus and Haemaphysalis inermis). Four major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks of Taiwan were genetically affiliated to a monophyletic group with highly homogeneous sequences (92.2–99.3% similarity), and can be discriminated from other Ixodes species and other genera of ticks with a sequence divergence ranging from 11.7 to 30.8%. Moreover, intraspecific analysis revealed that two distinct lineages are evident between the same species of I. granulatus ticks collected from Taiwan and Malaysia. Our results demonstrate that all these I. granulatus ticks of Taiwan represent a unique lineage distinct from the common vector ticks (I. ricinus complex) for Borrelia burgdorferi spirochetes.     

Phylogeny and form in fishes: Genetic and morphometric characteristics of dragonets (Foetorepus sp.) do not align

Measures of biodiversity are often hindered by a lack of methodological practices that distinguish cryptic or morphologically similar cohabiting species. This is particularly difficult for marine fishes where direct observations of the ecology and demography of populations are difficult. Dragonets (Foetorepus c.f. calauropomus) were collected as bycatch from research trawls deployed in waters off north-eastern Tasmania, Australia. Morphometric and genetic analyses were conducted on the 43 specimens recovered. Sequence analysis of two mitochondrial loci distinguished three genetic clusters, each having levels of dissimilarity consistent with species-level distinctions between other members of the Callionymidae. While clear morphological distinctions were observed between male and female fish, limited morphometric analyses could not differentiate between members of the three genetic groups. This finding highlights questions about the ability of genetically distinct but morphologically similar groups to occupy the same ecological niche, and points to additional and undescribed hidden biodiversity amongst cryptic species of fish.     

Glacial oceanographic contrasts explain phylogeography of Australian bull kelp

The evolutionary effects of Southern Hemisphere Pleistocene oceanographic conditions — marked by fluctuations in sea levels and water temperatures, and redirected currents — are poorly understood. The southeastern tip of Australia presents an intriguing model system for studying the biological impacts of palaeoceanography. In particular, contrasting oceanographic conditions that existed on eastern vs. western sides of the Bassian Isthmus during Pleistocene glacial periods allow for natural comparisons between putative refugial vs. re-invading populations. Whereas many western Tasmanian marine taxa were likely eliminated by cold subantarctic water during the last glacial period, eastern Tasmanian populations would have persisted in relatively warm temperatures mediated by the ongoing influence of the East Australian Current (EAC). Here we test for the effects of contrasting palaeoceanographic conditions on endemic bull kelp, Durvillaea potatorum , using DNA sequence analysis (COI; rbc L) of more than 100 individuals from 14 localities in southeastern Australia. Phylogenetic reconstructions reveal a deep (maximum divergence 4.7%) genetic split within D. potatorum , corresponding to the 'eastern' and 'western' geographical regions delimited by the Bassian Isthmus, a vicariant barrier during low Pleistocene sea levels. Concordant with the western region's cold glacial conditions, samples from western Tasmania and western Victoria are genetically monomorphic, suggesting postglacial expansion from a mainland refugium. Eastern samples, in contrast, comprise distinct regional haplogroups, suggesting the species persisted in eastern Tasmania throughout recent glacial periods. The deep east–west divergence seems consistent with earlier reports of morphological differences between 'western' and 'eastern' D. potatorum , and it seems likely that these forms represent reproductively isolated species.     

Cuticular hydrocarbon composition and phenotypic variability in sympatric populations of Ixodes ricinus ticks from Poland

Gas chromatography of cuticular hydrocarbons was used to assess genetic similarity and heterozygosity between 18 sympatric populations of adult Ixodes ricinus ticks collected from Poland. Sixteen n-alkanes, 45 monomethylalkanes, and 36 dimethylalkanes were identified. Forty-one compounds were detected in 90% of the specimens and nine hydrocarbons were present in all the specimens studied. Visual inspection of chromatograms revealed major differences in the relative abundance of hydrocarbons between populations of I. ricinus. The average proportion of heterozygotic hydrocarbons was 50.68%, while the average genetic similarity among all populations was 0.967. Two other methods were used to measure the intrapopulation variability: the number of isomeric forms for each hydrocarbon and the range of hydrocarbon abundance. It was found that some hydrocarbons manifested an unexpectedly high number of isomeric patterns, such as nonacosane (21 patterns), triacontane (23), pentacosane (26), and hexacosane (28). Our data suggest that I. ricinus ticks from Poland consist of several interbreeding, non-isolated populations, with a high degree of genetic flow between them.     

Isolation and characterization of ten polymorphic microsatellite loci in Ixodes arboricola, and cross-amplification in three other Ixodes species

We characterized ten polymorphic microsatellite loci from the tree-hole tick, Ixodes arboricola. Loci were screened in 11–18 individuals from three Belgian populations and five to ten alleles were found at each locus. Seven loci did not show deviations from Hardy–Weinberg equilibrium conditions and there were no indications for null alleles at these loci. The three other loci showed significant heterozygote deficiencies in at least one population, and a high potential for the occurrence of null alleles. We observed no effect of potential host DNA on the scoring of the microsatellites. Cross-amplification of the microsatellites was tested in eight specimens of three congeneric species: I. ricinus, I. hexagonus and I. frontalis. Depending on the species, six or seven of the loci were amplified in ≥4 of the 8 specimens and were polymorphic in each of these species (except for Ixaf 11 in I. frontalis and I. ricinus). These loci thus provide a tool for population genetic analysis of I. arboricola. The suitability of these markers needs to be further investigated in its congeners.     

Comparison of tick-borne microorganism communities in Ixodes spp. of the Ixodes ricinus species complex at distinct geographical regions

Characterizing the tick-borne microorganism communities of Ixodes ricinus (sheep tick) and Ixodes persulcatus (taiga tick) from the I. ricinus species complex in distinct geographical regions of Eastern Europe and European Russia, we demonstrated differences between the two ticks. Taiga ticks were more frequently mono- and co-infected than sheep ticks: 24.4 % (45/184 tested ticks) versus 17.5 % (52/297) and 4.3 % (8/184) versus 3.4 % (10/297), respectively. Ginsberg co-infection index values were significant at the various sites. Diversity of the tick-borne microorganism communities was estimated by the Shannon index, reaching values of 1.71 ± 0.46 and 1.20 ± 0.15 at the sheep-tick and the taiga-tick harbored sites, respectively. Richness of the tick-borne microorganism community in the sheep tick collection sites was about twice the value of the taiga tick collection sites. Future investigations are warranted to further characterize the peculiarities of the tick-borne microorganism communities among the ticks of the Ixodes ricinus complex.     

New Host and Locality Records for the Ixodes Auritulus (Acari: Ixodidae) Species Group, with a Review of Host Relationships and Distribution in the Neotropical Zoogeographic Region

New Neotropical records are presented for ticks belonging to the Ixodes auritulus Neumann, 1904, species group, together with a review of hosts and localities from which members of this complex have previously been collected. The range of the I. auritulus species group is now understood to include Colombia, and 15 bird species are listed as new hosts. From Guatemala to southern Argentina and Chile, specimens of the I. auritulus group have been found on birds belonging to the orders Ciconiiformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Piciformes, Procellariiformes and Tinamiformes. Passeriform birds are probably the principal hosts, sustaining tick populations throughout the Neotropics. Collection data have yielded four areas – southern South America (from 56° S to 51° S), southern Brazil (25° S–22° S), south-central Peru (14° S–10° S) and Central America (10° N–15° N) – where the I. auritulus group appears to commonly parasitize birds, but additional collections may show that the range of this complex is less discontinuous than currently perceived. Several morphological differences are described for ticks within and among these areas, but it is still unclear whether the I. auritulus group comprises more than one species.     

An electrophoretic comparison of Physaloptera Rudolphi, 1819 (Nematoda: Physalopteridae) from two species of Australian bandicoot (Marsupialia: Peramelidae)

An electrophoretic study was conducted on nematodes of the genus Physaloptera that occur in the stomachs of two species of Australian bandicoot, Perameles nasuta and P. gunnii. Each nematode was genetically characterised at 28 enzymes encoding a presumptive 30 loci. No fixed genetic differences were detected between the nematodes in P. gunnii from two localities. A comparison of nematodes from the two host species, however, revealed fixed genetic differences at 15 (50%) loci. This suggests that each host species is infected by a different species of Physaloptera.     

Some effects of tick infestations on juvenile northern brown bandicoot (Isoodon macrourus)

The effect of tick infestations on body weight and various blood parameters was monitored in juvenile northern brown bandicoots (Isoodon macrourus) after release into tick-infested or tick-free enclosures. Three species of ticks were observed in the enclosures, Haemaphysalis humerosa, Ixodes tasmani and Ixodes holocyclus. Bandicoots released into tick-infested enclosures showed a reduced growth rate (1.8 versus 2.5 g/day increase in body weight), a reduced haematocrit value (27.4 versus 40.0%) and an increased number of white blood cells when compared with bandicoots released into tick-free enclosures. These results suggest that tick infestations may influence the health of juvenile I. macrourus.     

VNTR loci reveal differentiation between and structure within populations of the eastern barred bandicoot Perameles gunnii

The Eastern Barred Bandicoot Perameles gunnii has declined in abundance within mainland south-eastern Australia, to a relict wild population of less than 100 individuals in Hamilton, Victoria. It is more common, but is also declining in Tasmania. Genomic DN A variability was compared within and between surviving populations of P. gunnii using variable number of tandem repeat (VNTR) markers in one of two ways. First, average percentage differences (APDs) were determined between profiles for two VNTR probe—endonuclease combinations. Secondly, because one of these combinations revealed two multiallelic VNTR loci, genotypes were assigned and analysed for homogeneity of allele frequencies among subpopulations, for deviation of heterozygosity from Hardy-Weinberg equilibrium within populations and for genetic structuring among individuals from different subpopulations. The results of both the APD and defined locus approaches showed consistent trends within and between populations. Genetic variability was higher among mainland P. gunnii than in Tasmanian populations (higher APDs, number of alleles, and heterozygosity at one locus), despite the known decline and subdivision of the Hamilton population. Eleven per cent of the variability detected in Hamilton was attributed to genetic differentiation between east and west subdivisions of the population. Departure from random mating indicating local inbreeding within collecting localities was evident for one locus in both north and south Tasmania, particularly at one locality. AH alleles at both loci were unique to either Hamilton or Tasmanian P. gunnii. The initial captive colony contains high heterozygosity for these loci. It is concluded that VNTR markers can be of benefit for use in studies of population differentiation and for conservation management.     

Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.     

Characterization of Borrelia Species Isolated from Ixodid Ticks,Ixodes ovatus

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.     

Molecular phylogenetic analyses indicate that the Ixodes ricinus complex is a paraphyletic group

The Ixodes ricinus species complex is a group of ticks distributed in almost all geographic regions of the world. Lyme borreliosis spirochetes are primarily transmitted by tick species within this complex. It has been hypothesized that the Lyme vector ticks around the world are closely related and represent a monophyletic group. This implies that vector competence in ixodid ticks for Lyme agents might have evolved only once. To test this hypothesis, we used a molecular phylogenetic approach. Two fragments of mitochondrial 16S ribosomal deoxyribonucleic acid were sequenced from 11 species in the I. ricinus complex and from 16 other species of Ixodes. Phylogenetic analysis using Bayesian methodology indicated that the I. ricinus complex is not a monophyletic group unless 3 additional Ixodes species are included in it. The known major vectors of Lyme disease agents in different areas of the world are not sister taxa. This suggests that acquisition of the ability to transmit borreliosis agents in species of Ixodes may have multiple origins.     

Dispersal patterns and population structuring among platypuses, Ornithorhynchus anatinus, throughout south-eastern Australia

Dispersal patterns can have a major impact on the dynamics and viability of populations, and understanding these patterns is crucial to the conservation and management of a species. In this study, patterns of sex-biased dispersal and waterway/overland dispersal are investigated in the endemic Australian platypus, Ornithorhynchus anatinus, a semi-aquatic monotreme. Analyses of over 750 individuals from south-eastern Australia at 13 microsatellite loci and two mitochondrial genes, cytochrome b and cytochrome oxidase subunit II, provide genetic insight into dispersal patterns. For the first time, platypuses of western Victoria are shown to be genetically distinct from other populations of the mainland. Despite distinct morphological differentiation either side of the Great Dividing Range, populations remain genetically similar between coastal and inland areas suggesting gene flow is likely to occur across these ranges. Landscape genetic analyses indicate variability in dispersal patterns between Victorian and Tasmanian platypuses with a greater avoidance of overland travel indicated in Victoria compared to Tasmania. Females appear to remain within their natal area or return to breed, maintaining greater genetic structure in maternally inherited mitochondrial DNA in comparison to nuclear DNA and sharing genetic similarity within a short river distance (i.e. ≤1.4 km). The results of this study provide a valuable spatial framework for the management of wild platypus populations within south-eastern Australia and a baseline for future monitoring of populations that are likely to be impacted by environmental and anthropogenic change.