Development and characterization of 12 polymorphic microsatellite loci in the American dog tick (Dermacentor variabilis)

Twelve polymorphic microsatellite loci were developed for the American dog tick (Dermacentor variabilis), an important vector of infectious diseases in humans and animals. Four multiplexed panels comprising the loci were developed and 45 ticks collected from two raccoons (Procyon lotor) were genotyped. The number of alleles per locus ranged from nine to 30, and single locus heterozygosities ranged from 0.18 to 0.93. Data generated using these markers will further our understanding of factors affecting gene flow in D. variabilis, thus helping to elucidate the transmission dynamics of diseases associated with this vector.     

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 F_s 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.     

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick,Dermacentor variabilis (Say)

Summary Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.     

Morphological evidence that salivary gland degeneration in the American dog tick,Dermacentor variabilis (Say), involves programmed cell death

During the preoviposition and oviposition periods of ixodid ticks, the salivary glands degenerate. It is unclear whether this is a necrotic or a programmed cell death event. We used an in situ TUNEL technique to determine if salivary gland degeneration involves apoptosis. Salivary glands were dissected from replete females at days 3, 5, 8, 11, 13, and 33 post-detachment. There were no differences in tick weight at detachment, suggesting that changes were not due to engorgement abnormalities. The onset of apoptosis began at day 5 and continued through oviposition at day 33. The greatest amount of nuclei containing fragmented DNA was observed on day 8 post-detachment, suggesting this was the peak occurrence of programmed cell death. Further, the temporal organization of programmed cell death suggests that the granule-secreting acini undergo apoptosis first, and that during the first week of oviposition the type I acini do not exhibit programmed cell death. These data suggest that the type I acini may still function in maintaining off-host hydration state of ovipositing females. These data provide morphological evidence that salivary gland degeneration involves a temporal programmed cell death event.     

Cockroach allatostatin-like immunoreactivity in the synganglion of the American dog tick Dermacentor variabilis (Acari: Ixodidae)

Using immunocytochemistry based on a monoclonal antibodyagainst Diploptera punctata allatostatin I and horseradishperoxidase-diaminobenzidine reaction, the presence of allatostatin-likeimmunoreactivity is demonstrated in the synganglion of Dermacentorvariabilis females. The immunoreactive cells are located in theprotocerebral, cheliceral, palpal, stomodeal, postesophageal, and opisthosomalregions of the synganglion. Strongly immunoreactive granules accumulate in theboundary area of the subganglia in the preesophageal part of the synganglion.This suggests that the immunoreactive materials may be released directly fromthere. In addition, a putative neurohemal area is found in the anterior area ofthe opisthosomal ganglion, where abundant immunoreactive materials are stored.Weak immunoreactivity and fewer immunoreactive cells are seen in newly moltedfemales compared with one month old, unfed females. Thus, the immunoreactiveproducts may be depleted during molting and synthesized in females beforefeeding.     

Internal and external mycoflora of the American dog tick,Dermacentor variabilis (Acari: Ixodidae), and its ecological implications

Scopulariopsis brevicaulis, the anamorph of Microascus brevicaulis (Microascaceae, Ascomycota), has been identified in the body contents of the tick Dermacentor variabilis. After topical application of the fungal inoculum, tick mortality was marked. This is the first account describing the internal mycoflora of D. variabilis with a novel technique used to recover potential biological control agents.     

The tick Ixodes ricinus: distribution and climate preferences in the western Palaearctic

In this study, multivariate spatial clustering on monthly normalized difference vegetation index (NDVI) maps is used to classify ecological regions over the western Palaearctic. This classification is then used to delineate the distribution and climate preferences of populations (clades) of the tick Ixodes ricinus L. (Acari: Ixodidae) from a geographically extensive dataset of tick records and a gridded 2.5-km resolution climate dataset. Using monthly layers of the NDVI, regions of similar ecological attributes were defined and nine populations with significant differences in critical climate parameters (P< 0.005) were detected. Grouping of tick records according to other categories, such as political divisions, a 4 degrees x 4 degrees grid overlying the study area, or the CORINE) and USGS) vegetation classification schemes did not provided significantly separated populations (P = 0.094-0.304). Factor analysis and hierarchical tree clustering provided an ecological overview of these tick clades: two Mediterranean and one Scandinavian (western) clades are clearly separated from a node that includes clades of different parts of central Europe and the British Isles, with contrasting affinities between the different clades. The capture records of these ecologically separated clades produce a clear bias when bioclimate envelope modelling is applied to the mapping of habitat suitability for the tick in the western Palaearctic. The best-performing methods (Cohen's kappa = 0.834-0.912) use partial models developed with data from each ecoregion, which are then overlapped over the region of study. It is concluded that the use of ecologically derived ecoregions is an objective step in assessing the presence of ecologically different clades, and provides a guide in the development of data partitioning for habitat suitability modelling.     

Evidence of a maternal effect that protects against water stress in larvae of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

We report that the ability to absorb water vapor from the air in larvae of the American dog tick, Dermacentor variabilis, changes depending upon moisture conditions where the eggs develop. When development occurs at lower relative humidities, resultant larvae can replenish water stores, maintain water balance, and survive at relative humidities as low as 75-85% RH, a range that agrees with previously published values for the critical equilibrium humidity or CEH. In contrast, exposure to high relative humidity conditions during development elevates the CEH to 93-97% RH. These larvae can survive only at relative humidities that are close to saturation, as 93% RH is a dehydrating atmosphere. For these larvae, absorption at 97% RH can be prevented by blocking the mouthparts with wax, indicating that an upward shift has occurred in the moisture threshold where the active mechanism for water vapor absorption operates. Based on transfer experiments between low and high relative humidities, the CEH of larvae is determined by the relative humidity experienced by the mother rather than the moisture conditions encountered by eggs after they are laid. The fact that no changes in body water content, dehydration tolerance limit and water loss rate were observed implies that adjustments to the CEH conferred by the mother have the adaptive significance of enabling larvae to maintain water balance by limiting the range of hydrating atmospheres.     

Absence of insect juvenile hormones in the American dog tick,Dermacentor variabilis (Say) (Acari:Ixodidae), and in Ornithodoros parkeri Cooley (Acari:Argasidae)

Synganglia, salivary gland, midgut, ovary, fat body and muscle alone and in combination from the ixodid tick, Dermacentor variabilis (Say), or the argasid tick, Ornithodoros parkeri Cooley, were incubated in vitro in separate experiments with L-[methyl-(3)H]methionine and farnesoic acid or with [1-(14)C]acetate. Life stages examined in D. variabilis were 3 and 72 h old (after ecdysis) unfed nymphs, partially fed nymphs (18 and 72 h after attachment to the host), fully engorged nymphs (2 d after detachment from host), 3 and 72 h old (after eclosion) unfed females, partially fed unmated females (12-168 h after attachment to host) and mated replete females (2 d after detachment from the host). Those from O. parkeri were third and fourth stadium nymphs and female O. parkeri, 1-2 d after detachment. Corpora allata from Diploptera punctata, Periplaneta americana and Gromphadorina portentosa were used as positive controls in these experiments. No farnesol, methyl farnesoate, JH I, JH II, JH III, or JHIII bisepoxide was detected by radio HPLC from any tick analysis while JH III, methyl farnesoate, and farnesol were detected in the positive controls. To examine further for the presence of a tick, insect-juvenilizing agent, Galleria pupal-cuticle bioassays were conducted on lipid extracts from 10 and 15 d old eggs, unfed larvae (1-5 d after ecdysis), unfed nymphs (1-7 d after ecdysis), and partially fed, unmated female adults (completed slow feeding phase) of D. variabilis. Whole body extracts of fourth stadium D. punctata and JH III standard were used as positive controls. No juvenilizing activity in any of the tick extracts could be detected. Electron impact, gas chromatography-mass spectrometry of hemolymph extracts from fed, virgin (forcibly detached 7 d after attachment) and mated, replete (allowed to drop naturally) D. variabilis and fully engorged (1-2 d after detachment) O. parkeri females also failed to identify the common insect juvenile hormones. The same procedures were successful in the identification of JH III in hemolymph of fourth stadium D. punctata. Last stadium nymphal (female) O. parkeri implanted with synganglia from second nymphal instars underwent normal eclosion to the adult. The above studies in toto suggest that D. variabilis and O. parkeri do not have the ability to make the common insect juvenile hormones, and these juvenile hormones do not regulate tick metamorphosis or reproduction as hypothesized in the literature.     

In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say)

Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1-50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50 x treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.     

Structure and anatomical relationships of the synganglion in the American dog tick,Dermacentor variabilis (Acari: Ixodiadae)

The synganglion of Dermacentor variabilis Say is a single nerve mass, condensed around the esophagus and within the periganglionic sinus of the ciculatory system. Protocerebral, cheliceral (including stomodeal bridge), and pedipalpal ganglia lie in the pre-esophageal portion of the nerve mass and bear optic, cheliceral, and pedipalpal nerves respectively. The unpaired stomodeal and the recurrent nerve which forms the hyper-esophageal ganglion arise from the stomodeal bridge. Paired primary and accessory nerves to the retrocerebral organ complex have mixed protocerebral-cheliceral origins. Pedal ganglia (including ventral olfactory lobes of pedal ganglia I) and composite opisthosomal ganglion lie in the post-esophageal nerve mass and bear pedal nerve trunks and two pairs of opisthosomal nerves respectively. Internally, the synganglion consists of cellular rind and fibrous core. A welldefined neurilemma with a laminar matrix covers nerve mass and peripheral nerves. The rind contains the somata of ganglionic neurons and ensheathing glial cells and is restricted to the synganglion mass. It is limited by two specialized glial layers, the external perineurium and internal subperineurium. Discrete glomerular formations are present within the protocerebrum and olfactory lobes. Olfactory glomeruli located in pedal ganglia I are associated with a pair of globuli cell groups. Possible physiological relationships between anatomical specializations of the synganglion, extraneural sinuses and circulating hemocytes are considered. The evolutionary significances of condensation in the stomatogastric neuropile regions and throughout the synganglion, together with the simplification and loss of glomerular formations, are discussed.     

Factors influencing in vitro infectivity and growth of Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni (Acari, Ixodidae)

Rickettsia peacockii, a spotted fever group rickettsia, is a transovarially transmitted endosymbiont of Rocky Mountain wood ticks, Dermacentor andersoni. This rickettsia, formerly known as the East Side Agent and restricted to female ticks, was detected in a chronically infected embryonic cell line, DAE100, from D. andersoni. We examined infectivity, ability to induce cytopathic effect (CPE) and host cell specificity of R. peacockii using cultured arthropod and mammalian cells. Aposymbiotic DAE100 cells were obtained using oxytetracycline or incubation at 37 degrees C. Uninfected DAE100 sublines grew faster than the parent line, indicating R. peacockii regulation of host cell growth. Nevertheless, DAE100 cellular defenses exerted partial control over R. peacockii growth. Rickettsiae existed free in the cytosol of DAE100 cells or within autophagolysosomes. Exocytosed rickettsiae accumulated in the medium and were occasionally contained within host membranes. R. peacockii multiplied in other cell lines from the hard ticks D. andersoni, Dermacentor albipictus, Ixodes scapularis, and Ixodes ricinus; the soft tick Carios capensis; and the lepidopteran Trichoplusia ni. Lines from the tick Amblyomma americanum, the mosquito Aedes albopictus, and two mammalian cell lines were non-permissive to R. peacockii. High cell densities facilitated rickettsial spread within permissive cell cultures, and an inoculum of one infected to nine uninfected cells resulted in the greatest yield of infected tick cells. Cell-free R. peacockii also were infectious for tick cells and centrifugation onto cell layers enhanced infectivity approximately 100-fold. The ability of R. peacockii to cause mild CPE suggests that its pathogenicity is not completely muted. An analysis of R. peacockii-cell interactions in comparison to pathogenic rickettsiae will provide insights into host cell colonization mechanisms.     

Comparison of novel and conventional methods of trapping ixodid ticks in the southeastern U.S.A.

Tick‐borne disease surveillance and research rely on resource‐effective methods for tick collection. This study compared the respective performances of several trapping methods in a mixed grassland–forest habitat in western Tennessee. To test for temporal differences in effectiveness, sites were sampled monthly (April–August 2013) using dry ice, dragging, flagging, sweep netting, carbon dioxide (CO₂) dragging and CO₂ flagging methods. To evaluate the effect of habitat on method effectiveness, four methods (dragging, CO₂ dragging, CO₂ flagging and dry ice) were compared in four habitat types (bottomland deciduous, upland deciduous, coniferous and grassland) in June 2014. In the temporal comparison, ticks were found to be most abundant in April and May, and there was a significant sampling period and method interaction, such that method effectiveness varied across sampling period. Sweep netting was significantly less effective than the other methods. In the habitat comparison, dry ice trap collections represented the most effective method in upland deciduous and coniferous habitats. Flagging using CO₂ was significantly less effective than CO₂ dragging and dragging in bottomland deciduous habitats. The success of the various collection methods did not differ significantly within grassland habitats. Overall, dry ice trapping and dragging were the most effective methods for tick collection across time and habitat.