An endosymbiotic conidial fungus, Scopulariopsis brevicaulis, protects the American dog tick, Dermacentor variabilis, from desiccation imposed by an entomopathogenic fungus

The functional role of an endosymbiotic conidial fungus (Scopulariopsis brevicaulis) prevalent within the integumental glands and hemocoel of the American dog tick (Dermacentor variabilis) was investigated to explore the nature of this tick/fungus association. D. variabilis is normally highly resistant to Metarhizium anisopliae, a widely-distributed entomopathogenic fungus, but when mature female ticks harboring S. brevicaulis were fed a solution containing a mycotoxin (Amphotericin B) to purge this mycobiont internally, the ticks inoculated with M. anisopliae displayed classic signs of pathogenicity, as evidenced by recovery of M. anisopliae from ticks by internal fungus culture, greatly accelerated net transpiration water loss rates (nearly 3x faster than ticks containing S. brevicaulis naturally) and elevation of critical equilibrium humidity (CEH) closer to saturation, implying a reduced capacity to absorb water vapor and disruption of water balance (water gain not equal water loss) that resulted in tick death. The presence of S. brevicaulis within the tick was previously puzzling: the fungus is transmitted maternally and there is no apparent harm inflicted to either generation. This study suggests that S. brevicaulis provides protection to D. variabilis ticks against M. anisopliae. Thus, the S. brevicaulis/tick association appears to be mutualistic symbiosis. Given that both organisms are of medical-veterinary importance, disruption of this symbiosis has potential for generating novel tools for disease control.     

Association of different genetic types of Francisella-like organisms with the rocky mountain wood tick (Dermacentor andersoni) and the American dog tick (Dermacentor variabilis) in localities near their northern distributional limits

Dermacentor andersoni and Dermacentor variabilis from allopatric and sympatric populations near their northern distributional limits were examined for the presence of Francisella species using molecular techniques that targeted 373 bp of the 16S rRNA gene. Although there was no evidence for the presence of Francisella tularensis in any tick, Francisella-like endosymbionts (FLEs) were common in D. andersoni and D. variabilis adults and immatures. A significantly greater proportion of female ticks contained FLEs compared to male ticks. In addition, significantly more D. variabilis adult individuals contained multiple FLE sequence types than did D. andersoni adults. Ten different types of FLEs were identified based on the sequence data, which has implications for diagnostic tests and epidemiological studies of F. tularensis in tick populations in Canada. The three most prevalent types of FLEs have been detected previously in D. andersoni or D. variabilis from other parts of their distributional ranges, whereas the other seven FLE types have not been reported previously. A comparison of the FLEs from both allopatric and sympatric populations of these two tick species provided insight into the relative host-specificity and the modes of transmission of these tick-borne bacteria. In general, each FLE type was specific for one tick species, suggesting vertical transmission of each bacterium. However, there were a few instances of potential cross-transfer of two FLE types to the other tick species at locations where D. andersoni and D. variabilis occurred in sympatry, suggesting that there may be occasional horizontal transmission of some FLEs.     

RdlDv,a novel GABA-gated chloride channel gene from the American dog tick Dermacentor variabilis

The American dog tick Dermacentor variabilis is a major transmitter of bacterial and viral pathogens in human and animal populations, and compounds active against this species would benefit both human and animal health. Invertebrate GABA-gated chloride channels are validated targets of commonly used insecticides and acaricides. We cloned a novel member of the invertebrate GABA-gated chloride channel gene family from Dermacentor variabilis, RdlDv. The closest homologue of the predicted gene product of RdlDv is the RDL protein encoded by the GABA-gated chloride channel gene Drosophila Rdl (Resistance to Dieldrin), with which it shares 64% amino acid identity. When expressed in Xenopus oocytes, RdlDv produces GABA-activated currents blocked by the known insecticides and RDL antagonists fipronil and picrotoxinin. These results suggest that RdlDv encodes a GABA-gated chloride channel subunit, making it a potential target for compounds active against the tick D. variabilis.     

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.     

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.     

Sequence and the developmental and tissue-specific regulation of the first complete vitellogenin messenger RNA from ticks responsible for heme sequestration

The first full-length mRNA for vitellogenin (Vg) from ticks was sequenced. This also represents the first complete sequence of Vg from the Chelicerata and of a heme binding Vg. The Vg cDNA from the American dog tick, Dermacentor variabilis was 5744nt in length (GenBank Accession number AY885250), which coded for a protein of 1843 aa with a calculated molecular weight of 208 kD. This protein had an 18 aa signal sequence, a single RXXR cleavage signal that would generate two subunits (49.5 and 157K in molecular weight) and lipoprotein N-terminal and carboxy von Willebrand factor type D domains. Tryptic digest MS analysis of vitellin protein confirmed the function of the cDNA as the tick yolk protein. Apparently, vitellin in D. variabilis is oligomeric (possibly dimeric) and is comprised of a mixture of the uncleaved monomer and subunits that were predicted from the single RXXR cleavage signal. The highly conserved GL/ICG motif close to the C-terminus in insect Vg genes was different in the tick Vg message, i.e., GLCS. This variant was also present in a partial sequence of Vg from Boophilus microplus. Phylogenic analysis showed that the full length Vg cDNA from D. variabilis and the partial cDNA from B. microplus were distinct from insects and Crustacea. The Vg message was not found in whole body RNA from unfed or fed males or in unfed and partially fed (virgin) females as determined by Northern blotting. The message was found in replete (mated) pre-ovipositional females, increased to higher levels in ovipositing females and was absent after egg laying was complete. The endocrine regulation of the Vg mRNA is discussed. The tissue sources of the Vg message are both the gut and fat body. Tryptic digest MS fingerprinting suggests that a second Vg mRNA might be present in the American dog tick, which needs further study.     

Active surveillance to update county scale distribution of four tick species of medical and veterinary importance in Oklahoma

The incidence of tick‐borne disease continues to increase in humans and companion animals in the United States, yet distribution maps for several tick vectors in Oklahoma, including Dermacentor variabilis, Dermacentor albipictus, Ixodes scapularis, and Amblyomma maculatum, are not available or are outdated. To address this issue, county‐scale tick records from peer‐reviewed literature and passive collections were reviewed for Oklahoma. Additionally, dry ice traps, tick drags, and harvested deer were utilized to actively collect adult ticks throughout the state. Through these methods, D. variabilis, D. albipictus, I. scapularis, and A. maculatum were identified in 88% (68/77), 45.4% (35/77), 66.2% (51/77), and 64.9% (50/77) of the counties in Oklahoma, respectively. Baseline maps were developed for the distribution of D. variabilis and D. albipictus and distribution maps were updated for I. scapularis and A. maculatum. This data confirms that these four species of ticks continue to be widespread within Oklahoma with a western expansion of the range of I. scapularis within the state. These results assist efforts to better understand the epidemiology of the different diseases caused by pathogens transmitted by these tick species within the Great Plains region.     

Disruption of pregnancy in rabbits serving as hosts for feeding ticks

The effect of tick infestation on reproductive success of mammals was studied by using rabbits bred after they had served as hosts for Dermacentor variabilis . Mating success, pregnancy incidence, litter size and neonatal viability were reduced in tick-exposed rabbits, and the proportional number of abnormalities found in preimplantation-stage embryos was much higher than in unexposed controls. Fecundity was restored by prolonging the interval between tick exposure and breeding or by treatment with an acaricide.     

Amblyomma americanum: identification of tick salivary gland antigens from unfed and early feeding females with comparisons to Ixodes dammini and Dermacentor variabilis

Salivary gland antigens involved in host resistance to tick feeding by Amblyomma americanum (lone star tick) have been identified. Gland extracts from unfed and partially fed 12-, 48-, 72-, 96-, and 120-hr females and their corresponding midgut tissues were analyzed by immunoblotting with sera from naturally immune and hyperimmune sheep and rabbits. Polypeptides at 90, 75, 58, 45, 33, and 23 kDa from the salivary glands of A. americanum females were consistently observed with antibodies from both sheep and rabbits. No antigens unique to tick midgut tissue were detected with immune sera. Female Dermacentor variabilis and Ixodes dammini shared 90- and 45-kDa salivary gland antigens with A. americanum, and these may represent conserved polypeptides. We speculate that some of the salivary gland antigens represent components of tick cement, while others are playing some other yet undetermined role in tick feeding.     

Tick paralysis in a grey fox.

One hundred seventeen Dermacentor variabilis were removed from the head and back of a grey fox (Urocyon cinereoargenteus) showing paresis and diminished motor reflexes of the hind limbs. Rapid and total recovery led to a diagnosis of tick paralysis.     

Molecular cloning,expression and isolation of ferritins from two tick species--Ornithodoros moubata and Ixodes ricinus

Genes encoding ferritins were isolated and cloned from cDNA libraries of hard tick Ixodes ricinus and soft tick Ornithodoros moubata. Both tick ferritins are composed of 172 amino-acid residues and their calculated mass is 19,667.2 Da and 19,974.5 Da for I. ricinus and O. moubata, respectively. The sequences of both proteins are closely related to each other as well as to the ferritin from another tick species Dermacentor variabilis (>84% similarity). The proteins contain the conserved motifs for ferroxidase center typical for heavy chains of vertebrate ferritins. The stem-loop structure of a putative iron responsive element was found in the 5' untranslated region of ferritin mRNA of both ticks. Antibodies against fusion ferritin from O. moubata were raised in a rabbit and used to monitor the purification of a small amount of ferritins from both tick species. The authenticity of ferritin purified from O. moubata was confirmed by mass-fingerprinting analysis. In the native state, the tick ferritins are apparently larger (~500 kDa) than horse spleen ferritin (440 kDa). On SDS-PAGE tick ferritins migrate as a single band of about 21 kDa. These results suggest that tick ferritins are homo-oligomers of 24 identical subunits of heavy-chain type. The Northern blot analysis revealed that O. moubata ferritin mRNA level is likely not up-regulated after ingestion of a blood meal.     

Dermacentor variabilis: characterization and modeling of macrophage migration inhibitory factor with phylogenetic comparisons to other ticks, insects and parasitic nematodes

We have identified and characterized the full length cDNA sequence of macrophage migration inhibitory factor (MIF) from the American dog tick, Dermacentor variabilis. The nucleotide and putative amino acid sequences from this study shared a high level of sequence conservation with other tick MIFs. The bioinformatics analysis showed across species conservation of the MIF amino acid sequence in ticks, insects and nematodes. The multiple sequence alignment identified Pro 1, 3, 55; Thr 7, 112; Asn 8, 72; Ile 64, 96; Gly 65, 110, Ser 63 and Leu 87 amino acids to be highly conserved among the sequences selected for this study. Tick MIF does not have the oxidoreductase domain as found in MIFs from other animals suggesting that tick MIF is not capable of performing as an oxidoreductase. The phylogenetic analysis revealed that tick MIFs share a closer evolutionary proximity to parasitic nematode MIFs than to insect MIFs.     

Seasonal activity and spatial distribution of host-seeking adults of the tick Dermacentor variabilis

The seasonal temporal and spatial distribution and emergence of overwintering and summer cohorts of host-seeking American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), adults were examined for 5 years in the Bluegrass region of Kentucky. Weekly drag cloth sampling was conducted from early March until late September 1992-1995 and early March until the end of May 1996. Overwintering adult cohorts became active in early to mid-April followed by bimodal host-seeking activity each year, with the duration and intensity of the first activity period being related to the previous year's summer cohort activity. The first activity peak extended from mid-April through late May and the second activity peak occurred in July. All host-seeking activity ceased by early August, except in 1995, when activity ended on 22 August. Laboratory survival of adults, collected weekly during 1993-1996, was used to estimate the emergence of summer adult cohorts. Summer adult cohorts became active in late May or early June and overlapped with overwintering adult host-seeking activity. The termination of host-seeking activity in overwintering adults could not be determined using the methods employed in this study and is most likely to have continued for the entire season. Host-seeking adults displayed contagious distribution patterns along trails bordering ecotone habitats, as determined by fitting a negative binomial frequency distribution to weekly sampling data. These patterns did not vary from year to year. In addition, the location of D. variabilis activity did not change from year to year, which suggests that microhabitats within apparently homogeneous environments enhance adult survival and lead to 'hot spots' of tick activity despite the ability of vertebrate hosts to disperse D. variabilis populations more widely. These data better define our understanding of D. variabilis adult seasonal and spatial host-seeking activity, estimate for the first time the intensity of summer cohort host-seeking activity, and relate summer cohort activity with spring activity the following year.     

Cloning and sequencing of putative calreticulin complementary DNAs from four hard tick species

Calreticulin (CRT) is a calcium-binding protein and has many functions in eukaryotic cells. CRT is possibly involved in parasite host immune system evasion. To better understand the molecular basis of CRT in ticks, we cloned and sequenced 4 full-length complementary DNAs (cDNAs) from the hard tick species, Dermacentor variabilis, Haemaphysalis longicornis, Ixodes scapularis, and Rhipicephalus sanguineus, using the technique of rapid amplification of cDNA ends. The deduced amino acid sequences share high identities (between 77 and 98%) with 3 known tick CRT sequences. The major characteristics of known CRTs are observed in all 4 of our deduced tick CRTs. These include 3 major domains, a signal peptide sequence at the beginning of the coding region, 2 triplets of conserved regions, cysteine sites providing disulfide bridges for N-terminal folding, and a nuclear localization signal. Remarkably, the replacement of the endoplasmic reticulum retention signal KDEL by HEEL, which is believed to be associated with secretion of CRT into the host during feeding and was previously recorded only in 2 ticks and a hookworm, is also present in all 4 of our tick putative CRTs. In addition, the CRT gene is potentially useful for tick phylogenetic reconstruction.     

Tick infestations of the eastern cottontail rabbit (Sylvilagus floridanus) and small rodentia in northwest Alabama and implications for disease transmission.

Studies were conducted over a four-county area of northwest Alabama to determine the association of eastern cottontail rabbits with Dermacentor variabilis, the eastern United States vector of Rocky Mountain spotted fever. A secondary objective was to compare infestations of this tick on rabbits with infestations on commonly encountered rodent species as a means of determining the relative importance of each in the disease transmission cycle. These epidemiologic surveys were conducted in response to reported fatal cases of Rocky Mountain Spotted Fever in two counties of the study area. From 202 eastern cottontail rabbits, 3,956 ticks were collected. Of this total, 79.87% were Haemphysalis leporispalustris, 9.15% Amblyomma americanum, 8.22% Ixodes dentatus, and 2.76% D. variabilis. Only immature stages of D. variabilis were collected from cottontail rabbits. Ticks were collected on rabbits in all months except November, and only one specimen was taken in January. Based on the average number of ticks per host collected in each month, April was the peak month for D. variabilis and I. dentatus. High values for H. leporispalustris also occurred at this time, but even higher values occurred in October and December. The heaviest infestation of A. americanum occurred during the month ofAugust and coincides with the activity period for the larvae of this species. Two hundred sixty-nine of the smaller Rodentia, comprising 13 species, yielded 264 ticks, all D. variabilis, and all but two were immature stages. Five rodent species, Microtus ochragaster Orozomys palustris, Peromyscus gossypinus, Peromyscus leucopus, and Sigmodon hispidus accounted for 95.83% of the ticks collected, and appeared to be preferred hosts for D. variabilis; all five had higher infestation levels per host than did the eastern cottontail rabbit. Data on host relationships in association with seasonal activity are presented.     

建立真菌透甲模型及红色毛癣菌和短帚霉透甲时间的比较

目的通过建立真菌透甲模型,观察真菌透甲时间,来探讨红色毛癣菌与短帚霉的相互作用。方法建立真菌透甲模型,分为红色毛癣菌组、短帚霉组、短帚霉-红色毛癣菌混合组,分析各组透甲时间。结果透甲模型中,红色毛癣菌的透甲时间为（9.46±1.89）d,短帚霉的透甲时间为（2.62±0.96）d,混合组中短帚霉的透甲时间为（2.54±0.78）d。结论体外透甲模型中,短帚霉比红色毛癣菌先穿透甲板。混合组中,红色毛癣菌对短帚霉的透甲时间无明显影响。     

Optimal sampling and spatial distribution of Ixodes pacificus, Dermacentor occidentalis and Dermacentor variabilis ticks (Acari: Ixodidae)

A common method for sampling tick populations is flagging, which is a method of dragging a white cloth over a plant substrate for a fixed distance along a transect. Flagging over rough physical surfaces or using long subtransect lengths could lead to the underestimation of tick densities. Using estimates of the drop-off rates of adult Ixodes pacificus in flag sampling, optimal sampling schemes (the length and number of subtransects) were examined using the relationships between the tick drop-off rates (c), the tick density and distribution patterns and the roughness of the sampled plant substrate. It was found that the optimal number of subtransects and Lmax, the longest subtransect length which did not significantly underestimate the tick density from c, were affected by the tick density, substrate roughness and tick distribution pattern. This study also showed that the density and distribution of I. pacificus and Dermacentor occidentalis varied greatly over time in the populations sampled, while the Dermacentor variabilis densities were low and showed no significant changes over time. Both I. pacificus and D. occidentalis had clumped distributions along trails and these clumps were aggregated. However, the clump sizes (or individual clump areas) changed significantly over time because of density fluctuation or the movement of ticks. Finally, a positive association between the number of I. pacificus and D. occidentalis adults was observed from 2 m subtransect flag collections in March 1995; no relationship was found in 1994. © Rapid Science Ltd. 1998     

Dermacentor variabilis: acquired resistance to ticks in BALB/c mice

To assess the ability of Dermacentor variabilis larvae to feed on mice during repeated infestations, known numbers of larvae were confined to the skin of BALB/c mice within plastic capsules, and different skin sites were used in four successive infestations. In secondary infestations larval feeding appeared to be enhanced, but in subsequent infestations the mice expressed acquired tick resistance, manifested by reduction in numbers of engorged larvae and in weights of fed larvae. Resistance, once established, was still demonstrable four weeks after the previous infestation. Histological examination of skin biopsies from BALB/c mice undergoing repeated infestations revealed slight reactions to primary infestations and increasingly severe responses in subsequent infestations. The latter reactions included prominent participation of mast cells and eosinophils, many of which were degranulated close to tick bite sites. It is suggested that these cells might play a role in the expression of murine tick resistance.