Morphometric Analysis of Chemoreception Organ in Male and Female Ticks (Acari: Ixodidae).

The Haller's organ plays a crucial role in a tick's ability to detect hosts. Even though this sensory organ is vital to tick survival, the morphology of this organ is not well understood. The objective of this study was to characterize variation in the morphological components of the Haller's organ of three medically important tick species using quantitative methods. The Haller's organs of Ixodes scapularis Say (Ixodida: Ixodidae) (black-legged tick), Amblyomma americanum (L.) (Ixodida: Ixodidae) (lone star tick), and Dermacentor variabilis (Say) (Ixodida: Ixodidae) (American dog tick) were morphologically analyzed using environmental scanning electron microscopy and geometric morphometrics, and the results were statistically interpreted using canonical variate analysis. Our data reveal significant, quantitative differences in the morphology of the Haller's organ among all three tick species and that in D. variabilis the sensory structure is sexually dimorphic. Studies like this can serve as a quantitative basis for further studies on sensor physiology, behavior, and tick species life history, potentially leading to novel methods for the prevention of tick-borne disease.

Infrared light detection by the haller's organ of adult american dog ticks, Dermacentor variabilis (Ixodida: Ixodidae).

The Haller's organ (HO), unique to ticks and mites, is found only on the first tarsus of the front pair of legs. The organ has an unusual morphology consisting of an anterior pit (AP) with protruding sensilla and a posterior capsule (Cp). The current thinking is that the HO's main function is chemosensation analogous to the insect antennae, but the functionality of its atypical structure (exclusive to the Acari) is unexplained. We provide the first evidence that the HO allows the American dog tick, Dermacentor variabilis, to respond to infrared (IR) light. Unfed D. variabilis adults with their HOs present were positively phototactic to IR. However, when the HOs were removed, no IR response was detected. Ticks in these experiments were also attracted to white light with and without the HOs, but were only positively phototactic to white light when the ocelli (primitive eyes) were unobstructed. Covering the eyes did not prevent IR attraction. A putative TRPA1 receptor was characterized from a D. variabilis-specific HO transcriptome we constructed. This receptor was homologous to transient receptor potential cation channel, subfamily A, member 1 (TRPA1) from the pit organ of the pit viper, python, and boa families of snakes, the only receptor identified so far for IR detection. HO scanning electron microscopy (SEM) studies in the American dog tick showed the AP and Cp but also novel structures not previously described; the potential role of these structures in IR detection is discussed. The ability of ticks to use IR for host finding is consistent with their obligatory hematophagy and has practical applications in tick trapping and the development of new repellents.

First description of the nymph and larva of Dermacentor compactus Neumann, 1901 (Acari: Ixodidae), parasites of squirrels (Rodentia: Sciuridae) in southeast Asia.

Recent reexamination of collection lots stored in the United States National Tick Collection revealed adult specimens of Dermacentor compactus Neumann, 1901 (Acari: Ixodidae) reared from field-collected nymphs, which allowed us to associate field-collected unidentified nymphs and larvae with this species. Nymphs of D. compactus can be easily distinguished from those of other congeneric species by the shape of the scutum and spiracular plate, the hypostome dentition, and the size of the spurs on the coxae. Larvae of this species can be distinguished by the shape and sculpture of the scutum, the shape of basis capituli, the absence of auriculae, and the size of the spurs on coxae II and III. Both nymphs and larvae feed mostly on various species of squirrels (Rodentia: Sciuridae). Considerably fewer nymphs and larvae were found on murid rodents (Rodentia: Muridae), domestic dogs (Carnivora: Canidae), and a snake (Squamata: Colubridae).

Embryo Development and Morphology of the Rocky Mountain Wood Tick (Acari: Ixodidae).

Dermacentor andersoni Stiles embryogenesis was observed using fluorescent and scanning electron microscopy for eggs held under laboratory conditions (25°C and at 93% relative humidity). Early embryonic cell divisions appeared to be synchronous and holoblastic, giving rise to a uniform blastoderm surrounding the yolk. The cells of the blastoderm became concentrated on one side of the embryo, forming the segmented germ band. Distinct opisthosomal and prosomal segment morphologies, which are characteristic of chelicerate embryos, were observed during germ band elongation. Mouth and leg appendages grew from the prosomal segments. As development progressed, the segments were fused into the idiosoma and capitulum of the free-living larval form. An embryo staging system was established based on embryo developmental morphology and the timing of morphogenetic events. The staging system will serve as a basis for future studies directed at understanding morphogenetic mechanisms or for observing the impact of abiotic factors, such as temperature or humidity, on tick development.

Molecular and functional characterization of vacuolar-ATPase from the American dog tick Dermacentor variabilis.

Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases.

First description of the nymph and larva of Dermacentor raskemensis (Acari: Ixodidae), parasites of pikas and other small mammals in Central Asia.

Dermacentor raskemensis Pomerantzev, 1946 is one of the rare Asian species in this genus. The immature stages of this species have never been described. Reexamination of D. raskemensis holdings stored in the United States National Tick Collection revealed a collection lot containing reared nymphs and larvae of this species. This collection made it possible for us to find numerous nymphs and larvae of D. raskemensis among previously unidentified material collected in the field. Both immature stages of D. raskemensis are described here for the first time. Nymphs of D. raskemensis can be distinguished from those of other Dermacentor species in the region by small spiracular plate, relatively short and obtuse lateral projections of basis capituli dorsally, relatively short spurs on coxa I and the internal spur is characteristically very broadly rounded at its apex, and very small spur on coxa IV, whereas larvae of D. raskemensis can be distinguished from other Dermacentor by relatively short and obtuse lateral projections of basis capituli, approximately 6 denticles in the median files on hypostome, and relatively short, broad, and rounded spur on coxa I. The nymphs and larvae of D. raskemensis studied originate from Afghanistan, India, Iran, and Pakistan, where they were collected from pikas and other small mammals.

Reinstatement of Dermacentor kamshadalus Neumann (Acari: Ixodidae) as a valid species parasitizing mountain goats and sheep in the United States, Canada, and Russia.

Reexamination of Dermacentor albipictus (Packard, 1869) holdings stored in the United States National Tick Collection revealed several collections of a morphologically distinct Dermacentor species. Comparison of these specimens with other Dermacentor taxa showed that they are identical to an old taxon originally described as Dermacentor variegatus kamshadalus Neumann, 1908. For more than a century, this taxon was known only from the male holotype specimen collected in Russia, and the name was considered a junior synonym of D. albipictus. D. kamshadalus is reinstated here to a full species rank, and its male is redescribed and its female and nymph are described for the first time. Adults of D. kamshadalus can be distinguished from those of D. albipictus by a short spur on trochanters I, shorter spurs on coxae I, shorter dorsal cornua, more numerous perforations on spiracular plates, less numerous and shorter setae on idiosoma, especially around spiracular plates, and considerably paler coloration of the conscutum and scutum. The nymph of D. kamshadalus can be differentiated from that of D. albipictus by shorter spurs on coxae I and the numerous perforations on the spiracular plates. Adults and nymphs ofD. kamshadalus are recorded from the United States, Canada, and Russia, where they have been collected from mountain goats, Oreamnos americanus (de Blainville), bighorn sheep, Ovis canadensis Shaw, and sheep, Ovis sp. of which the species was not stated.

[Fine structural features of the sensory system in the ixodid tick Dermacentor niveus (Parasitiformes, Ixodidae)].

The main sensory organs (eyes, Haller's organ, and palpal organ) of Dermacentor niveus were examined in scanning and transmitting electron microscopes. The Haller's organ consists of a capsule with five porous SW-WP olfactory sensilla and pleomorphs; anterior pit with porous SW-WP, two grooved DW-WP, two thin NP-SW, and one conical DW-NP sensilla; four posterior DW-WP sensilla; and anterior group of SW-WP and DW-UP sensilla. Duct orifices of two dermal glands are situated near the capsule. Contact chemosensory sensilla of palps are represented by 4 DW-UP and 6 SW-UP sensilla. Eyes possess flattened lenses without the system of radial channels typical of eyes of other ixodid ticks examined. Unipolar photoreceptor cells with microvilli of rhabdomeres running in parallel to longitudinal cells axis contain basal bodies treated as rudiments of the cilia. On the whole, sensory system of D. niveus, dweller of semi-exposed habitats (floodland meadows, tugai forests, and shrub thickets of arid zones) is characterized by combination of features typical of species dwelling in exposed (deserts) and concealed (forests) habitats.

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.

Morphology and structural organization of gené's organ in Dermacentor reticulatus (Acari: Ixodidae).

Scanning and transmission electron microscopical investigations revealed that Genés organ in unfed and ovipositing females of Dermacentor reticulatus is formed as a double-sac-structure consisting of an outer epithelial and an inner cuticular sac. In ovipositing ticks the latter emerges through the camerostomal aperture to the exterior. Genés organ in unfed ticks consists of a corpus, two posterior horns and a pair of undeveloped glands at each side, which differentiate in ovipositing ticks to compound, branched tubular glands with a main efferent duct for each gland opening into the lumen between the epithelial and the cuticular sac. Genés organ of egg-laying females corresponds basically in morphology and structural organization to that of unfed ticks. Compared with unfed ticks, however, in ovipositing ticks the corpus and horns are longer and broader, the glands are fully developed and the cuticular sac is evertable. The epithelial sac as the outermost part of Genés organ is continuous with the hypodermis of the basis capituli and the scutum, arises at the camerostomal aperture, forms the corpus and the two blind-ending horns, passes into the epithelium of the main excretory ducts of the glands and envelops the cuticular sac. The cuticular sac passes into the cuticle of the basis capituli and the scutum, arises at the camerostomal aperture, is folded, expands into the horn tips and consists inwards of a smooth epicuticula and outwards of a fibrous endocuticula. Muscles originating from the scutum pass caudomedially through the epithelial sac and are inserted into the cuticular sac. The entire surface of the maximally everted cuticular sac is covered with an amorphous mass. In cleaned samples, ledge-like structures appear on the lateral surface. These ledges turn into balloon-like structures which extend over the medial and dorsal surface. The entire surface including the balloon-like structures and the ledges are provided with numerous cribrate pits.

Characterization of an endosymbiont infecting wood ticks, Dermacentor andersoni, as a member of the genus Francisella.

A microorganism (Dermacantor andersoni symbiont [DAS]) infecting Rocky Mountain wood ticks (D. andersoni) collected in the Bitterroot Mountains of western Montana was characterized as an endosymbiont belonging to the genus Francisella. Previously described as Wolbachia like, the organism's DNA was amplified from both naturally infected tick ovarial tissues and Vero cell cultures by PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA) and Francisella membrane protein genes. The 16S rDNA gene sequence of the DAS was most similar (95.4%) to that of Francisella tularensis subsp. tularensis. Through a combination of Giménez staining, PCR assay, and restriction fragment length polymorphism analysis, 102 of 108 female ticks collected from 1992 to 1996 were infected. Transovarial transmission to female progeny was 95.6%, but we found no evidence of horizontal transmission.

Evidence for the reproductive isolation of Dermacentor marginatus and Dermacentor reticulatus (Acari: Ixodidae) ticks based on cross-breeding, morphology and molecular studies.

The species status of Dermacentor marginatus and Dermacentor reticulatus was evaluated by scanning electron microscope (SEM) examination of adult ticks, cross-breeding experiments and molecular biological analysis of eggs derived from transspecific pairings. The SEM investigations including the morphometric quantification of phenotypic features resulted in an unequivocal differentiation of adult D. marginatus and D. reticulatus ticks. The cross-breeding experiments demonstrated that irrespective of whether female ticks of both species were applied with con- or transspecific male ticks or without males to sheep, they engorged and laid eggs. The larvae, however, developed only in eggs which originated from conspecific matings. A nested polymerase chain reaction (PCR) of the second internal transcribed spacer (ITS2) using the DNA of eggs from transspecific pairings and sequencing of the PCR products revealed two different genotypes. The genotypes of eggs originating from D. marginatus and D. reticulatus females of these pairings differed. However, the eggs deposited by D. marginatus always possessed the same two genotypes as did the eggs produced by D. reticulatus. These results argue for a strict reproductive isolation of D. marginatus and D. reticulatus and, therefore, for a separate species status.

Developmental studies of Anaplasma marginale (Rickettsiales:Anaplasmataceae) in male Dermacentor andersoni (Acari:Ixodidae) infected as adults by using nonradioactive in situ hybridization and microscopy.

The development of Anaplasma marginale Theiler was studied in ticks using a nonradioactive in situ hybridization method developed in our laboratory. Male Rocky Mountain wood ticks, Dermacentor andersoni Stiles, were infected intrastadially by allowing them to feed for 7 d on an infected calf (acquisition feeding). The ticks were then removed and held in a humidity chamber for 5 d before being fed on a 2nd susceptible call for 10 d (transmission feeding). Two groups of 10 ticks were collected daily during the 22-d experiment. In one group one-half of each tick was processed and embedded in paraffin and in the other group one-half of each tick was embedded in LR White for in situ hybridization. The companion tick halves from each group were fixed and embedded in Dow Epoxy Resin resin for routine light and electron microscopy. As detected by in situ hybridization on LR White- and paraffin-embedded sections and by microscopy, initial infection of A. marginale in ticks occurred in gut tissues either on the 7th d of acquisition feeding or the 1st d of the held period and infection persisted throughout transmission feeding. The highest number of ticks with gut infection was observed on the 5th d of transmission feeding. Salivary glands became infected with A. marginale on the 1st day of transmission feeding and remained infected throughout the transmission feeding period. Peak infection was observed on day 4 of transmission feeding. After the beginning of transmission feeding, A. marginale infection was also observed in interstitial, reproductive, skeletal muscle, fat body, and Malpighian tubule tissues. Although A. marginale infection of ticks clearly originates in midgut epithelial cells, many tissues eventually become infected during transmission feeding, resulting in a generalized infection. The infection of multiple tissues may contribute to the ability of A. marginale infection to persist in intrastadially infected male ticks.

Evidence of a myoepithelial cell in tick salivary glands.

Type III acini from feeding female Dermacentor variabilis varied in size during in vitro and in vivo fluid production. As the type III acinus enlarged, its lumen enlarged and the adlumenal cell became thinner. As the acinus contracted, its lumen became smaller while the adlumenal cell became wider. Actin was demonstrated in salivary glands using an immunoblot technique. Actin was localized in the adlumenal cells of type III acini with fluorescent microscopy using rhodamine-phalloidin and with electron microscopy using heavy meromyosin to decorate actin filaments. Pre-treatment of salivary glands with cytochalasin D abolished fluorescence in adlumenal cells subsequently treated with rhodamine-phalloidin. These results support the hypothesis that the adlumenal cell in type III acini functions as a myoepithelial cell.

Toxic effect of endocytobionts from Dermacentor reticulatus ticks in three tick species after in vivo application.

The endocytobionts from ovaries of partially engorged female Dermacentor reticulatus ticks, inoculated introcoelomally into females of three tick species, D. reticulatus, Ixodes ricinus and Haemaphysalis inermis, caused considerable morphological alterations in the examined cells and tissues of the synganglion, fat body, tracheal complex and ovary of these recipients.

Development of Anaplasma marginale in male Dermacentor andersoni transferred from parasitemic to susceptible cattle.

The development and transmission of Anaplasma marginale was studied in Dermacentor andersoni males. Laboratory-reared male D andersoni were allowed to feed for 7 days on a calf with ascending A marginale parasitemia. The ticks were then held in a humidity chamber for 7 days before being placed on 2 susceptible calves. Anaplasmosis developed in the calves after incubation periods of 24 and 26 days. Gut and salivary glands were collected from ticks on each day of the 23-day experiment and examined with light and electron microscopy. Colonies of A marginale were first observed in midgut epithelial cells on the sixth day of feeding on infected calves, with the highest density of colonies found in gut cells while ticks were between feeding periods. The first colonies contained 1 large dense organism that subsequently gave rise to many reticulated organisms. Initially, these smaller organisms were electron-lucent and then became electron-dense. On the fifth day after ticks were transferred to susceptible calves for feeding, A marginale colonies were found in muscle cells on the hemocoel side of the gut basement membrane. A final site for development of A marginale was the salivary glands. Colonies were first seen in acinar cells on the first day that ticks fed on susceptible calves, with the highest percentage of infected host cells observed on days 7 to 9 of that feeding. Organisms within these colonies were initially electron-lucent, but became electron-dense.

Mixed Rickettsia-virus infection in Dermacentor reticulatus imago.

Electron microscopic examination revealed replication and accumulation of Rickettsia sibirica in the fat body of experimentally infected Dermacentor reticulatus ticks. Rickettsia are released from the fat body cells by budding being surrounded with cytoplasm and plasmalemma of the host cell. Eukaryotic cell structures have been detected consisting of lamella layers whirled around the intact rickettsiae. In addition to rickettsia, microorganisms morphologically resembling Francisella tularensis and an orbivirus were found in tick tissues at morphological examination. The morphology of the virus and stages of its morphogenesis are described. Mixed viral and rickettsial infection has been shown to develop in the same ticks and even in the same fat body cells in a very close association.

Fine structure of the fat body and nephrocytes in the life-stages of Dermacentor variabilis.

The fine structure of the fat body and associated nephrocytes of the American dog tick, Dermacentor variabilis (Say), was described in unfed larvae, unfed nymphs, and in unfed and fed adults of both sexes. The fat body consisted of one type of cell, the trophocyte. Morphological changes that occurred in the trophocytes of both sexes were dependent on feeding. The ultrastructure of feeding male trophocytes was distinct from trophocytes of feeding females. In the feeding female, the trophocyte developed an ultrastructure characteristic of cells that produce secretory proteins. A type of scalariform cell junction was found associated with rough endoplasmic reticulum of the trophocytes. Nephrocytes were closely associated with trophocytes but were not part of the fat body. Nephrocyte ultrastructure was unaltered throughout the life-stages we examined, except at the end of oviposition. Organelles in the nephrocytes were not randomly distributed, but were found in distinct regions of the cytoplasm. Slit diaphragms at the surface of the nephrocytes were extracellular specializations that had a periodic ultrastructure.

Anaplasma marginale in tick cell culture.

Anaplasma marginale was propagated in a tick cell line derived from Dermacentor variabilis embryos. The rickettsial organism was identified and monitored in culture by transmission electron microscopy and the indirect immunofluorescence technique, using specific monoclonal antibodies. Inoculation of the embryonic tick cell line with midguts of infected adult ticks (culture 1), nymphal ticks (culture 2) and adult ticks that were infected as nymphs and dissected as adults (culture 3) resulted in 3 continuous cultures of A marginale. Culture 1 had been maintained through 22 passages over a 11-month period; cultures 2 and 3 had been maintained for 18 passages over a 9-month period. Growth of A marginale in the cell line began in the area of the nuclear membrane at approximately 4 days after inoculation or transfer. Thereafter, the organisms were observed in inclusions scattered throughout the cytoplasm of the host cells. Maximal growth of the organism occurred at 7 to 14 days, after which numbers of inclusions rapidly decreased to minimal or undetectable levels. The organism began new cycles of growth with each 1:5 to 1:10 split and transfer of the host cells. Electron microscopy of recently infected cells revealed a morphology of the organism that closely resembled that observed in marginal bodies of infected erythrocytes. After several passages, A marginale organisms had a varied morphology and resembled the organism described in midgut cells of naturally infected ticks.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental infection with Rickettsiella phytoseiuli in adult female Dermacentor reticulatus (Ixodidae): an electron microscopy study.

Rickettsiella phytoseiuli naturally occurring in Phytoseiulus persimilis mites was cultivated in adult female Dermacentor reticulatus ticks. It demonstrates all six known developmental stages: dense, intermediate, bacterial, giant, crystal-forming and small dark particles. These stages of rickettsiae were found in salivary glands, Malpighian tubules, synganglion, ovaries, tracheal complex, haemolymph, fat body and alimentary tract. Rickettsiella phytoseiuli did not infect the Gené's organ. It multiplied in female ticks in a manner similar to that in the typical host mite, P. persimilis.