How much pilocarpine contaminates pilocarpine‐induced tick saliva?

Pilocarpine is often applied or injected into ticks to induce salivation, and the resulting saliva used to test for various pharmacological, biochemical and immunological activities. To measure the amount of pilocarpine in pilocarpine-induced tick saliva, an HPLC-MS/MS method, based on capillary strong cation exchange chromatography online with an ion trap mass spectrometer, was used to measure pilocarpine in the pg to ng range. Results indicate large concentrations of pilocarpine in Ixodes scapularis Say and Amblyomma americanum (Linnaeus) (Acari: Ixodidae) saliva, ranging from 3 to 50 mm. Due to the known effects of pilocarpine on smooth muscle and immune cells, appropriate controls are proposed and discussed for proper interpretation of results using this saliva preparation.     

Fine structural analysis of palpal receptors in the tick Amblyomma americanum (L.)

Summary Palps of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) were studied by scanning and transmission electron microscopy. The terminal palp segment (IV) bears the so-called palpal organ, a cluster of 10 short, blunt-tipped sensilla. All sensilla (except for the center sensillum) receive a dual innervation: 2 mechanoreceptive dendrites which terminate in the socket membrane plus several chemoreceptive dendrites (4–12) which enter the lumen. The thick-walled cuticular shaft possesses 2–3 small pore openings (100 Å) below the tip, thus establishing communication between dendrites and environment. Two structurally different types of palpal sensilla exist: The A-type has a characteristic doublelumen and always contains 4 dendrites, the B-type features a single lumen and a specially layered cuticular shaft with 6–12 dendrites. The fine structure of the tick palpal receptors corresponds closely to that of known contact chemoreceptors in insects.This research was supported in part by a contract with the Office of Naval Research (R. C. Axtell, principal investigator), and by NIH Training grant ES 00069. Paper no. 3700 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.     

Effects of octopamine on fluid secretion by isolated salivary glands of a feeding ixodid tick

Octopamine elicited a dose-related secretory response by salivary glands isolated from the feeding female tick Amblyomma americanum. Half-maximal stimulation occurred at about 60 μM. Phentolamine (10 μM) failed to inhibit the octopamine-mediated response; however, thioridazine (50 μM) inhibited both octopamine (1,000 μM) and dopamine-stimulated (0.1 μM) secretion. Maximal stimulation by dopamine (1.0 μM) showed no further increase in the rate of secretion after adding octopamine (1,000 or 0.1 μM). Glands responded to octopamine (100 μM) with rates significantly lower than controls following exposure to amphetamine (1,000 μM). Octopamine receptors do not appear to mediate the secretory response, and octopamine may stimulate secretion by releasing catecholamines from presynaptic neurons. These results support the hypothesis that dopamine is the natural transmitter mediating fluid secretion in the feeding tick salivary gland.     

一种收集蜱类唾液的方法

建立了一种收集蜱类唾液的方法。注射多巴胺(20mgmL)10μL于森林革蜱DermacentorsilvarumOlenev吸血雌蜱血腔内,可使唾液腺分泌唾液,用毛细管连续收集30min,可得唾液15～20μL蜱。     

Modeling the biotic and abiotic factors that describe the number of active off‐host Amblyomma americanum larvae

Amblyomma americanum (L.) is a three‐host tick that spends most of its life off‐host and is an important vector of pathogens in the eastern United States. Our objectives were to develop a predictive statistical model that describes the number of active, off‐host larvae from 2007 to 2011 and to determine the environmental variables associated with this pattern. Data used in this study came from monitoring conducted in northeast Missouri in which off‐host ticks were collected from a permanent plot in a forest and an old field habitat every other week from approximately February to December. Variables examined were day length, degree days, total precipitation prior to sampling, wind speed, saturation deficit, number of adults prior to sampling, and collection site. Of the four regression models tested, the negative binomial model was selected. Fitted candidate models were compared relative to one another using values of eight model selection criteria and model averaging was used to develop a predictive model. The residual plots indicated that the weighted average model performs well in describing the number of larvae. Of the variables considered, the number of larvae was most strongly associated with increasing degree days, the number of active adults prior to sampling, and the forested site.     

Responses of Amblyomma americanum and Dermacentor variabilis to odorants that attract haematophagous insects

Carbon dioxide (CO₂), 1‐octen‐3‐ol, acetone, ammonium hydroxide, L‐lactic‐acid, dimethyl trisulphide and isobutyric acid were tested as attractants for two tick species, Amblyomma americanum and Dermacentor variabilis (Acari: Ixodidae), in dose–response bioassays using Y‐tube olfactometers. Only CO₂, acetone, 1‐octen‐3‐ol and ammonium hydroxide elicited significant preferences from adult A. americanum, and only CO₂ was attractive to adult D. variabilis. Acetone, 1‐octen‐3‐ol and ammonium hydroxide were separately evaluated at three doses against CO₂ (from dry ice) at a field site supporting a natural population of A. americanum nymphs and adults. Carbon dioxide consistently attracted the highest number of host‐seeking ticks. However, for the first time, acetone, 1‐octen‐3‐ol and ammonium hydroxide were shown to attract high numbers of A. americanum. Further research is needed to determine the utility of these semiochemicals as attractants in tick surveillance and area‐wide management programmes.     

Amblyomma americanum salivary gland homolog of nSec1 is essential for saliva protein secretion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in "knocking down" nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands.     

Essential oils of Cupressus funebris, Juniperus communis, and J. chinensis (Cupressaceae) as repellents against ticks (Acari: Ixodidae) and mosquitoes (Diptera: Culicidae) and as toxicants against mosquitoes

Juniperus communis leaf oil, J. chinensis wood oil, and Cupressus funebris wood oil (Cupressaceae) from China were analyzed by gas chromatography and gas chromatography-mass spectrometry. We identified 104 compounds, representing 66.8-95.5% of the oils. The major components were: α-pinene (27.0%), α-terpinene (14.0%), and linalool (10.9%) for J. communis; cuparene (11.3%) and δ-cadinene (7.8%) for J. chinensis; and α-cedrene (16.9%), cedrol (7.6%), and β-cedrene (5.7%) for C. funebris. The essential oils of C. funebris, J. chinensis, and J. communis were evaluated for repellency against adult yellow fever mosquitoes, Aedes aegypti (L.), host-seeking nymphs of the lone star tick, Amblyomma americanum (L.), and the blacklegged tick, Ixodes scapularis Say, and for toxicity against Ae. aegypti larvae and adults, all in laboratory bioassays. All the oils were repellent to both species of ticks. The EC(95) values of C. funebris, J. communis, and J. chinensis against A. americanum were 0.426, 0.508, and 0.917 mg oil/cm(2) filter paper, respectively, compared to 0.683 mg deet/cm(2) filter paper. All I. scapularis nymphs were repelled by 0.103 mg oil/cm(2) filter paper of C. funebris oil. At 4 h after application, 0.827 mg oil/cm(2) filter paper, C. funebris and J. chinensis oils repelled ≥80% of A. americanum nymphs. The oils of C. funebris and J. chinensis did not prevent female Ae. aegypti from biting at the highest dosage tested (1.500 mg/cm(2) ). However, the oil of J. communis had a Minimum Effective Dosage (estimate of ED(99) ) for repellency of 0.029 ± 0.018 mg/cm(2) ; this oil was nearly as potent as deet. The oil of J. chinensis showed a mild ability to kill Ae. aegypti larvae, at 80 and 100% at 125 and 250 ppm, respectively.     

A conceptual model of the Amblyomma americanum life cycle in northeast Missouri

The geographic distribution of Amblyomma americanum (the lone star tick) has increased as has its role as a pathogen vector. The objectives of this study were to determine seasonal activity patterns of each life stage of A. americanum in the northwestern part of the species range and the relationship of these activity patterns among life stages and degree days. Tick activity was monitored over four years since 2007 in a forest and old field habitat located in northeast Missouri. Every other week from February to December, ticks were collected using bait and drag methods. Autocorrelations demonstrated yearly seasonal patterns in each life stage and cross‐correlations between life stages depicted a relationship between activity at a life stage and the previous stage's activity. Cross‐correlations indicated that degree days were related to activity. These data indicated that A. americanum generally complete their life cycle in a minimum of two years in northeast Missouri, with overwintering occurring predominantly in the nymphal and adult stages. These data provide a baseline to compare the life cycle of A. americanum in northeast Missouri to populations in different parts of the species range or at different times in the region.     

Ecdysteroids in females and eggs of the Ixodid tick Amblyomma hebraeum

Summary

A mated Amblyomma hebraeum female will engorge on a host for about 8 days before detaching and beginning the maturation of its single egg batch which is laid during a period of about 30 days. The feeding period is characterized by an important synthesis of endocuticular material occurring before the rapid feeding phase. This latter phase, correlated with an enormous weight uptake, shows an increase of ecdysteroid levels measured in the whole animal by RIA. However, the hemolymphatic levels of ecdysteroids remain very low (12 pg 20-hydroxyecdysone equivalent (20-OH-E eq.) per μ1. Within 4 days after detachment, the salivary glands degenerate. Ecdysteroid levels in the whole animal continue to increase, reaching high values (about 500 ng 20-OH-E eq./tick) at the moment of oviposition which begins 10–14 days after dropping. During the same period, hemolymphatic ecdysteroid levels increase, rising to a peak (600 pg 20-OH-E eq./μ1) 1 day prior to the beginning of oviposition. Then, the levels decrease and stabilize around 250 pg 20-OH-E eq./μl during egg-laying. Freshly laid eggs contain large amounts of ecdysteroids (2744 pg 20-OH-E eq./mg).

20-Hydroxyecdysone and ecdysone have been found to be the major free ecdysteroids in hemolymph, ovaries and eggs (verified by the HPLC-RIA technique and GC-MF of silylated HPLC fractions). Helix juice (or esterase) labile ecdysteroid conjugates do not seem to be present to any noticeable extent in hemolymph, ovaries and eggs.     

Application of RNA interference in tick salivary gland research.

Ticks are obligate ectoparasites that feed on a variety of hosts including mammals, birds and reptiles. Prolonged attachment on the host and an ability to transmit a wide variety of pathogens are the special features of tick feeding. Salivary glands are the major route for secretion of excess fluid, several proteins, and factors that counteract the host immune response and hence play a significant role in the success of tick feeding. RNA interference (RNAi) enables scientists to silence genes encoding proteins in an absolutely sequence specific manner at the mRNA level. This technique has already been successfully employed in analyzing roles of proteins of important functions or in assigning roles to several proteins of unknown functions in a variety of animals. In this review, we outline the process of RNAi and the applicability of RNAi in tick salivary gland research.     

Failure by engorged stages of the lone star tick, Amblyomma americanum, to react to assembly pheromone, guanine and uric acid

Response to tick-exposed filter paper discs (tick excreta) by fed and non-fed stages of the lone star tick Amblyomma americanum (L.) (Acari: Ixodidae) was examined from larva to adult. By contrast with engorged stages, each non-fed stage exhibited a classic arrestment response to assembly pheromone, characterized by lack of movement, retraction of legs and formation of small groups; this response was seen in approximately 75% of ticks that made contact with pheromone-treated surfaces. Less pronounced arrestment (by approximately 66% of ticks) was elicited by (0.001-0.005 M) guanine as the active pheromonal ingredient and by uric acid, a chief excretory product of birds. Lack of response to arrestment cues post-engorgement suggests that this response is kairomonal where guanine mimics uric acid as a host cue that signals immature A. americanum's preferred bird host. The functional overlap simultaneously favours tick retention in areas with an abundance of successful ticks, signalled by a heavy bout of excretion (guanine) soon after hatching and moulting. Control significance pertains to the use of these compounds as trap arrestants, but not attractants, with advantages in effectiveness against any non-fed stage.     

Attachment kinetics of the adult tick Amblyomma variegatum to cattle

At the beginning of the 1999 rainy season, three traditional cattle herds were monitored for 48 days while grazing in the bushy savannah of southwestern Burkina Faso. Cattle in each herd were caught on several occasions each day and the attached ticks were counted. This confirmed that Amblyomma variegatum Fabricius (Acari: Ixodidae) adults picked up in the pastures mainly attach to the interdigital areas (87% of the 791 ticks captured), and reach the predilection sites later (chest and udder/inguinal area) when the animals lie down. As many females as males attached to the hosts, but the seasonal distribution was very heterogeneous, with only a few females attaching as long as the humidity rate remained low. It is suggested that this prevents eggs from being laid when conditions are not optimal for their survival and that of the larvae. Ticks attached all day but the number picked up hourly and daily varied greatly according to their density on the pasture. As a general trend, confirmed by another study carried out in 2005, the number of ticks picked up daily increased from less than one tick/animal/day, before the onset of the rainy season, to 6.5 (+/- 1.5) ticks/animal/day on average during the infestation peak, which lasted 6-8 weeks, until early or mid-July. The number then decreased to less than one tick/animal/day from the end of July onwards. The infestation on the predilection sites followed the same trend, with a daily tick burden increase of three to five A. variegatum adults, depending on herd and year, during the infestation peak.     

Integrated Management Strategies for Amblyomma Americanum (Acari: Ixodidae) in Non-Agricultural Areas

The simulation model LSTSIM was revised and adapted to estimate the effects of different management strategies on populations of the lone star tick, Amblyomma americanum (Linnaeus), in non-agricultural areas. The control technologies evaluated were area-wide acaricide application, acaricide self-treatment of white-tailed deer, vegetation reduction and reductions in the density of white-tailed deer. Estimated changes in tick populations using these technologies compared favorably with results from actual integrated tick management studies at five different geographic locations. Area-wide acaricide application and vegetation reduction, as well as combinations of the two technologies proved to be useful for short-term, seasonal management of ticks in small non-agricultural areas and residential sites. Self-treatment of deer with acaricide, applied topically or as a systemic, appears to be the most cost-effective technology for use in area-wide, long-term programs for tick control. The results of simulations suggest that reductions in deer density should be considered as a component of any tick management program.     

Ultrastructure of Haller's organ in the tick Amblyomma americanum (L.)

Summary Haller's organ on the tarsus of the tick Amblyomma americanum (L.) (Acarina: Ixodidae; nymphal stage) was studied by scanning and transmission electron microscopy. It consists of a distal bristle group, (the anterior pit), and a proximal capsule which encloses several sensilla. The seven sensilla of the anterior pit (A1–A7) are all thick-walled and multi-innervated (2–9 neurons), but at least three different types can be differentiated. Sensilla A1 and A2 possess large, plugged pores (>1000 Å) and are the only sensilla with branching dendrites. A3 and A5 are characterized by a spoke-wheel arrangement of the cuticle wall and very fine pores (100–200 Å) penetrating the spokes centrally; A4, A6, and A7 do not exhibit any pore system but a single opening at the bristle tip is assumed.The capsule contains seven thin-walled, blunt-tipped sensilla, and several non-sensory cuticular projections (pleomorphs). All of these sensilla possess large plugged pores in the cuticle wall and numerous dendritic branches of several neurons (3–5) in the lumen. Glandular openings were found inside the capsule; their significance is discussed.The fine structure of Haller's organ supports the functions postulated by Lees (1948), namely olfaction for the capsule and humidity reception (among others) for the anterior pit.This research was supported in part by the Office of Naval Research, and by NIH Training grant ES 00069. Paper no. 3459 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh.     

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.     

A case of Amblyomma testudinarium tick bite in a Korean woman

A case of tick bite was found in the inguinal region of a 74-year-old Korean woman. She was attacked by the tick while working in her vegetable garden in the vicinity of mountain located in Suncheon City, the southern coastal area of the Korean Peninsula. On admission she complained of mild discomfort and itching around the bite area. The causative tick was 23 mm long and had slender pedipalps. The scutum was quite ornate and had eyes at the edge. The genital aperture was located anterior to the level of the coxa II. The spiracular plate was comma-shaped and the anus was surrounded posteriorly by the anal groove. The coxa I had subequal 2 spurs; the external one slightly larger. The spur of coxa IV was slightly longer than those of coxae II and III. The tarsus IV had 2 distinct subapical ventral spurs. It was identified as the fully engorged adult female of Amblyomma testudinarium. This is the first human case of Amblyomma bite in Korea.     

Invasion of West African cattle by the tick Amblyomma variegatum

Studies in Cameroon and Burkina Faso examined the invasion process of cattle by adult Amblyomma variegatum Fabricius (Acari: Ixodidae) ticks. Nearly all the ticks picked up in the pasture during grazing were found on the limb ends, near the hooves, where they temporarily attached. Then when the cattle lay down, the ticks moved from the feet towards the predilection sites, where they attached definitively. Many ticks disappeared during this displacement. All the female ticks and approximately 70% of the males were thus unable to attach to the predilection sites as long as the pioneer males had not attached and started to produce attraction-aggregation-attachment pheromones. Nevertheless, A. variegatum females and males attached to the feet in similar numbers during grazing, whether the cattle were already infested or not, indicating that stimuli originating from the hosts are powerful enough to attract both sexes. After attachment of the pioneer males, the number of ticks that successfully reached the predilection sites increased. However, even on infested animals, 40-50% of A. variegatum ticks found near the hooves after the grazing periods disappeared during the night following their capture. When moving from the temporary attachment sites towards the final ones, one-third of the ticks changed the individual host. Considering this two-stage infestation process, it is suggested that a targeted tick control, using a foot-bath, might greatly reduce cattle infestation. In particular, it could be effective in traditional herds, with animals grazing permanently during the day, lying down only once back in the night pen.     

<Emphasis Type="Italic">In vitro</Emphasis> splenocyte proliferation responses of BALB/c mice to salivary gland extracts of three ixodid tick species (Acari: Ixodidae)

In vitro proliferation and cytokine production were investigated in BALB/c mice splenic cell cultures that were stimulated with concanavalin A (ConA) and lipopolysaccharide (LPS) and simultaneously exposed to salivary gland extracts (SGE) of unfed and partially fed adult ixodid ticks (Ixodes ricinus, Rhipicephalus appendiculatus, Amblyomma variegatum). Generally, tick SGE enhanced proliferation of unstimulated splenocytes and SGE of unfed ticks suppressed mitogen induced proliferation. Partially fed R. appendiculatus and A. variegatum suppressed ConA responses, while partially fed I. ricinus stimulated both ConA and LPS induced proliferation. A. variegatum and R. appendiculatus females slightly enhanced LPS responses 2 days after attachment but suppressed them at the end of the slow feeding phase. In 72 h ConA induced cell cultures, interferon gamma (IFN-γ) production was suppressed by SGE of all ticks, interleukin (IL)-10 production was enhanced by unfed I. ricinus and partially fed A. variegatum males and IL-5 production was enhanced by feeding R. appendiculatus females and A. variegatum males. The study revealed variability in the responsiveness of murine splenocytes to SGE of different ixodid tick species, whereby patterns of host immunomodulation within one tick species differed between sexes and changed during feeding.