Characterization of protective antigens from the midgut of Amblyomma variegatum ticks

Separation of midgut membrane proteins from the tick,Ambylomma variegatum, using a non-ionic detergent (TritonX-114), resulted in two protein fractions, namely DET (detergent) and AQ (aqueous). In immunoblotting analysis with polyclonal antibodies against these fractions, 4 proteins (M_r ∼27,000, 67,000, 86,000 and 95,000,)and 2 proteins (M_r ∼54,000 and 67,000) were detected in the DET and AQ fractions, respectively. Three of the DET fraction proteins M_r∼27,000, 67,000 and 95,000 were glycosylated since they bound to the lectin, concanavalin A. In 2-dimensional gel electrophoresis, the AQ and DET fraction proteins were found to be acidic in nature. In a series of bioassay experiments, rabbits were first immunised with both DET and AQ fractions and then infested with ticks. The egg batch weights of these ticks were reduced by 50% compared to control ticks. Furthermore, there was a significant reduction in the hatchability of eggs laid by ticks fed on rabbits previously immunised with both DET (14%) and AQ (33%) fractions. Based on the egg hatchability, there productive capacity of ticks was reduced by 77 and 48% by DET and AQ fractions, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick,Amblyomma americanum (L.)

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.     

Anteroposterior gradient during nymphal-adult moulting cycle of the tropical bont tick,Amblyomma variegatum (Acarina: Ixodidae)

Summary The structure of the extensible (alloscutum) and inextensible (scutum) integument of the nymph, Amblyomma variegatum was examined during the whole bloodmeal and the nymphal-adult moulting cycle. Integumental events were tentatively correlated with the ecdysteroid levels measured by radioimmunoassay. We observed that all the integumental events were realised along an anteroposterior gradient. During the 5 days corresponding to the bloodmeal, although the hormone concentration was low, a new endocuticle was deposited on both the alloscutum and scutum. Furthermore, mitoses were initiated in the capitulum. On days 1–2 after the meal, ecdysteroid titres began to increase and reached a first peak corresponding to 4.1 ng 20-hydroxyecdysone equivalents/tick on the 4th day after the ticks dropped off their host. At this time the epidermis of the capitulum was detached and the outline of the adult capitulum was already visible. Mitotic activity in the alloscutum was initiated. On day 6 post-drop, the frontal apolysis was achieved and the ecdysteroid titres declined to basal values. A second peak much higher than the first one (maximum value of 33.7 ng/tick) and identified principally as 20-hydroxyecdysone by HPLC/RIA was noted on the 13th day post-drop. During the period of increase in the ecdysteroid levels (days 9–10 post-drop), the mitotic phase ended in the alloscutum and the apolysis began. Epicuticle was deposited after day 12 postdrop. Then, while the titre fell to low values (about 1.6 ng/tick, days 16–20 post-drop), the exocuticle was deposited and the nymphal cuticle was digested. All adult structures were functional 3 days before ecdysis. In young male as in female adults the mean value of the ecdysteroid levels corresponded to about 2.5 ng/tick. Finally, hydrolysis of tick whole extracts with esterase demonstrated a low increase of RIA-positive material, demonstrating the probable presence of natural ecdysteroid fatty-acid conjugates in this species.     

A tick B-cell inhibitory protein from salivary glands of the hard tick, Hyalomma asiaticum asiaticum

Some studies done to date suggest that B-cell inhibitory factor occurred in tick saliva. In this study, a novel protein having B-cell inhibitory activity was purified and characterized from the salivary glands of the hard tick, Hyalomma asiaticum asiaticum. This protein was named B-cell inhibitory factor (BIF). The cDNA encoding BIF was cloned by cDNA library screening. The predicted protein from the cDNA sequence is composed of 138 amino acids including the mature BIF. No similarity was found by Blast search. The lipopolysaccharide-induced B-cell proliferation was inhibited by BIF. This is the first report of the identification and characterization of B-cell inhibitory protein from tick. The current study facilitates the study of identifying the interaction among tick, Borrelia burgdorferi, the causative agent of Lyme disease, and host.     

Progress Towards the Eradication of Amblyomma variegatum from the Caribbean

Progress in the Caribbean Amblyomma Program (CAP) is reviewed since its inception in 1995 when regional eradication activities for Amblyomma variegatum ticks were initiated using Bayticol pour-on. Technical achievements in the various islands were slow initially, and showed a wide diversity in attaining eradication targets. St. Kitts, considered as a model program, eliminated the tropical Bont tick (TBT) from most of the island in less than 3 years. However, the elimination of very low numbers of residual adult TBT that persisted in three ‘hot-spots’ took another 3 years. A similar problem was faced in St. Lucia, although the approach there was to cull the wild cattle in the last remaining hot-spot. Both islands were certified as ‘Provisionally free from TBT’ in November 2001. In 2002, Anguilla and Montserrat attained the same status, and Barbados and Dominica also qualify for certification. Certification is based on strict, three-monthly, surveillance criteria: essentially, two successive TBT-free surveillance rounds on a statistically acceptable sample of the livestock population. Within the responsibility of the CAP, three islands remain TBT-infested, Antigua, Nevis, and St. Martin/St. Maarten. Nevis is of concern because it has been operational since late 1995, and appears unable to resolve the problem of treating stray small ruminants. Current funding should be adequate to complete eradication on Nevis and in St. Martin/St. Maarten. The major outstanding challenge is Antigua. In comparison to other islands they have the largest number of livestock, and have much larger populations than originally reported: sheep and goats are 4–5-fold higher, and cattle are 1.35-fold higher. The cost for Bayticol is, therefore, almost double and an additional US$ 1.5 million is required for that commodity alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Attraction of Amblyomma variegatum (ticks) to the attraction-aggregation-attachment-pheromone with or without carbon dioxide

The responses of adult Amblyomma variegatum ticks released from various distances to different doses of the synthetic attraction-aggregation-attachment pheromone (AAAP) (made up of ortho-nitrophenol, methyl salicylate and nonanoic acid in paraffin oil), dispensed from the center of circular field plots, were studied in the presence or absence of elevated levels of CO₂. Up to 90% of the ticks released were attracted to the pheromone source in the presence of CO₂ within 3h. CO₂ alone was unattractive, similar to previous findings in Zimbabwe, but unlike results from a Caribbean A. variegatum population, which was significantly attracted to this signal. In the absence of CO₂, smaller but significant proportions of the released ticks were attracted to the pheromone, albeit more slowly, suggesting another variation in the responses of this bont tick to inter- and intra-specific signals. Our results are interpreted in the light of a study undertaken elsewhere demonstrating relatively high heterozygosity among tick populations. Possible directions of further research to explore the use of the pheromone in off-host control of the tick are also highlighted.     

Purification and cloning of a novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin-B was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY by Edman degradation. The cDNA encoding ixosin-B was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 89 amino acids including mature ixosin-B. Purified ixosin-B exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide. It is also the fourth family of antimicrobial peptide from hard ticks.     

New member of the protein disulfide isomerase (PDI) family identified in Amblyomma variegatum tick

Ticks belonging to arthropoda are blood feeding, geographically widespread ectoparasites of mammals, reptiles and birds. Their saliva contains active substances that protect them from host immune attack and allow for transmission of various pathogens during the feeding process. Characterization of tick saliva components can therefore contribute to the development of effective methods for the control of tick-borne diseases.

Here we describe the identification and basic characterization of a gene encoding a 55 kDa protein found in the salivary glands (SG) of Amblyomma variegatum tick. Based on the primary structure and homology to the family of protein disulfide isomerases (PDI; EC 5.3.4.1) the gene was named AvPDI. The 1461 nt long AvPDI open reading frame codes for a 487 amino acid protein. In vitro expressed AvPDI was exclusively localized in the endoplasmic reticulum. RT-PCR and Western blot analysis revealed that AvPDI expression is not restricted to the SG of the tick. More detailed analysis on tissue slides from SG detected an AvPDI specific signal in granular cells of the acini type II and III. Finally, reductase activity of AvPDI was confirmed in an insulin assay. The structural and functional characteristics suggest that AvPDI is another member of the PDI protein family and represents the first more closely characterized PDI in the ticks.     

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.     

Factors affecting the distributions of the ticks Amblyomma hebraeum and A. variegatum in Zimbabwe: implications of reduced acaricide usage

The ticks Amblyomma hebraeum and A. variegatum are the main vectors of heartwater, a disease of ruminants caused by Cowdria ruminantium, in the agricultural areas of Zimbabwe. At present, A. hebraeum is widely distributed in the dry southern lowveld, and occurs in at least seven foci in the higher rainfall highveld. Amblyomma variegatum occurs in the Zambezi valley and surrounding dry lowveld areas in the northwest. The distribution of A. hebraeum has changed considerably over the past 70 years, while that of A. variegatum appears to have remained fairly static. The distribution patterns of both species in Zimbabwe display anomalous features; the ticks occur in areas of lowest predicted climatic suitability for survival and development and in areas where the densities of cattle, the most important domestic host, are lowest. The only factor favouring the survival of the species in the lowveld habitats in which they occur is the presence of alternative wildlife hosts for the adult stage. Their absence from more climatically favourable highveld habitats appears to have been the result of intensive acaricide treatment of cattle over a long period and a historic absence of significant numbers of wildlife hosts. Eradication of A. hebraeum and A. variegatum by intensive acaricide treatment of cattle can be achieved in the absence of significant numbers of alternative hosts, because of the long attachment and feeding periods of the adults of these tick species. However, eradication becomes impossible when alternative hosts for the adult stage are present, because a pheromone emitted by attached males attracts the unfed nymphal and adult stages to infested hosts. The unfed ticks are not attracted to uninfested hosts, such as acaricide-treated cattle.Regular acaricide treatment of cattle is expensive and so, for economic reasons, the Government of Zimbabwe is no longer enforcing a policy of strict tick control. It is likely that reduced tick control will result in the spread of Amblyomma ticks to previously uninfested areas. Added to this, recent introductions of various wildlife species to highveld commercial farming areas have created conditions in which the ticks could become established in higher rainfall areas. Amblyomma hebraeum is more likely to spread than A. variegatum, because its adults parasitize a wider range of wildlife hosts (warthogs, medium to large-sized antelope, giraffe, buffalo and rhinoceros), whereas adults of A. variegatum appear to be largely restricted to one wildlife species (buffalo) in Zimbabwe, the distribution of which is now confined to very limited areas of the country, as part of foot and mouth disease control measures. A model to predict the rate of spread of A. hebraeum through the highveld is described.Possible control options for dealing with the spread of Amblyomma ticks and heartwater to previous unaffected highveld areas, include (1) continuation of intensive acaricide treatment of cattle to prevent the spread, (2) establishment of a buffer zone of intensive tick control around affected areas to contain the spread and (3) allow the spread to occur and control heartwater by means of immunization. An economic analysis to determine the costs and benefits of the control options, which takes into account the development of Amblyomma-specific tick control technologies and improved heartwater vaccines, is recommended.Deceased.     

Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum

Earlier work from our laboratory indicated that injection of 20-hydroxyecdysone (20E) into non-vitellogenic female Amblyomma hebraeum ticks stimulates the synthesis of vitellogenin (Vg), but not its uptake into oocytes [Friesen, K., Kaufman, W.R., 2004. Effects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum. Journal of Insect Physiology 50, 519-529]. In contrast, Thompson et al. [Thompson, D.M., Khalil, S.M.S., Jeffers, L.A., Ananthapadmanaban, U., Sonenshine, D.E., Mitchell, R.D., Osgood, C.J., Apperson, C.S., Roe, M.R., 2005. In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say). Journal of Insect Physiology 51, 1105-1116] demonstrated that injection of 20E into virgin female Dermacentor variabilis ticks stimulated both vitellogenesis and Vg uptake into oocytes. In addition to the species difference in the two studies there were substantially different methods for injecting 20E. In our earlier work we injected small partially fed ticks after removing them from the host. Thompson et al. injected the females while they remained attached to the host. So in this study we repeated our earlier experiments on A. hebraeum using on-host injection. We also injected 20E into off-host ticks with or without haemolymph collected from engorged ticks (days 2-10 post-engorgement), or from large partially fed mated ticks in the rapid phase of engorgement, to see whether we might detect a 'vitellogenin uptake factor' (VUF) in haemolymph. Off-host injection of 20E (0.45mug/g body weight (bw)) did not induce ovary development beyond that of vehicle-injected controls. But ticks in this study, receiving 20E plus haemolymph from engorged ticks, showed a significant increase in ovary weight beyond that of 20E alone (1.31+/-0.05% bw; 34 for 20E plus haemolymph and 1.03+/-0.05% bw; 25 for 20E alone). However, in normal engorged A. hebraeum, the ovary exceeds 7% bw at the onset of oviposition. As in our earlier work, in this study 20E stimulated Vg-synthesis (3.9+/-0.5mgVt-equivalents/ml) beyond that occurring in vehicle-injected ticks (0.76+/-0.14mgVt-equivalents/ml), and there was a further increase in ticks injected with 20E plus haemolymph from engorged ticks (8.9+/-1.0mgVt-equivalents/ml). On-host injection of 20E alone (6mug20E/g bw) did not produce a statistically significant increase in oocyte length over that of vehicle-injected controls, whereas on-host injection of 20E plus engorged haemolymph resulted in significantly larger oocytes (261+/-57mum) compared to vehicle-injected controls (132+/-11mum), compared to 20E alone (131+/-12mum), or haemolymph alone (124+/-24mum). There was a marked stimulation of Vg-synthesis by 31mug20E/g bw (6.0+/-1.5mgVt-equivalents/ml) compared to vehicle-injected controls (1.02+/-33mgVt-equivalents/ml). Vt accumulation by ovaries was significantly greater in ticks treated with haemolymph (12+/-3mugVt/mg ovary) or 20E plus haemolymph (56+/-26mugVt/mg ovary) compared to vehicle-injected controls (5.1+/-1.5mugVt/mg ovary). There was also a significant effect of 6mug20E/g bw plus engorged haemolymph on ovary weight (1.74+/-0.29% bw) compared to vehicle-injected ticks (0.95+/-0.10% bw), but not compared to ticks injected with 20E alone (1.25+/-0.19% bw). We conclude that at least some of the differences observed between the two laboratories relate to the species difference, and that there is some evidence that the engorged haemolymph of A. hebraeum contains a VUF.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

An Inhibitor of Thrombin-Stimulated Blood Platelet Aggregation from the Salivary Glands of the Hard Tick Amblyomma variegatum (Acari: Ixodidae)

The tropical bont tick, Amblyomma variegatum can cause intense skin irritation and inflammation and bites that often develop into septic wounds or abscess in their host. Crude salivary gland extract (SGE) of partially engorged A. variegatum females as well as SGE protein fractions purified by three-step reverse phase HPLC procedure were tested for their anti-aggregatory effect on isolated human blood platelets stimulated with thrombin and compared with the effect of recombinant hirudin. At concentrations 10⁻³ and 5 × 10⁻³ μg protein/ml the following rank order of antiplatelet activity was detected: AV 16/3 (inhibitor purified from AV-III, third purification) > SGE > AV-II (fraction from first purification) > AV-III (fraction from first purification) > hirudin. The effect of all fractions tested was dose-dependent. For fraction AV 16/3, the inhibitory effect was 49 and 61% for 10⁻³ and 5 × 10⁻³ μg protein/ml, respectively. The results suggest that protein fractions from A. variegatum SGE possess an antithrombin effect on human blood platelets with hirudin-like activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Attachment sites of the tick Amblyomma marmoreum on its tortoise host,Geochelone pardalis

Leopard tortoises (n=162) were collected over a period of 19 months at the National Zoological gardens, Pretoria, South Africa and the attachment sites of larvae, nymphs and adults of A. marmoreum monitored. Most of the larvae (80%) and nymphs (87%) were found on the anterior parts of the tortoise while adults (90%) were found mainly on the posterior regions of the host body. The immature stages were predominant in soft skinned areas protected by the carapace especially the neck and upper leg areas. Male and female ticks were most commonly found on the posterior armpit. posterior legs and the areas surrounding the base of the tail. Interstadial competition as a major factor influencing attachment site segregation is doubtful. Anterior attachment site selection of the immature stages is believed to result largely from ticks lying passively in wait and then climbing onto the host as it moves through the vegetation and the requirement for soft skinned and protected areas to allow for blood feeding. Adult females usually attach posteriorly in areas that allow them to fully engorge without being dislodged by mechanical disturbance, while males prefer to attach at sites where they are protected but still remain in close proximity to female pheromone signals.     

Cytotoxic effects of permethrin in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

Because of the medical and veterinary importance of ticks and the wide use of synthetic chemical substances such as permethrin (active ingredient of Advantage® Max3 – Bayer)for their control, this study evaluated the effects of different concentrations (206, 1031 and 2062 ppm) of the acaricide on the salivary glands of Rhipicephalus sanguineus semi-engorged females. Results showed that permethrin is a potent substance that acts morpho-physiologically in the tick glandular tissue, causing changes in the acini shape intense vacuolation in acinar cells, and disruption of the tissue by cell death process, with subsequent formation of apoptotic bodies, especially at higher concentrations, thus precluding the accurate identification of different types of acini. Importantly, it is demonstrated that permethrin acts on salivary gland tissue, as well as affecting the nervous system, accelerating the process of glandular degeneration, and interfering with the engorgement process of female ticks, preventing them from completing the feeding process.     

Quantification and cellular localization of dopamine in the salivary gland of the ixodid tick Amblyomma hebraeum

Dopamine (DA) content of the salivary glands in partially fed female and fed male ticks, Amblyomma hebraeum Koch (Acari: Ixodidae), was measured by high-performance liquid chromatography with electrochemical detection or by a radioenzymatic assay for catecholamines following experimental treatment. Some glands were held in vitro for up to 3 days. Other preparations (backless explants) allowed one side to be surgically denervated, the contralateral side serving as control. Normal ticks were sampled for up to 4 days post-removal from the host (rabbits). In the backless explants, there was little if any difference in DA content between denervated and control sides, even after 4 days in vitro, indicating that unilateral denervation did not eliminate the major salivary gland pool of DA. High doses of reserpine (333 g per g body weight) and 6-hydroxydopamine (1000 g per g body weight) did not significantly reduce the DA content of the salivary gland, also suggesting that only a minor component of the DA pool is within axons innervating the salivary gland. A dispersed population of cells rich in tyrosine hydroxylase immunoreactivity (an enzyme marker for catecholamine-synthesizing cells) was found in close association with the granular acini. This further suggests that the major DA pool in the salivary gland may be in cells other than the dopaminergic nerves arising from the central nervous system. © Rapid Science Ltd. 1998     

Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick,Amblyomma hebraeum

Ovaries of the ixodid tick, Amblyomma hebraeum Koch, grew rapidly after engorgment as a result of yolk uptake. At 26 °C, oviposition began by day 10 post-engorgement, plateaued on days 16-18, and ended by day 38. Vitellin (Vt) was partially purified from ovaries of day 10 engorged ticks by gel filtration and ion exchange chromatography. This Vt comprises seven major and several minor polypeptides. Two polypeptides (211 and 148 kD) from haemolymph of engorged female ticks corresponded to minor polypeptides of similar molecular weight in the ovary. The haemolymph titre of the 211 and 148 kD polypeptides increased up to the onset of oviposition. These polypeptides were absent in males and non-vitellogenic females (day 0 engorged or day 10 partially-fed females), and were thus designated as vitellogenin (Vg). Antibodies raised against haemolymph Vg211 and 148 recognized these polypeptides in partially purified Vt, as well as six of the seven major polypeptides. Using these antibodies we developed an indirect, competitive ELISA to quantify Vg. Rise in haemolymph Vg-concentration lagged slightly behind the rise in haemolymph ecdysteroid (ES)-concentration, and Vg-synthesis was stimulated by injections of 20E into non-vitellogenic females. These observations indicate that an ES is the vitellogenic hormone in A. hebraeum.