A comparison of the cuticular properties of argasid and ixodid ticks: Ornithodoros moubata (Argasidae) vs. Amblyomma hebraeum and Ixodes pacificus (Ixodidae).

Ticks (Chelicerata, Ixodida) are blood-feeding ectoparasites believed to have evolved at least about 120 millions of years ago and found worldwide. However, many aspects of their unique life cycle and anatomy, including their mechanical properties, remain to be understood. Here, we compared the mechanical properties of the cuticle of the argasid tick Ornithodoros moubata to those of two species of ixodid tick, Amblyomma hebraeum and Ixodes pacificus that we explored in our earlier studies of the tick exoskeleton. Significant differences were expected given the substantial difference in life cycle, including a five-fold increase during the repeated adult blood meal for female O. moubata vs. 70- to 120-fold during the single feeding of the adult female A. hebraeum and I. pacificus. We demonstrate here that the layered structure and mechanical properties (stiffness and viscosity) of the cuticle show minor differences, but the difference in cuticle thickness is substantial. Ductility is lost during feeding; reduced pH restores ductility. Previous work suggests that this occurs in vivo in engorged ixodid ticks; there is no evidence of this occurring in vivo in O. moubata. Thinning of cuticle in O. moubata fed females is consistent with the predicted stretch of cuticle due to the blood meal; there is no evidence of cuticle synthesis during the short feeding period. Dimensional analysis suggests that the soft feel of argasid ticks is related to cuticle thickness, not cuticle stiffness. Relative to argasid ticks, the hard ixodid ticks accommodate a ca. 20-fold higher size of blood meal by starting with a thicker cuticle and growing much additional cuticle during engorgement.

A comparison of the cuticular properties of the female ticks Ixodes pacificus and Amblyomma hebraeum (Acari: Ixodidae) throughout the feeding period.

The mechanical properties of the cuticle of Ixodes pacificus (Ip) are compared to those of Amblyomma hebraeum (Ah) from our earlier work. The 10-fold size difference between the species is expected to lead to significant differences in mechanical properties, because cuticular stretch depends on high internal hydrostatic pressure during the rapid phase of engorgement. We demonstrate here: (1) The cuticle of partially fed Ip is less stiff and viscous than that of Ah. (2) A stretch-recoil cycle in both ticks consists of recoverable deformation (ESv) and permanent deformation (ESp); ESp is higher in Ip, and increases sharply during the slow phase of engorgement, but not in Ah. (3) Injected dopamine (DA) increases ESp and reduces all measures of stiffness and viscosity, suggesting that a catecholaminergic neurotransmitter plays a fundamental role in modulating mechanical properties of the cuticle. However, unlike Ah, DA's effect was not different from that of the control (1.2% NaCl). Mere insertion of the needle may have punctured the gut, causing the release of perhaps a catecholamine that increases ESp, an hypothesis supported by the fact that inserting a needle without any injection also caused an increase in ESp. (4) Stretch reduces ESp, but subjecting loops to pH 6.5 in vitro restores it. (5) Despite the smaller size of Ip, later onset of the rapid phase of engorgement, a thinner cuticle and different mechanical properties all reduce the internal pressure needed for stretch.

Cuticle expansion during feeding in the tick Amblyomma hebraeum (Acari: Ixodidae): The role of hydrostatic pressure.

Female Amblyomma hebraeum ticks (Acari: Ixodidae) increase their weight ∼10-fold during a 'slow phase of engorgement' (7-9 days), and a further 10-fold during the 'rapid phase' (12-24h). During the rapid phase, the cuticle thins by half, with a plastic (permanent) deformation of greater than 40% in two orthogonal directions. A stress of 2.5 MPa or higher is required to achieve this degree of deformation (Flynn and Kaufman, 2015). Using a dimensional analysis of the tick body and applying the Laplace equation, we calculated that the tick must achieve high internal hydrostatic pressures in order to engorge fully: greater than 55 kPa at a fed:unfed mass ratio of ∼20:1, when cuticle thinning commences (Flynn and Kaufman, 2011). In this study we used a telemetric pressure transducer system to measure the internal hydrostatic pressure of ticks during feeding. Sustained periods of irregular high frequency (>20 Hz) pulsatile bursts of high pressure (>55 kPa) were observed in two ticks: they had been cannulated just prior to the rapid phase of engorgement, and given access to a host rabbit for completion of the feeding cycle. The pattern of periods of high pressure generation varied over the feeding cycle and between the two specimens. We believe that these pressures exceed those reported so far for any other animal.

Mechanical properties of the cuticle of the tick Amblyomma hebraeum (Acari: Ixodidae).

Female ticks of the family Ixodidae increase their mass up to 100-fold during the 7-10 day feeding period. We determined the material properties of the alloscutal cuticle of female Amblyomma hebraeum from the time of moulting through to full engorgement. The material properties of the cuticle were evaluated by a Kelvin-Voigt analysis of compliance determined from the stretch of loops of cuticle under stress. There was a 3-fold increase in cuticle dry mass during the first 3 weeks post-moult, during which the ductility and stiffness of the cuticle increased substantially. Under stress, the cuticle displayed time-dependent stretch, with a plastic (non-recoverable) and viscoelastic (recoverable) component. Plastic deformation was reasonably constant in the range 10-15% over a wide range of induced stress above ∼ 0.6 MPa. The plastic component of tick alloscutal cuticle was about 5-10 times higher than that of unsclerotized insect cuticle. Tick cuticle is far more ductile than unsclerotized insect cuticle. Material properties of the cuticle did not change significantly as a function of cuticular water content over the normal range throughout the feeding cycle (13-37% wet mass). Injected dopamine (DA) reduced one measure of the viscosity of the cuticle by 38%. Plastic deformability of the cuticle was reduced by 70% after an in vitro stretch, but restored in fully engorged ticks, and in in vitro stretched loops by treatment with DA and reduced pH. Thinning of the cuticle by half during the rapid phase of engorgement requires plastic deformation (irreversible strain) in two orthogonal dimensions in excess of 40%. Treatment with DA increased plastic deformation and enabled extensibility (strain at the point of rupture) above 40%.

Molecular characterization of two vitellogenin genes from the tick, Amblyomma hebraeum (Acari: Ixodidae).

We have identified full-length cDNAs encoding two vitellogenins (Vg) from the African bont tick Amblyomma hebraeum Koch (1844). Vg is a large storage protein that is the precursor to vitellin (Vn), the major yolk protein found in eggs. The first Vg cDNA is 5866 bp long, with a 5715 bp reading frame encoding a 1904 amino acid protein. The second Vg cDNA is 5963 bp long, with a 5781 open reading frame encoding a 1926 amino acid protein. Both proteins possess a short N-terminal signal peptide of 21 and 16 amino acids respectively, which following cleavage result in 213.8 kDa Vg1 and 215.9 kDa Vg2 proteins. The conceptual amino acid translations for both proteins show the N-terminal lipid binding domain, the internal DUF1943 domain and the C-terminal von Willebrand factor type D domain common to all other known Vgs. In addition, these sequences do not resemble any of the conserved sequences that are the hallmarks of the highly similar tick storage protein (carrier protein; CP). Phylogenetic analysis indicates that the Vgs isolated in this study cluster together with other tick Vgs. Using RT-PCR, both Vg1 and Vg2 were expressed only in mated females, and only after they had fed to repletion. In situ hybridizations indicated that both Vgs were expressed only in the midgut and fat body of these females, and was not present in any other female tissues, nor in either fed or unfed males.

Salivary gland degeneration and ovarian development in the Rocky Mountain wood tick, Dermacentor andersoni Stiles (Acari: Ixodidae). I. Post-engorgement events.

It is well known that female ixodid ticks undergo salivary gland degeneration and the greater part of ovarian development following engorgement. The process has been particularly well studied in Amblyomma hebraeum, including the hormonal control of these processes. The purpose of this study, and the second one in this series, is to compare the processes in Dermacentor andersoni with those of A. hebraeum. A major difference between the two species is that virgin female D. andersoni feed to a much higher fed-to-unfed weight ratio than do virgin female A. hebraeum, the former achieving an average of 67× the unfed weight in this study, the latter normally achieving an average of only about 10-15× the unfed weight. We show here that although engorged virgins (defined here as those that exceed a fed-unfed weight ratio of 50, even though they do not detach from the host spontaneously) degenerate their salivary glands and develop their ovaries, these processes proceed more slowly in virgins that they do in mated females, as is the case for A. hebraeum.

Salivary gland degeneration and ovary development in the Rocky Mountain wood tick, Dermacentor andersoni Stiles (Acari: Ixodidae). II. Determination of the 'critical weight'.

The feeding cycle of female ixodid ticks is divided into preparatory, slow, and rapid feeding phases. When a female Amblyomma hebraeum is removed from the host after achieving a minimum size of about 10-13× the unfed weight, various physiological and behavioural changes occur: (a) haemolymph ecdysteroid concentration rises dramatically, (b) the tick does not reattach to the host when given the opportunity, (c) its salivary glands degenerate within about 4 days (if mated) or 8 days (if virgin), and (d) oocyte maturation and oviposition occur (Kaufman and Lomas, 1996; Invert. Repr. Devel. 30: 191-198). None of these changes occur if the tick is removed from the host at smaller sizes. This transition, which occurs when the tick enters the rapid phase of engorgement, has been named the 'critical weight'. To date, the critical weight has been determined for A. hebraeum only. The present study established that, in both mated and virgin D. andersoni, the critical weight is similar to that of A. hebraeum. Although a small percentage of virgin A. hebraeum do exceed the critical weight, achieving perhaps 20× the unfed weight, virgin D. andersoni regularly fed well beyond their CW (>50× the unfed weight) and occasionally engorged completely (100× the unfed weight), although they did not detach spontaneously from the host within 21 days of attachment.

Antimicrobial activity in the egg wax of the tick Amblyomma hebraeum (Acari: Ixodidae) is associated with free fatty acids C16:1 and C18:2.

Untreated eggs of the tick Amblyomma hebraeum Koch (Acari: Ixodidae) exhibited antimicrobial activity (AMA) against Gram-negative but not Gram-positive bacteria; eggs denuded of wax by solvent extraction showed no AMA. The unfractionated egg wax extract, however, showed AMA against Gram-positive but not Gram-negative bacteria, as also shown by Arrieta et al. (Exp Appl Acarol 39: 297-313, 2006). In this study we partitioned the egg wax into various fractions, using a variety of techniques, analyzed their compositions, and tested them for AMA. The crude aqueous extract exhibited AMA. However, although more than 30 metabolites were identified in this extract by nuclear magnetic resonance analysis, none of them seemed likely to be responsible for the observed AMA. In the crude organic extract, cholesterol esters were the most abundant lipids, but were devoid of AMA. Fatty acids (FAs), with chain lengths between C13 and C26 were the next most abundant lipids. After lipid fractionation and gas chromatography/mass spectroscopy, free FAs, especially C16:1 and C18:2, accounted for most of the AMA in the organic extract. The material responsible for AMA in the crude aqueous extract remains unidentified. No AMA was detected in the intracellular contents of untreated eggs.

Female ixodid ticks grow endocuticle during the rapid phase of engorgement.

Lees (Proc Zool Soc Lond 121:759-772, 1952) concluded that the ixodid tick Ixodes ricinus grows endocuticle during the slow but not during the rapid, phase of engorgement, a conclusion supported by Andersen and Roepstorff (Insect Biochem Mol Biol 35:1181-1188, 2005) for the same species. In this study analysis of dimensional data and cuticle weight measurements from female ixodid ticks (Amblyomma hebraeum) were used to test this hypothesis. Both approaches showed that endocuticle growth continues during the rapid phase, tapering to zero at a fed/unfed weight ratio of ~60. Of the total mass of cuticle in the engorged tick 32-43% was formed during the rapid phase. We demonstrate that if cuticle growth stopped at the end of the slow phase, there would not be sufficient cuticle to account for the thickness of cuticle observed at the end of engorgement. This finding is consistent with prior studies of Rhipicephalus (Boophilus) microplus, and with a dimensional analysis of the cuticle thickness data of Lees for I. ricinus, in contradiction to his conclusion from an analysis of tick cuticle weight measurements. An examination of cuticle weight measurements for I. ricinus by Andersen and Roepstorff similarly supports the finding of cuticle growth during the rapid phase. All ixodid ticks undergo major body expansion, typically tenfold or more, during a rapid phase of engorgement and require sufficient cuticle at the end of that process to contain their body. The fact that cuticle grows during the rapid phase of engorgement in three species suggests that this is a general characteristic of the family Ixodidae.

Cuticular plasticization in the tick, Amblyomma hebraeum (Acari: Ixodidae): possible roles of monoamines and cuticular pH.

The degree of plasticization of the alloscutal cuticle of a 'hard' (ixodid) tick, Amblyomma hebraeum, and a 'soft' (argasid) tick, Ornithodoros moubata, was assessed throughout the blood-feeding period. Cuticle viscosity was calculated from rate of creep of cuticle under constant load using a Maxwell model. Feeding-related plasticization (i.e. increased rate of extension under a constant load) occurred in A. hebraeum but not in O. moubata. Maxwell viscosity of unfed A. hebraeum cuticle was relatively high (approximately 720 GPa s) but was significantly lower in feeding ticks. Small partially fed ticks displayed a viscosity of approximately 108 GPa s. Still lower values (42 GPa s) were observed in the largest of the engorged ticks. Following cessation of feeding, there was a significant but limited reversal in viscosity back to approximately 100 GPa s. The water content of cuticle of unfed A. hebraeum (23.4% of wet mass) rose sharply after the onset of feeding and reached a plateau value of 34.0% at a fed/unfed weight ratio of 3 and beyond. Ixodid ticks lay down new endocuticle during the feeding period. The observed increase in cuticle hydration suggests that both old and new cuticles are hydrated during feeding. Monoamines may play an important role in controlling cuticle viscosity. Dopamine (DA) injected into partially fed A. hebraeum caused plasticization. 5-Hydroxytryptamine (serotonin, 5-HT), which induces plasticization in the blood-sucking insect Rhodnius prolixus, had no statistically significant effect on tick cuticle. Octopamine (OA) and tyramine both caused cuticle stiffening (i.e. opposed plasticization). This suggests a possible inhibitory effect but co-injection of OA with DA did not reduce DA-induced plasticization. The mechanism leading to plasticization of tick cuticle may involve a change in cuticular pH. The viscosity of tick cuticle loops was highest at pH 8.0 (389 GPa s) and fell precipitously in the acidic range to a low value of 2.2 GPa s at pH 5.5-5.7. A cuticular pH of approximately 6.5 would account for the lowest viscosity observed under physiological conditions (42.4 GPa s for large, day 0, engorged ticks). The V-ATPase inhibitor, concanamycin A, was a potent inhibitor of DA-induced plasticization. These results are consistent with a model in which DA acts to cause plasticization through transport of H(+) ions into the cuticle. Measurement of cuticular ion (Na(+), K(+), Ca(2+), Mg(2+)) content did not suggest that plasticization is caused by any of these ions. Taken together, our results suggest that the mechanism of cuticular plasticization in feeding A. hebraeum is related to hydration, and involves the transport of H(+) ions into the sub-cuticular space by cells in the hypodermis. Feeding-induced plasticization was not observed in the rapid feeding tick, O. moubata.

Salivary gland degeneration and vitellogenesis in the ixodid tick Amblyomma hebraeum: Surpassing a critical weight is the prerequisite and detachment from the host is the trigger.

The normal engorged body weight of female ixodid ticks (Acari: Ixodidae) is about 100x the unfed weight. Virgin female Amblyomma hebraeum normally do not feed beyond 10x the unfed weight. However, about 10-20% of a population of virgins will feed to perhaps 20x the unfed weight, but not much beyond that. In A. hebraeum, when females surpass about 10x the unfed weight, the following changes in physiology occur if they are removed from the host: (a) they will not reattach if given the opportunity, (b) their salivary glands (SGs) will undergo autolysis within 4 days if they are mated or 8 days if they are virgin, and (c) egg maturation and oviposition will occur in due course. Mated or virgin female ticks removed from the host below about 10x the unfed weight do not experience the latter changes (Kaufman, W.R., Lomas, L., 1996. 'Male Factors' in ticks: their role in feeding and egg development. Invertebrate Reproduction and Development 30, 191-198). In 1984 we named this transitional weight, the 'critical weight' (CW). Its absolute value is probably a species-specific characteristic (Kaufman, W.R., 2007. Gluttony and sex in female ixodid ticks: how do they compare to other blood-sucking arthropods? Journal of Insect Physiology 53, 264-273). Although mated females tend to engorge within a day of surpassing the CW, virgin females surpassing the CW can remain attached to the host for at least several weeks more. It is not known whether the physiological changes in the SGs and ovaries listed above occur in those large virgins that remain attached, although we suppose that this would be maladaptive. Instead, we hypothesize in this study that surpassing the CW is only a prerequisite for inducing these changes, and that detachment is the actual trigger. We support our hypothesis by demonstrating that large virgins, remaining attached to a host for 8 days, did not undergo SG degeneration nor complete egg maturation during the attachment period. Those changes occurred only within 8 days following detachment. So some type of sensory information associated with attachment to the host, and still undefined, inhibits expression of the physiological changes hitherto associated merely with surpassing the CW.

The impact of RNA interference of the subolesin and voraxin genes in male Amblyomma hebraeum (Acari: Ixodidae) on female engorgement and oviposition.

Reducing or replacing the use of chemical pesticides for tick control is a desirable goal. The most promising approach would be to develop vaccines that protect hosts against tick infestation. Antigens suitable for the development of anti-tick vaccines will likely be those essential for vital physiological processes, and in particular those directly involved in feeding and reproduction. In this study genes from Amblyomma hebraeum Koch that encode for subolesin and voraxin were studied in male ticks by RNA interference (RNAi). Males (unfed or fed) were injected with dsRNA of (1) subolesin, (2) voraxin, (3) subolesin plus voraxin or (4) injection buffer, after which they were held off-host overnight and then allowed to feed on rabbits together with normal female A. hebraeum. Females that fed together with male ticks injected with subolesin or subolesin + voraxin dsRNA had a higher rate of mortality, weighed substantially less and produced a smaller egg mass than the controls. However, females feeding with males injected with voraxin dsRNA alone were not significantly different from the controls with respect to mortality, engorged weight or fecundity. However, as assessed by semi-quantitative RT-PCR, voraxin was not silenced in this study, the reasons for which remain unknown. The results of this study suggest that A. hebraeum subolesin is worthy of further testing as a candidate tick vaccine antigen.

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 (0.45microg/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 (6microg20E/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+/-57microm) compared to vehicle-injected controls (132+/-11microm), compared to 20E alone (131+/-12microm), or haemolymph alone (124+/-24microm). There was a marked stimulation of Vg-synthesis by 31microg20E/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+/-3microgVt/mg ovary) or 20E plus haemolymph (56+/-26microgVt/mg ovary) compared to vehicle-injected controls (5.1+/-1.5microgVt/mg ovary). There was also a significant effect of 6microg20E/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.

Antimicrobial activity in the egg wax of the African cattle tick Amblyomma hebraeum (Acari: Ixodidae).

Eggs of the tick Amblyomma hebraeum Koch (Acari: Ixodidae) inhibited the growth of Escherichia coli and Serratia marcescens (Gram-negative bacteria) in solid culture, but not the growth of Staphylococcus epidermidis, and only marginally the growth of Bacillus subtilis (Gram-positive bacteria). When egg wax was extracted with chloroform/methanol (2:1), the extract contained antibacterial activity, but the denuded eggs did not. When assayed against bacteria in liquid culture, the aqueous phase inhibited the growth of S. epidermidis. However, the activity against E. coli was lost during extraction. The antimicrobial component of the aqueous phase was heat stable (100 degrees C for 10 min), resistant to proteinase K (15 min at 55 degrees C) and to pronase (30 min at 37 degrees C). The antibacterial activity in the aqueous phase increased the permeability of the cell membrane of susceptible bacterial cells within 30 min. However, lysis of the cells was detected by optical density measurements (OD(600 nm)) only after 1.5 h. The most evident cytological changes observed by transmission electron microscopy were a thickening of the cell wall and the appearance of numerous electron lucent areas within the cytoplasm of treated bacteria. Gené's organ, the egg-waxing organ in ticks, grew enormously during the first 16 days post-engorgement, and gained antimicrobial activity by day 10 (when oviposition began). This suggests that Gené's organ is the major source of the antibacterial substance in the egg wax. The vitellogenic hormone in A. hebraeum, 20-hydroxyecdysone, when injected into recently engorged females, did not stimulate growth of Gené's organ or precocious secretion of antimicrobial activity.

Pharmacological characterization of the ergot alkaloid receptor in the salivary gland of the ixodid tick Amblyomma hebraeum.

Female ticks of the family Ixodidae osmoregulate by secreting the excess fluid of the blood meal back into the host's circulation via the salivary glands. At least three receptors control salivary fluid secretion in the tick Amblyomma hebraeum: (1) dopamine (DA) stimulates fluid secretion via a DA receptor, (2) ergot alkaloids (ErAs) stimulate fluid secretion via an ErA-sensitive receptor (the natural ligand of which has not been identified), and (3) a GABA receptor potentiates the action of DA and ErAs. Here we present some pharmacological properties of the ErA-sensitive receptor. Of the 11 ErAs we tested, (i) four were complete agonists (approximate concentration eliciting 50% maximum response is given in parentheses): dihydroergotamine (0.02 micromol l(-1)), ergonovine (ErN; 0.06 micromol l(-1)), methylergonovine (0.1 micromol l(-1)) and alpha-ergocriptine (0.9 micromol l(-1)); (ii) three were ;incomplete agonists' (approximate concentration eliciting 20% maximum response is given in parentheses): ergocorninine (3.5 micromol l(-1)), ergocristinine (7.5 micromol l(-1)) and ergocristine (10 micromol l(-1)); (C) three were partial agonists (approximate concentration eliciting the respective maximum response in parentheses): ergocornine (50% maximum by 1 micromol l(-1)), methysergide (28% maximum by 10 micromol l(-1)) and bromocriptine (22% maximum by 10 micromol l(-1)); and (D) one had no activity up to 1 mmol l(-1): ergothioneine. Bromocriptine and methysergide did not antagonize the action of DA, but were effective competitive antagonists of ErN, with K(i)s of approximately 0.3 micromol l(-1) and 11 micromol l(-1), respectively. Ergothioneine was not an antagonist at either the DA- or ErA-sensitive receptor. The putative protein kinase C activators, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DiC8), neither stimulated salivary fluid secretion nor potentiated the action of DA or ErN. The putative protein kinase C inhibitors, bisindolymaleimide (BIM) and calphostin C did not inhibit the action of DA or ErN, although low concentrations of calphostin C (10 nmol l(-1)) appeared to potentiate the action of DA but not ErN. The ion transport inhibitors, furosemide and amiloride (both up to 1 mmol l(-1)), had no significant effect on DA-stimulated or ErN-stimulated fluid secretion.

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.

Partially fed adult female Amblyomma hebraeum ticks were injected with 20-hydroxyecdysone (20E; up to 43 microg/g body weight (bw)), juvenile hormone III (JH III; up to 100 microg/g bw), bovine insulin (up to 2000 mU/g bw), or triiodothyronine (up to 200 ng/g bw) in an attempt to stimulate vitellogenesis. Of these, only 20E stimulated synthesis and release of vitellogenin (Vg). Immunoblot analysis revealed that Vg-synthesis occurred in the fat body. However, consistent with earlier observations suggesting that a distinct signal may be required for Vg-uptake, there was no significant Vg-uptake by oocytes of partially fed, 20E-treated ticks. Because Vg-uptake commonly occurs via receptor-mediated endocytosis (i.e., a specific Vg-receptor), we attempted to identify a vitellin (Vt)-binding protein in ovaries of engorged female ticks. A single 86 kDa Vt-binding protein was identified, even under reducing conditions (2-mercaptoethanol), by a ligand-blotting technique. Sodium salt of suramin (5 mM) inhibited binding of Vt to the 86 kDa protein. However, this protein was also detected in ovaries from small partially fed ticks (50-100 mg), suggesting that the inability of 20E to stimulate Vg-uptake in partially fed ticks may not have been due to the absence of a Vg-receptor.

Two feeding-induced proteins from the male gonad trigger engorgement of the female tick Amblyomma hebraeum.

Most female ixodid ticks, once mated, feed to repletion within 6-10 days. Previous studies indicate that an engorgement factor (EF), passed to the female during copulation, may be the stimulus for engorgement. Here, we show that extracts of the testis/vas deferens of fed (but not unfed) male Amblyomma hebraeum contain EF bioactivity when injected into the hemocoel of feeding virgins. We have produced recombinant proteins (recproteins) from 28 feeding-induced genes in the male gonad and have identified a recombinant A. hebraeum engorgement factor (recAhEF) among these recproteins. recAhEF is a combination of two peptides, recAhEFalpha (16.1 kDa) and recAhEFbeta (11.6 kDa), neither of which has bioactivity on its own. recAhEF also stimulates salivary gland degeneration and partial development of the ovary, suggesting that it may be the same material as another male gonadal protein from this tick, male factor. We propose the name "voraxin" for the natural EF of ticks. When normal mated females were put on a rabbit immunized against recAhEF, 74% failed to feed beyond one-tenth the normal engorged weight within 14 days whereas all mated ticks put on a control rabbit engorged normally (mean duration of 8.8 +/- 0.8 days). This result constitutes preliminary evidence that an anti-tick vaccine might be developed from voraxin.

Rhipicephalus appendiculatus (Acari: Ixodidae): dynamics of Thogoto virus infection in female ticks during feeding on guinea pigs.

Engorged nymphs (Rhipicephalus appendiculatus) were inoculated parenterally with Thogoto (THO) virus (approximately 1 microl per nymph; 10(6)-10(7) PFU/ml). The adult females which resulted were used as the source of infected ticks for this study. Hemolymph, salivary glands, synganglion, gut, ovary, and Malpighian tubules were collected on each day of the blood meal and titrated for THO virus by plaque assay. The percent of tissues infected with virus was 16% or less on the day of attachment. Percent infection rose for all tissues throughout 6-7 days of feeding, reaching 40-100% infection during the rapid phase of engorgement. For the first 4 days of feeding, virus titer in the synganglion was higher than in salivary glands (means of 6.4-34.7 PFU/synganglion and 1.6-8.8 PFU/salivary gland pair). From days 5-7, virus titer was generally higher in the salivary gland than the synganglion (means of 422, 408, and 817 PFU/gland pair and means of 62, 811, and 9 PFU/synganglion). However, because a salivary gland pair is much heavier than a synganglion, the virus concentration in the synganglion was much higher than in the salivary gland during the slow phase of feeding. During the rapid phase of feeding, the difference in virus titer between the synganglion and salivary gland reduced. This difference between the early and late stages of feeding may explain why a previous study [J. Gen. Virol. 70 (1989) 1093], using immunofluorescence and immuno-gold labelling, failed to detect virus in the salivary gland early in feeding. These data provide evidence to explain that R. appendiculatus can transmit THO virus within 24h of attachment, an important epidemiological finding.

Identification and characterization of genes differentially expressed in the testis/vas deferens of the fed male tick, Amblyomma hebraeum.

Most ixodid ticks must feed for at least a few days to complete gonad maturation. Substances produced by the mature male gonad, and carried in the spermatophore, induce physiological changes in the female that lead to engorgement and oviposition. To begin defining the molecular phenotype at this stage of male development, we differentially cross-screened a cDNA library made from the testis and vas deferens of fed ticks (Amblyomma hebraeum Koch) and isolated 35 genes that were putatively up-regulated in tissues of fed compared to unfed animals. While the majority of these were novel, two clones, AhT/VD16 and AhT/VD146, yielded homologies (53 and 44%) to known genes (acylphosphatse and 9.0 kD Drosophila melanogaster basic protein, respectively). Results of Northern blot analysis of AhT/VD16 and AhT/VD146 demonstrate that both clones hybridized with mRNA transcripts that were up-regulated in the testis/vas deferens of fed compared to unfed males. In addition, hybridization of clone AhT/VD16 to water strider (Gerris argentatus) genomic DNA, and sequence similarities to mammalian acylphosphatase, suggest that it represents an evolutionarily conserved sequence.