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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.

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.

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.

In brief Analyses of core and peripheral temperatures in young men and women dressed in cold-weather clothing at various levels of cold indicate that the distribution of thermal insulation over the body can complement or confuse physiological heat conservation mechanisms. For short exposures, heavily insulating the hands and feet prevents cold discomfort. However, overinsulating the hands and feet for longer exposures may result in a decreased core temperature and shivering. More thermal insulation over the torso than the hands and feet complements physiological heat conservation mechanisms and maintains core temperature.

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.

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.

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.

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.

Ticks have attracted a great deal of scientific attention primarily because of their role as vectors of numerous pathogens. The majority of tick researchers worldwide focus primarily on microbiological and clinical issues relating to these pathogens, and on methods (pesticidal and biological) for controlling tick populations. Unfortunately, it is often forgotten that ticks are also interesting in their own right to the general biologist because of their unusual physiological (and other) adaptations. Here I review some of these adaptations relating primarily to osmoregulation. (i) I outline their ability to take up water vapour directly from the atmosphere, an adaptation that enables them to withstand desiccation for extended periods while unfed and, in the case of larvae and nymphs, following engorgement. (ii) I present the remarkable filtration-resorption mechanism of the argasid tick coxal organ, analogous to that of the vertebrate glomerular kidney, that enables them to regulate haemolymph fluid volume and composition following the blood meal. (iii) I then turn attention to the salivary glands of female ixodid ticks, which serve the on-host osmoregulatory function in this family of ticks, (iv) and I discuss the pharmacological control of salivary fluid secretion. (v) Finally, I link the latter to the mechanism of pathogen transmission by the salivary glands, using the tick-borne Thogoto virus as a specific example.

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., This research was supported by most generous grants from NSERC Canada (STPG Program) and from the Alberta Science and Research Investments Program (ASRIP) to WRK, the Oklahoma Agricultural Experiment Station (project 1669) and the Walter R. Sitlington Endowed Chair for Food Animal Research (KM Kocan, Oklahoma State University). V Naranjo was funded by Consejería de Educación, JCCM, Spain and AD Smith gratefully acknowledges the Province of Alberta for the award of a Queen Elizabeth II Graduate Scholarhip (Master’s).

Female ticks of the family Ixodidae increase their weight 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 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 weight during the first 3 weeks post moult, during which the ductility and stiffness of the cuticle increased substantially. Under stress, cuticle displayed time-dependent stretch, with a plastic (non-recoverable) and viscoelastic (recoverable) component. Plastic deformation was reasonably constant in the range of 10 to 15% over a wide range of induced stress above ∼0.6 MPa. The plastic component of tick alloscutal cuticle was about five to ten times higher than that of unsclerotized insect cuticle. Tick cuticle is also 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 weight). Injected dopamine (DA) reduced one measure of the viscosity of cuticle by 38%. Plastic deformability of 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 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%.

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.

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.

Injection of the avermectin analogue, MK-243, into engorged female Amblyomma hebraeum Koch resulted in reduced ovary weight, oocyte length, and ovary vitellin content. There was no significant reduction in hemolymph vitellogenin concentration in MK-243 treated ticks. Although MK-243 was previously shown to markedly reduce hemolymph 20E-concentration, injection of 20E, the vitellogenic hormone in this tick, did not reverse the effects of MK-243 on ovary development. These data suggest that MK-243 may exert its inhibition of egg development more at the level of vitellogenin uptake than vitellogenin synthesis. MK-243 also reversed salivary gland degeneration slightly, probably via its inhibitory effect on 20E-synthesis.

The pyrethroid insecticide, cypermethrin (CyM), stimulates vitellogenesis in Ornithodoros moubata (an argasid tick) by stimulating the release of the normal vitellogenesis-inducing factor (a neuropeptide) and subsequent release of the vitellogenic hormone [19]. Here we examine the effects of CyM on egg development in the ixodid tick, Amblyomma hebraeum. Ovary weight, oocyte size, and vitellin content of the ovary were measured after CyM treatment; in partially fed ticks, none of these parameters were affected significantly. However, CyM treatment caused an inhibition of ovary development, as well as reduction of both hemolymph 20-hydroxyecdysone (20E; the vitellogenic hormone in this species) and vitellogenin (Vg)-concentrations in engorged ticks. In addition, the degree of salivary gland degeneration (which is triggered by 20E) was slightly reduced in CyM-treated engorged ticks. These results indicate that CyM acts differently in Amblyomma compared to Ornithodoros. Instead of stimulating vitellogenesis, CyM inhibits egg development perhaps in part as a result of inhibiting release of 20E.

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.

The tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) naturally infests many host species. However, the mechanisms that enable it to feed on such a wide range of hosts are unclear. One possibility is that a tick population maintains molecular (genotypic and/or phenotypic) diversity among individuals such that individuals vary in their competency in taking bloodmeals under different feeding conditions. As a first step in testing this hypothesis, we showed that the polymorphism of salivary gland proteins, previously demonstrated in unfed ticks, was maintained during feeding on guinea-pigs. We then compared feeding performance under standard laboratory rearing conditions: one instar (adults or nymphs) feeding on guinea-pigs, with three changed conditions: (1) two instars (adults and nymphs) feeding together on guinea-pigs; (2) one instar (adults or nymphs) feeding on hamsters; and (3) two instars (adults and nymphs) feeding together on hamsters. The mean engorged weight of adult females was significantly reduced under all changed conditions, indicating that most of the adult individuals were significantly challenged by the changed conditions. However, some individuals achieved successful engorgement, indicating competence to the changed condition, and demonstrating variation in adaptive ability among individuals. Engorged females produced egg masses positively correlated to the engorged weights. More interestingly, the correlation coefficient (R) increased when feeding condition was changed. This may lead to more efficient selection for population adaptation under the changed conditions. As the feeding success of ixodid ticks depends on the efficiency of the cocktail of immunomodulatory saliva, the relevance of the polymorphism of salivary gland proteins and host adaptation is discussed.

THE FEEDING CYCLE OF ADULT FEMALE TICKS (ACARI: Ixodidae) is divided into preparatory, slow and rapid feeding phases. At the transition from slow to rapid feeding, Amblyomma hebraeum (Koch) females reach a 'critical weight' (CW; approx. 10x the unfed weight) that is characterized by several behavioural and physiological changes. Five of these changes were used as criteria to establish a more precise estimate of CW than we have to date. The CW as defined by re-attachment to the host was 9x the unfed weight, while for haemolymph ecdysteroid titre, salivary gland degeneration, ovary weight, oocyte length and oocyte vitellin content the CW was 10x, 10x, 12x, 12x and 13x, respectively. CW thus varies depending on the parameter measured. Although previous studies have established the influence of ecdysteroids on salivary gland degeneration and vitellogenesis, here we demonstrate a further effect of ecdysteroids: inhibiting re-attachment to the host.

Adult Rhipicephalus appendiculatus ticks, infectedwith Thogoto (THO) virus or control, were fed on guinea pigs and removed atintervals throughout the feeding cycle. Salivary fluid secretion was measuredbyan in vitro technique. The salivary glandsof infected, partially-fed ticks secreted fluid in vitro at about 75% the rateof controls, but the difference between infected and controls among engorgedticks was not statistically significant. Basal and DA-stimulated levels ofcyclic AMP (cAMP) were determined in isolated glands and were significantlyaffected by THO virus infection. The differences in secretory rate amongcontroland infected ticks could not be explained in terms of altered cAMP levels.Haemolymph volume was measured by a tracer-dilution technique using3H-inulin. The mean haemolymph volume for both THO-infected andcontrol groups was between 23–24% body weight throughout the feedingcycle, indicating that infection by this arbovirus did not influence salivaryfluid secretion via altered haemolymph volume. The mechanism by which THO virusaffects secretory activity of its tick vector remains unknown.

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.

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 μg per g body weight) and 6-hydroxydopamine (1000 μg 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

We examined the hemolymph ecdysteroid titer (by radioimmunoassay) and profile of the ecdysteroid receptor (EcR/USP; by [3H]ponasterone A binding, gel mobility shift assay, Western blot) in the salivary gland of the ixodid tick, Amblyomma hebraeum Koch (Acari: Ixodidae) throughout the tick feeding period and first 6 days post-engorgement. Throughout the slow phase of feeding, the hemolymph ecdysteroid titer was approximately 18 pg/microliter. The titer peaked at approximately 52 pg/microliter during the rapid phase of feeding, falling back to approximately 22 pg/microliter on the day of engorgement. Ecdysteroid titer rose again to approximately 750 pg/microliter by day 6 post-engorgement. EcR was undetectable by any of the three assays in unfed ticks. Following the onset of feeding, there appeared both specific ponasterone A binding and two major EcR bands detected by Western blot analysis. Both measurements were sustained throughout the feeding period, but declined after detachment when the salivary glands were degenerating. After ticks reached about 100 mg (by which time most females are mated), a discrete DNA-binding band was shown by gel mobility shift assay using Drosophila hsp27 EcRE as a probe. Moreover, the band intensified when hemolymph ecdysteroid titer reached its peak during the rapid phase of feeding; it declined along with decreasing EcR/USP levels, and with specific ligand binding activity following engorgement. This study suggests a role for the small hemolymph ecdysteroid peak during the rapid phase of feeding in initiating salivary gland degeneration.

Pooled tissue samples are frequently used in biochemical studies involving parasites in order to ensure that there is sufficient material for experimentation. A pooled sample is considered to represent the overall phenotypic characteristics of the investigated population. However, this will not be the case if there is a significant degree of molecular polymorphism among individuals in the sampled population. Here we demonstrate marked differences in the protein profile of salivary glands among individuals from three species of ixodid tick (Rhipicephalus appendiculatus, Amblyomma variegatum, Ixodes ricinus), and show that pooling the tissue of several individuals masks substantial qualitative differences among the individuals. Our observations indicate that much greater caution is needed in general when using pooled samples if the molecular diversity within the population is not clearly defined.

Using an rf solenoid magnet, we studied the depolarization of a stored 104.1 MeV vertically polarized proton beam. The two primary rf depolarizing resonances were properly centered around the protons{close_quote} circulation frequency f{sub c}, at f{sub c}(3{minus}{nu}{sub s}) and f{sub c}({nu}{sub s}{minus}1), where {nu}{sub s} is the spin tune; moreover, each resonance was roughly consistent with the expected width of about 720 Hz. Each primary rf resonance had two synchrotron sideband resonances at the expected frequencies. The two {nu}{sub s}{minus}1 sidebands were deep dips while the two 3{minus}{nu}{sub s} sidebands were very shallow; this was not expected. Moreover, all four sideband resonances were unexpectedly wider than the two primary resonances. {copyright} {ital 1998} {ital The American Physical Society}

We investigated the effect of physiological state of adult ticks (Amblyomma hebraeum Koch; Acari: Ixodidae) on their responses to a CO2-enriched atmosphere. Ticks were exposed to CO2 or air in an experimental chamber; we measured the effects on questing, movement and latency (the time after exposure to CO2 or air when ticks began questing or moving). This study confirmed the hypothesis that responsiveness to CO2 is enhanced during host-seeking periods of the life cycle and reduced at other times. Thus, whereas newly moulted ticks quested infrequently when exposed to CO2, questing increased over the 6 week period following moulting. Likewise, in 6 week old ticks, movement in CO2 was significantly greater than in air. When a partially fed female below approximately ten times its unfed weight is removed from a host, it reattaches if subsequently presented to another host. Such females quested vigorously in CO2 but not in air. When a partially fed female above approximately ten times its unfed weight is removed from a host, it usually does not subsequently reattach. Such females quested infrequently, there being no significant difference between air and CO2. Males removed from a host at any stage of feeding reattach readily when given the opportunity. They quested frequently, moved readily and the latency was very low in air or CO2. Exp Appl Acarol 22: 667–681 © 1998 Kluwer Academic Publishers

We have identified the full-length cDNA encoding a vitellogenin receptor (VgR) from the African bont tick Amblyomma hebraeum Koch (1844). VgRs are members of the low-density lipoprotein receptor superfamily that promote the uptake of the yolk protein vitellogenin (Vg), from the haemolymph. The AhVgR (GenBank accession No. JX846592) is 5703 bp, and encodes an 1801 aa protein with a 196.5 kDa molecular mass following cleavage of a 22 aa signal peptide. Phylogenetic analysis indicates that AhVgR is highly similar to other tick VgRs. AhVgR is expressed in only the ovary of mated, engorged females, and is absent in all other female tissues and in both fed and unfed males. Unfed, adult females injected with a VgR-dsRNA probe to knock-down VgR expression experienced a significant delay in ovary development and started oviposition significantly later than controls. These results indicate that the expression of AhVgR is important for the uptake of Vg and subsequent maturation of the oocytes.

Summary Female ticks of the family Ixodidae utilize their salivary glands as the major organs for fluid balance, secreting back into the host a dilute saliva. Feeding is composed of three phases: a preparatory phase (1–2 days) during which the tick establishes the feeding lesion, a slow phase (~7 days) during which body weight increases 10-fold, and a rapid phase (~1 day) in which body weight increases a further 10-fold. Following engorgement, the salivary glands are resorbed by an autolytic process triggered by an ecdysteroid hormone. If a female is removed from the host prior to repletion, her subsequent behaviour depends mostly on two factors: the degree of engorgement achieved and whether or not she has mated. If removed during the preparatory or slow phase of engorgement, the salivary glands are not resorbed, the tick will lay virtually no eggs and she will reattach to a host if given the opportunity, all of this irrespective of whether she is virgin or mated. If removed during the rapid phase of eng...

Ecdysteroids cause salivary gland degeneration in female ixodid ticks. We tested the effects of the following compounds on salivary gland degeneration in the ixodid tick Amblyomma hebraeum Koch: HHCS (22 S ,23 S -homocastasterone), SSBR (22 S ,23 S -homobrassinolide), STGM (2α,3α(OH) 2 -Δ 22 -stigmasten-6-one), RH 5849, and RH 5992. The first three are brassinosteroids (putative ecdysone antagonists) and the last two are nonsteroidal mimics of ecdysone in a variety of insects. In vitro, HHCS (up to 4 μg/ml, 8.4 μ M ) did not attenuate degeneration caused by 20-hydroxyecdysone; on the contrary, it enhanced the degree of salivary gland degeneration. SSBR (up to 4.5 μg/ml, 9 μ M ) likewise did not reduce 20-hydroxyecdysone mediated degeneration. RH 5849 up to 15 μg/ml (51 μ M ) and RH 5992 up to 10 μg/ml (28 μ M ) had no ecdysone-mimicking effect. In vivo, both RH compounds had an ecdysone-mimicking effect. RH 5849 (but not RH 5992) at 10 μg/tick increased ovary wet weight slightly. None of the brassinosteroids displaced a significant amount of [ 3 H]ponasterone A (PoA) from the ecdysone receptor at 6 μg/ml (12–14 μ M ) or below. RH 5849 (14 μg/ml, 47 μ M ) displaced 8% of (PoA) binding; at 68 μg/ml (230 μ M ) displacement was 30%. For RH 5992 inhibition of PoA binding was 16, 33, and 43% at 4.5, 16, and 79 μg/ml (13, 45, and 79 μ M ), respectively. Overall, the brassinosteroids and the RH compounds do not act on the tick salivary gland ecdysteroid system in a way similar to the way they act on some insect systems.

We assayed a variety of tick (Amblyomma hebraeum Koch; Acari, Ixodidae) tissues for a number of amino acids throughout the feeding and early reproductive periods. Our HPLC assay could detect as little as 2–5 pmol per sample of the following: GABA, glycine, serine, glutamine, alanine, taurine, glutamate and aspartate. All of these amino acids could be detected in the salivary gland, synganglion (=total CNS in acarines), haemolymph, Gene's organ, seminal receptacle and ovary. GABA reached high levels in the salivary gland of freshly engorged ticks (685 nmol g-1) and in the synganglion it exceeded 1000 nmol g-1 throughout most of the feeding cycle and the first week post-engorgement. GABA also reached a peak titre in the haemolymph of 40 nmol ml-1. Taurine levels peaked at 1065 nmol g-1 in the salivary gland from large partially fed ticks. Glutamate and aspartate were likewise found in the salivary gland and synganglion at high concentrations. For most of the amino acids there is insufficient information to correlate these titres (and fluctuations of titres) to neuromodulatory functions. It is possible, however, that the high GABA titre in the salivary gland of engorged ticks is correlated with an augmented level of fluid secretion.

Salivary gland degeneration in ixodid ticks is triggered by an ecdysteroid hormone. We used [ 3 H]ponasterone A (PoA) as a specific ligand to detect the ecdysteroid receptor in the salivary glands of large partially fed female ticks ( Amblyomma hebraeum Koch; Acari; Ixodidae). Binding of [ 3 H]PoA was thermolabile and sensitive to pronase, but not to DNase or RNase, indicating that the ligand binds to a protein. Scatchard analysis of [ 3 H]PoA binding strongly suggested the presence of an ecdysteroid receptor in cytosolic and nuclear extracts of the tissue. The K d and B max for PoA binding in cytosol were 0.72 ± 0.09 n M and 175 ± 12 fmol/mg protein, respective ( n = 8). Corresponding figures for neclear extract were 1.1 ± 0.5 n M and 282 ± 35 fmol/mg protein, respectively ( n = 3; P > 0.05 compared to cytosol). The relative ability of unlabeled ecdysteroids to compete for [ 3 H]PoA binding was (in descending order): PoA > muristerone A > makisterone A > 20-hydroxyecdysone > mesylinokosterone > ecdysone. The K d estimated for 20-hydroxyecdysone (probably the natural hormone) correlates very well with its physiological potency in inducing salivary gland degeneration in vivo and in organ culture. None of the vertebrate steroids tested (estradiol, testosterone, progesterone, and corticosterone) was able to displace PoA binding at a concentration 10 5 times higher than PoA. The cytosolic form of the receptor migrated to the 3.2 S region of a 10-40% sucrose density gradient.

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.

We formed the first ``atomic-optics'' beam of electron-spin-polarized hydrogen atoms using a quasiparabolic polished copper mirror coated with a hydrogen-atom-reflecting film of superfluid $^{4}\mathrm{He}$. The mirror was located in the gradient of an 8-T solenoidal magnetic field and mounted on an ultracold cell at 350 mK. After the focusing by the mirror surface, the beam was again focused with a sextupole magnet. The mirror, which was especially designed for operation in the magnetic field gradient of our solenoid, increased the focused beam intensity by a factor of about 7.5.

We investigated two methods of producing a continuous free beam of electron-spin polarized atomic hydrogen, using a 7.5 T solenoid magnetic field and a helium film-coated cell. The first method involves accumulating H ↓ at high field in a 300 mK storage cell and flipping the electron spin by using microwaves to drive a hyperfine transition. The resulting H↑ atoms are accelerated and focused by the solenoid gradient to form an extracted beam. The second method uses the helium film-coated cell as an ultra-cold nozzle. Unpolarized hydrogen is thermalized in high field by collisions with the cell walls; the field gradient subsequently separates the atoms of different spin states to produce an H↑ beam.

Summary Hormonal control of egg development in the ixodid tick Amblyomma hebraeum Koch was investigated as follows: ovaries from small partially fed or fully engorged females were transplanted into the haemocoel of engorged females that were undergoing vitellogenesis; the transplants responded to this milieu by completing oocyte maturation. The following treatments all failed to stimulate egg development within eight days in small partially fed ticks: (a) slow fusion of 20-hydroxyecdysone (up to 7.2 μg) over 24 h; (b) topical application or injection of up to 50 μg juvenile hormone III; (c) topical application of 10 μg of methylfarnesoate and juvenile hormone III bisepoxide; (d) a combination of 20-hydroxyecdysone and juvenile hormone III. Also, none of the latter treatments accelerated oocyte maturation in engorged females. Injections (up to 100 μg per tick) or topical application (1 mg per tick) of precocene II did not inhibit egg development in engorged females which survived this treatment. Thus, alth...

Analytic calculations and particle tracking simulations are presented for a polarized atomic hydrogen beam produced by extraction from a superfluid helium coated cell in a large solenoidal magnetic field. Compression of the beam by the solenoidal field and subsequent focusing by a sextupole are considered.

In the adult female tick, Amblyomma hebraeum Koch (Acari: Ixodidae), salivary gland degeneration is triggered by an ecdysteroid, provided the female is above a critical weight (about 300–400 mg). In mated females, salivary gland degeneration is complete within 4 days of detachment from the host. In virgin females, salivary gland degeneration is delayed by 4 days. This delay in salivary gland degeneration could be prevented in virgin females by injecting a homogenate of male gonad into the haemocoel. All “male factor” activity associated with the male gonad resides exclusively within the testis/vas deferens/ejaculatory duct; no male factor activity was associated with the spermatozoa or the accessory gland portion of the male gonad. Male factor activity within the gonad increases markedly after males have fed for 5 days. Male factor can be destroyed by either heat treatment or by proteinase K digestion, suggesting that it is a protein. Some male factor activity was also found in the synganglia of either fed, mated males or females, and the ovary from fed mated females, but not in male salivary glands.

A dose-dependent reduction in weight of total egg mass and a slightly increased oviposition latency were observed following injection of the avermectin analogue MK-243 (4"-epi-methylamino-4"-deoxyavermectin B1) directly into the haemocoel of engorged female Amblyomma hebraeum Koch (Acari: Ixodidae). Egg laying was almost completely inhibited at 100 micrograms/kg body weight. MK-243 markedly inhibited ovary development and vitellogenesis. Ticks treated with 100 micrograms MK-243/kg also had one-tenth the haemolymph ecdysteroid concentration compared to controls 10 days post-engorgement. Thus, among its other effects on ticks, the avermectins also inhibit the process of vitellogenesis.

The normal engorged body weight of female ixodid ticks (Acari: Ixodidae) is about 100× the unfed weight. Virgin female Amblyomma hebraeum normally do not feed beyond 10× the unfed weight. However, about 10–20% of a population of virgins will feed to perhaps 20× the unfed weight, but not much beyond that. In A. hebraeum, when females surpass about 10× 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 10× 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 avermectins, broad-spectrum anti-parasitic agents which are effective against ticks, act principally by stimulating gamma-aminobutyric acid (GABA) receptors directly or indirectly. GABA is known to potentiate dopamine-induced fluid secretion in the salivary glands of female ixodid ticks. We thus tested whether 10 microM ivermectin would have an effect similar to GABA in the isolated salivary gland preparation of Dermacentor andersoni. Ivermectin was not an agonist at this GABA receptor.

Two phytecdysteroids (abutasterone, makisterone A) and five synthetic ecdysteroid analogues, all at 1 microgram/ml, were tested on salivary glands from the female tick, Amblyomma americanum L. (Acari:Ixodidae), held in organ culture for four days. All of these substances caused a significant reduction in fluid secretory competence of salivary glands in vitro. This constitutes further evidence that the structural requirements for causing salivary-gland degeneration in ticks are similar to those generally required for ecdysteroid activity in other arthropods. Although vertebrate steroids are known to augment fluid secretory competence by salivary glands in organ culture, beta-estradiol was not able to attenuate the degenerating effect of 20-hydroxyecdysone, supporting the suggestion that ecdysteroids and vertebrate steroids have distinct sites/mechanisms of action on this tissue.

The storage of ultracold spin-polarized hydrogen atoms offers the possibility of producing a high intensity nuclear polarized atomic hydrogen jet. We stored electron spin polarized atomic hydrogen at 0.4 K in an open 5 T magnetic storage cell. We also observed directly, for the first time, the extraction of hydrogen atoms from the storage cell by flipping their spins using a microwave driven transition. The results are being used to design a high intensity jet of nuclear polarized atomic hydrogen to be used as an internal target in the 400 GeV to 3 TeV UNK accelerator.

In the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae), the salivary glands degenerate and vitellogenesis begins, provided the tick exceeds a critical weight (approx. 300–350 mg). The following were measured in ticks: (1) at various times following normal engorgement (1000–3000 mg) and (2) at various stages (weights) of the feeding cycle, 7 days after removal from the host: salivary fluid secretory competence and ecdysteroid content (by radioimmunoassay) of haemolymph and ovary extracts. (1) Haemolymph ecdysteroid titre rose from

INTRODUCTION There is a broad tendency among female insects for fecundity to increase with mating frequency (Ridley, 1988). A female's eggs carry forward to the next generation her genes and those of her mate(s). Hence, one might guess that promiscuity should be of selective advantage for both sexes. On closer inspection it is not as simple as that. The survival of a female's offspring is as much a function of her mate's fitness as it is of her own, so she has some interest in determining the paternity of her offspring (Birkhead, 1998; Eberhard, 1998; Wedell & Karlsson, 2003). On the other hand, how can the male's paternity be assured if his spermatozoa have to compete with those of other males in the same arena? In this chapter some of the mechanisms used by male insects that assure their paternity in this ‘promiscuous world’ are reviewed. These will be compared to what is known from the less extensive literature on ticks, with the object of determining whether there are lessons from insects that might point our way to future enquiries in ticks. SPERM PRECEDENCE AND PATERNITY Because of the proverbial battle of the sexes, as well as that among competing males, one encounters numerous mating strategies among terrestrial arthropods, and a fascinating literature on sperm competition to match. The degree to which sperm competition occurs in any instance is determined by a number of factors (reviewed extensively by Simmons & Siva-Jothy, 1998, and by Parker, 1998): (1) How readily will a previously mated female accept further males (sperm pre-emption)?

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.

We measured the analyzing power A out to ${\mathit{P}}_{\mathrm{\ensuremath{\perp}}}^{2}$=7.1 (GeV/c${)}^{2}$ with high precision by scattering a 24-GeV/c unpolarized proton beam from the new University of Michigan polarized proton target; the target's 1-W cooling power allowed a beam intensity of more than 2\ifmmode\times\else\texttimes\fi{}${10}^{11}$ protons per pulse. This high beam intensity together with the unexpectedly high average target polarization of about 85% allowed unusually accurate measurements of A at large ${\mathit{P}}_{\mathrm{\ensuremath{\perp}}}^{2}$. These precise data confirmed that the one-spin parameter A is nonzero and indeed quite large at high ${\mathit{P}}_{\mathrm{\ensuremath{\perp}}}^{2}$; most theoretical models predict that A should go to zero.

Progress is reported on t l~e p~uiluction of an intense polarized atolnic hydrogen beam using microtx,nre rli iven cs t rac t io~~ of stal)ilizrd a ttsnlic 1lgdlr)gcn. Electrc.)n ~ ~ i n ~ ~ o l i ~ r i z e t l a.t.olnic ytlrog~n call 1,s stored at 11igl1 drilsit,ies over rela.t,ivelg lol~g taillie 1)eriods in a higll nlz1.gpet.i~ field (2 5 T ) nntl a.t 1t)tv tcmpera.tmes (2 0.8 Ii). D. Iileppner and T. 0. ~iinikoskil proposed t,ll;l.t. R hiall tl(msit,y a.to~llic h~-drogen beam could I,e prod~~cetl I,y using clect,rol~ spill rcsona.~lce (ESR.) plulllping to est,r:~.ct 11)-dmge~i atollls froill a. nlagiletic storage l>ottlc. Such a ~~~ic ro~vave dri~ien est~mction process cvulrl l,rovitle a.11 i~ltc,lisc lonv velocity sl>in-pola.rized atomic bcaln with il, na.rrom relocit,y tlist,ribut.ion; it should be llossil,le t,o at,t,;l.in a, d(,i~sit,y of about 1014 atoms/cln2 l>>? using ll~aglletic focusil~g. We to use such a. sl,i~l-l)olarizetl jet t.o st ,~~tly l ) ~ elast,ic sca.tt.eriilg at 400 GcV and 3 TeV in t,lle NEPTUN-A experilnellt at, U N I ~ " 011r al,llaratus'~ was designetl specifically t,o develop the t,ecll~~iql~e of proclucillg a high density aton~ic l1ydrogffn 11eam by nlicrr)xvas-e tlriven estractio~l. Tlle vertical sectioll cont,a,inetl a, conti~l~~ous flow dilution refrigerat,or xt-ibh R, cooling poxxler of 27 ~ I F V at 0.35 I