Assuring paternity in a promiscuous world: are there lessons for ticks among the insects ?

In this article I begin with a few current ideas on some physiological factors that influence mating choice in insects. Emphasis is placed on those proteins produced by the male reproductive accessory glands which increase female fecundity and reduce her receptivity to subsequent males. Strategies used by late-arriving males to favour their paternity are also mentioned. With a number of insect models as background, I then review what is currently known about several male factors in ticks (a capacitation factor, a male factor, an engorgement factor and a vitellogenesis stimulating factor) and suggest where we might focus our experimental activities in the future.

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection.

Adult Rhipicephatus appendiculatus ticks, infected with Thogoto (THO) virus or control, were fed on guinea pigs and removed at intervals throughout the feeding cycle. Salivary fluid secretion was measured by an in vitro technique. The salivary glands of infected, partially-fed ticks secreted fluid in vitro at about 75% the rate of controls, but the difference between infected and controls among engorged ticks was not statistically significant. Basal and DA-stimulated levels of cyclic AMP (cAMP) were determined in isolated glands and were significantly affected by THO virus infection. The differences in secretory rate among control and infected ticks could not be explained in terms of altered cAMP levels. Haemolymph volume was measured by a tracer-dilution technique using 3H-inulin. The mean haemolymph volume for both THO-infected and control groups was between 23-24% body weight throughout the feeding cycle, indicating that infection by this arbovirus did not influence salivary fluid secretion via altered haemolymph volume. The mechanism by which THO virus affects secretory activity of its tick vector remains unknown.

Molecular individuality and adaptation of the tick Rhipicephalus appendiculatus in changed feeding environments.

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.

Profile of the ecdysteroid hormone and its receptor in the salivary gland of the adult female tick, Amblyomma hebraeum.

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.

Molecular individuality: polymorphism of salivary gland proteins in three species of ixodid tick.

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.

Ecdysteroid regulation of salivary gland degeneration in the ixodid tick, Amblyomma hebraeum: a reconciliation of in vivo and in vitro observations.

Salivary gland degeneration in the female tick Amblyomma hebraeum Koch is triggered by an ecdysteroid (ES) hormone. Under both in vivo and in vitro conditions, degeneration requires 4 days for completion. In partially fed females that have fed beyond a "critical weight," the commitment period for salivary gland degeneration occurs between 24 and 48 h after removal from the host. Although tissue degeneration begins within 24 h postengorgement, ES titer as measured by radioimmunoassay (RIA) does not rise to threshold levels until 48 h postengorgement. To explain this anomaly we examined two hypotheses: (1) there is an early hormonal signal (e.g., 3-dehydroecdysone; 3DE) that is an ES not detectable by the antibody used in our RIA; and (2) the low hemolymph titer during the first 2 days postengorgement is not an accurate reflection of the ES concentration within the tissue itself. 3-Oxoecdysteroid 3 beta-reductase (ketoreductase) was present in salivary glands, but neither ketoreductase nor 3DE was detected in hemolymph. The ES concentration of salivary gland homogenates was similar to that of hemolymph, while that of saliva was undetectable. Together, these results support our second hypothesis that the metabolically active tissue of the salivary gland experiences a suprathreshold concentration of hormone even though the concentration in hemolymph is below threshold levels.

DNA binding properties of the ecdysteroid receptor in the salivary gland of the female ixodid tick, Amblyomma hebraeum.

Salivary gland degeneration in the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae) is controlled by an ecdysteroid hormone. In an earlier study (Mao, H., McBlain, W.A., Kaufman, W.R., 1995. Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum. Gen. Comp. Endocrinol. 99, 340-348), we demonstrated that a protein component of a salivary gland extract binds to ponasterone A (Pon A) with high affinity (Kd-1 nM), suggesting a tick ecdysteroid receptor (EcR). In this study, the Pon A binding protein bound to calf thymus DNA; this binding could be dissociated by Drosophila hsp27 EcRE. The binding protein shifted the [32P]hsp27 EcRE band on a gel mobility shift assay; formation of the complex with hsp27 EcRE required KCl (optimal concentration was approximately 75 mM). A number of physiologically effective ecdysteroids enhanced the binding with the following order of potency: Pon A > Mur A > Mak A > 20E > ecdysone, whereas vertebrate steroids (estradiol, cholesterol, corticosterone, progesterone, testosterone) had no such effect. Using monoclonal antibodies against Drosophila EcR and USP, we found that AG 10.2 recognized three bands (90.5, 87.3 and 84 kDa for EcR) and AB11 recognized at least two major bands (50.3 and 47.1 kDa for USP) in the salivary gland extract by western blot analysis. In addition, AB11 supershifted the tick EcR-hsp27 EcRE band on a gel mobility shift assay, indicating that the tick EcR heterodimerized with a USP-like protein for DNA binding. Furthermore, selective mutations to the 15-basepair palindrome of hsp27 EcRE at positions-5, + 2, or adding a base to the spacer, resulted in considerably reduced affinity to the tick EcR/USP. We thus propose a sequence similarity of EcREs between A. hebraeum and its insect counterpart.

Amblyomma variegatum (Acari: Ixodidae): mechanism and control of arbovirus secretion in tick saliva.

Saliva is considered to be the conduit by which pathogens are transmitted from blood-sucking arthropod vectors to their vertebrate hosts, but supporting evidence for this is fragmentary. To determine if Thogoto (THO) virus, a tick-borne member of the influenza virus family, is transmitted via tick saliva, and whether virus replication is a prerequisite for such transmission, two experimental conditions were compared: (1) "biological transmission" and (2) "mechanical transmission." In (1), THO virus was allowed to infect and replicate in a natural vector, Amblyomma variegatum: virus was detected in saliva collected from 3/22 (14%) ticks. In (2), virus was inoculated directly into the hemocoel with the drug used to induce salivation and saliva was collected immediately to preclude the possibility of virus replication: virus was detected in saliva collected from 31/170 (18%) ticks. The results demonstrate that THO virus is secreted in tick saliva and that virus can pass from the hemolymph to the salivary glands independently of viral replication within the tick. The comparatively low numbers of ticks that yielded virus-positive saliva samples together with the results from assays of serial saliva samples suggested that virus secretion may not be a continuous process during salivation. Ticks in which THO virus had established an infection showed an impaired secretory response compared with uninfected ticks and ticks used for mechanical transmission.

Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum.

Salivary gland degeneration in ixodid ticks is triggered by an ecdysteroid hormone. We used [3H]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 [3H]PoA was thermolabile and sensitive to pronase, but not to DNase or RNase, indicating that the ligand binds to a protein. Scatchard analysis of [3H]PoA binding strongly suggested the presence of an ecdysteroid receptor in cytosolic and nuclear extracts of the tissue. The Kd and Bmax for PoA binding in cytosol were 0.72 +/- 0.09 nM and 175 +/- 12 fmol/mg protein, respectively (n = 8). Corresponding figures for nuclear extract were 1.1 +/- 0.5 nM and 282 +/- 35 fmol/mg protein, respectively (n = 3; P > 0.05 compared to cytosol). The relative ability of unlabeled ecdysteroids to compete for [3H]PoA binding was (in descending order): PoA > muristerone A > makisterone A > 20-hydroxyecdysone > mesylinokosterone > ecdysone. The Kd 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.

Adaptations of arboviruses to ticks.

Arboviruses differ from other viruses in their need to replicate in both vertebrate and invertebrate hosts. The invertebrate is a blood-sucking arthropod that is competent to transmit the virus between susceptible animals. Arboviruses transmitted by ticks must adapt to the peculiar physiological and behavioral characteristics of ticks, particularly with regard to blood feeding, bloodmeal digestion, and molting. Virus imbibed with the blood meal first infects cells of the midgut wall. During this phase the virus must contend with the heterophagic bloodmeal digestion of ticks (an intracellular process occurring within midgut cells) and overcome the as yet undefined "gut barrier" to infection. Genetic and molecular data for a number of tick-borne viruses indicate ways in which such viruses may have adapted to infecting ticks, but far more information is needed. After infection of midgut cells, tick-borne viruses pass to the salivary glands for transmission during the next blood-feeding episode. To do this, the virus must survive molting by establishing an infection in at least one cell type that does not undergo histolysis. Different tick-borne viruses have different strategies for surviving the molting period, targeting a variety of tick tissues. The infection can then persist for the life span of the tick with little evidence of any detrimental effects on the tick. Transmission to a vertebrate host during feeding most probably occurs via saliva that contains virus secreted from infected salivary gland cells. The virus then enters the skin site of feeding, which has been profoundly modified by the pharmacological effects of tick saliva. At least three tick-borne viruses exploit such tick-induced host changes. This phenomenon (saliva-activated transmission) is believed to underlie "nonviremic transmission," whereby a virus is transmitted from an infected to an uninfected cofeeding tick through a host that has an undetectable or very low viremia. Thus tick-borne viruses that have adapted to the feeding characteristics of their tick vectors may not need to induce a virulent infection (with high viremia) in their natural vertebrate hosts. Efficient transmission of tick-borne viruses between cofeeding ticks may be a means of amplifying virus infection prevalence in F1 generations infected by transovarial transmission.

Differential recognition of saliva antigens from the ixodid tick Amblyomma hebraeum (Acari: Ixodidae) by sera from infested and immunized rabbits.

We determined the protein composition and antigenic content of saliva from Amblyomma hebraeum female ticks of different weight classes. The mean protein concentration of saliva of small partially fed ticks (< 100 mg) was 333 +/- 83 micrograms/ml and that of large partially fed ticks (150-420 mg) was 59 +/- 14 micrograms/ml. The reduction in concentration mostly was caused by the significantly higher fluid volume per minute secreted by large ticks. Polypeptide analysis of saliva indicated the presence of a protein (14 kilodaltons [kD]) only in ticks weighing < 60 mg. Other saliva proteins of 21 and 26 kD were present only in ticks weighing < 150 mg, whereas 68-kD protein was absent or very faint in ticks > 100 mg. Immunoblot analysis indicated that sera from rabbits infested with ticks recognized 13 saliva antigens ranging in size from 23 to 200 kD. The antigens were present in detectable quantities in the saliva of small ticks only. In contrast, the sera from rabbits immunized with tick saliva recognized only four antigens from 63 to 200 kD. The 63-kD antigen was not present in the saliva of large ticks. These data indicate that the saliva of A. hebraeum from small ticks is antigenically more complex than that of large ticks and that the route of immunization influences the humoral immune response of the host to the saliva antigens.

Modification of the skin feeding site by tick saliva mediates virus transmission.

A tick vector of Thogoto (THO) virus was shown to secrete a factor in saliva which potentiates the transmission of THO virus to uninfected ticks feeding on an apparently non-viraemic host. The effect of the saliva activated transmission (SAT) factor on the virus occurred at the site of inoculation in the skin and was apparent even when the virus was introduced 3 days after the SAT factor. The results suggest that tick saliva can play an important role in disease transmission by virtue of host modification at the site of feeding.

Effects of the avermectin analogue MK-243 on vitellogenesis and reproduction in the ixodid tick, Amblyomma hebraeum.

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.

An indirect mechanism by which a protein from the male gonad hastens salivary gland degeneration in the female ixodid tick, Amblyomma hebraeum.

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 (approximately 300-400 mg). In mated females, salivary gland degeneration is virtually complete within 4 days of detachment from the host. In virgin females, salivary gland degeneration is delayed by 4 days. This delay can be reversed by the injection of a male reproductive tract homogenate directly into the hemocoel. In this study, we consider a possible mechanism of action for this "male factor." Once mated, male factor likely gains access to its target tissue(s) as a humoral factor. Male factor, however, appears not to act by sensitizing the salivary glands to the action of ecdysteroids. Instead, it appears to act by accelerating the appearance of ecdysteroids in the hemolymph.

Ivermectin is not an agonist at a GABA receptor in tick salivary glands.

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.

Further investigations on the action of ecdysteroids on the salivary glands of the female tick Amblyomma americanum.

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.

Organ culture of ixodid-tick salivary glands.

This work describes an organ-culture method for isolated salivary glands of the ixodid tick Amblyomma hebraeum Koch. The support medium is a TC 199-1% agarose gel with no hormonal or undefined supplements. I used a fluid-transport assay for measuring viability of salivary glands. When cultured at 26 degrees C, female glands secreted fluid at 65% on day 2, 59% on day 4 and 42% on day 6 post-culture compared to glands tested on day 0. Fluid secretory rates in culture were higher than rates of glands from partially fed ticks (about 180-250 mg) on days 2, 4 or 6 post-removal from the host. Salivary glands from fed males cultured for 4 days lost only 5% of their fluid secretory competence, whereas glands dissected from males 4 days post-removal lost 63% of their fluid secretory competence.

Effect of 20-hydroxyecdysone on the salivary glands of the male tick, Amblyomma hebraeum.

Female ticks (Acari: Ixodidae) feed only once in the adult stage, dying after laying a large batch of eggs. During the early post-engorgement stage, haemolymph ecdysteroid titre rises, which is probably responsible for autolysis of the salivary glands that takes place at this time. Males, on the other hand, can re-attach and feed numerous times during the adult stage. Males were fed on rabbits for either 7 or 14 days. Haemolymph was collected either the day of removal from the host or 4 days later, and ecdysteroid titre was measured by radioimmunoassay. The approximate titre in all 4 groups was 20 ng of 20-hydroxyecdysone (20-OHE) equivalents/ml haemolymph. Fluid secretory competence in vitro can be used as an index of salivary-gland degeneration. The glands dissected from fed males which had been left off the host for 4 days lost 62% of their fluid secretory competence compared to glands dissected shortly after the males were removed. This loss in fluid secretory competence was reversed by allowing ticks left off the host of 4 days to resume feeding. Male salivary glands lost fluid secretory competence when exposed for 4 days in organ culture to 20-OHE; the effect was maximal at the lowest concentration tested (20 ng/ml). Thus, although male salivary glands were highly sensitive to 20-OHE, it is still not clear whether this hormone causes the tissue to degenerate.

Tick-host interaction: a synthesis of current concepts.

Ixodid ticks are recognized world-wide as major vectors of arboviruses, rickettsiae, spirochaetes and parasitic protozoa of man and domestic animals. Some ticks also inject a debilitating, sometimes fatal, paralytic toxin in their saliva. All these factors are transmitted via the salivary glands and mouthparts of the feeding tick. Tick feeding is a prolonged and complex process. Major developmental events occurring within the tick during feeding, as well as extensive tick-host interactions, all influence the likelihood of pathogen transmission. In this review, Reuben Kaufman discusses the sequence and complexity of these interactions.