Moulting

There is a good body of evidence that ecdysteroids, particularly 20-hydroxyecdysone, regulate moulting in ticks as they do in insects and crustaceans (Fig. 1). It has been shown that feeding in vitro of nymphs of the argasid, Ornithodoros porcinus, with porcine blood containing 20-hydroxyecdysone, accelerated moulting (cited in Solomon, Mango & Obenchain, 1982). Surprisingly, similar treatment of nymphs of O. parkeri with 20-hydroxyecdysone (Campbell & Oliver, 1984) or O. moubata with the incompletely hydroxylated ecdysteroid, 22,25-dideoxyecdysone (Diehl et al. 1986) did not affect the moulting period, although the ticks underwent additional moults without taking a further blood meal. However, as with O. porcinus, in nymphs of the ixodid, Hyalomma dromedarii, topical application of 20-hydroxyecdysone speeded up moulting (Khalil et al. 1984). As might be expected, the sensitivity of ticks is highly dependent upon the dose of ecdysteroid administered, the precise developmental time, and the method of exposure (Sonenshine, 1991). By injection of 20-hydroxyecdysone into female O. parkeri, a dose-dependent induction of apolysis and cuticle formation has been demonstrated (Pound, Oliver & Andrews, 1984).

There is also substantial evidence for a close temporal correlation between haemolymph ecdysteroid titres and the moulting process. Haemolymph and whole-body ecdysteroid titres in fifth-stage nymphs of the argasid O. moubata have been carefully correlated with the moulting processes which are induced by the blood meal (Germond, Diehl & Morici, 1982). Between days 2 and 3 post-feeding, when the ecdysteroid titres were low, a few procuticle lamellae were deposited and mitosis initiated. Ecdysteroid titres then began to increase (days 3–4), with the mitotic period ending on day 4. The hormone titres then rose sharply simultaneously with apolysis and the formation of the exuvial space (days 4–5), with highest titres being observed during deposition of the new epicuticle (days 5–6). The titres began to decrease concomitantly with the beginning of procuticle deposition and digestion of the old cuticle (day 6), and started to decrease to low values shortly before ecdysis (days 9–10). Similar correlations have been demonstrated in nymphs of O. parkeri (Zhu et al. 1994) and in the ixodids, Amblyomma hebraeum (Diehl, Germond & Morici, 1982) and A. variegatum (Stauffer & Connat, 1990), with probably a similar situation in larvae of O. moubata (cited in Diehl et al. 1986; Dotson, Connat & Diehl, 1991).

Using well-synchronised A. variegatum, all the integumental events were realised along an anterior-posterior gradient. Of particular significance was the fact that a small peak of ecdysteroid preceded the major peak of hormone during this nymphal-adult moulting cycle. Many structural changes were observed in the integument, salivary glands and dermal glands during this moulting cycle, but additionally, the muscles of the mouthparts and the pharynx underwent a temporary de-differentiation at the end of the first small peak of ecdysteroids. At the end of the second peak of hormone, when the ecdysteroid titre dropped to basal levels, these muscles reappeared in an adult differentiated state (Stauffer & Connat, 1990). This occurrence of two ecdysteroid peaks is reminiscent of the larval-pupal moult in holometabolous insect species.

In all tick species investigated, at high ecdysteroid titres, the principal hormone is 20-hydroxyecdysone, the presumed major active hormone, accompanied by ecdysone (see Delbecque, Diehl & O'Connor, 1978; Dees, Sonenshine & Breidling, 1984a). As in insects, the capacity to transform ecdysone into 20-hydroxyecdysone is widely distributed in tissues, including the midgut (cited in Diehl et al. 1986) and fat body (Zhu, Oliver & Dotson, 1991a).

Certain argasids, but not ixodids, have been induced experimentally to supermoult by administration of ecdysteroids, although adult ticks do not normally moult (Diehl et al. 1986). Apparently, sensitivity to induction of supermoulting depends on the species of tick, the hormone and the type and timing of application. Supermoulting in O. moubata and O. parkeri frequently resulted in failure of ecdysis, which rendered subsequent feeding impossible (Connat et al. 1983a; Campbell & Oliver, 1984). However, in O. porcinus, supermoulting resulted in healthy specimens which could go on to feed, supermoult and oviposit (Mango, Odhiambo & Galun, 1976). Interestingly, the speculation was made in this case that supermoulting could occur in nature when the host feeds on ecdysteroid-rich plants.

Termination of larval diapause

It has been demonstrated that administration of ecdysteroids leads to termination of larval diapause in the ixodids, Dermacentor albipictus and Rhipicephalus sanguineus, although the significance of this to the in vivo situation is unclear (Wright, 1969; Sannasi & Subramoniam, 1972).

Ecdysteroid receptors

In insects, where the molecular action of ecdysteroids is best understood of the Arthropods, this action is mediated by a heterodimeric receptor composed of two members of the nuclear superfamily of receptors, the ecdysone receptor (EcR) and ultraspiracle (USP) (Henrich, Rybczynski & Gilbert, 1999; Koslova & Thummel, 2000; Riddiford, Cherbas & Truman, 2001; Henrich, 2004). USP is the insect homologue of the vertebrate Retinoid X Receptor (RXR) genes, which have a critical role in many nuclear signalling pathways (Mangelsdorf & Evans, 1995). In the case of numerous nuclear receptor ligands, such as ecdysteroids, they exert diverse effects on development, growth and physiological processes (Thummel, 2001, 2002). Such a diversity of effects is frequently due to the existence of multiple forms of the receptor protein that differentially transduce the ligand signals. Multiple forms of receptors can arise from expression of related gene families, to yield receptor sub-types, from alternative RNA processing of a single gene to yield receptor isoforms, or from a combination of both mechanisms (Leid, Kastner & Chambon, 1992).

All nuclear receptors possess a characteristic modular structure (Fig. 2) that includes a variable N-terminal domain (A/B), a highly conserved DNA-binding domain, C, a variable hinge region, D, a conserved ligand-binding domain, E, together with a variable C-terminal region (F) of unknown significance and may be present in some, but not all nuclear receptors (Fig. 2).

Fig. 1. Formulae of ecdysone and 20-hydroxyecdysone.

Fig. 2. The modular structure characteristic of all nuclear receptors. A/B, variable N-terminal domain; C, a highly conserved DNA-binding domain; D, a variable hinge region; E, a conserved ligand-binding domain, and; F, a variable C-terminal region that is present in some, but not all nuclear receptors.

cDNAs encoding three ecdysteroid receptor isoforms that have common DNA- and ligand-binding domains linked to distinct amino termini have been isolated from the ixodid tick, A. americanum (Guo et al. 1997). The DNA- and ligand-binding domains share an average of 86 and 64% identity, respectively, with such domains from insect EcR proteins. In these A. americanum EcRs, the amino termini are highly divergent and they also lack the ‘F’ C-terminal domain occurring in the insect EcRs. Analysis of the cDNAs showed that RNA processing is complex and in addition to producing transcripts with unique amino termini, produces EcR transcripts with different 5′ and 3′ untranslated regions, as well as splicing variants having incomplete open reading frames. Results of Northern blot analyses suggested both stage- and tissue-specific regulation of the EcR mRNA expression.

The vertebrate RXRs are encoded by a multigene family, whereas the insect RXR homologue, Ultraspiracle (USP), is encoded by a single gene. To determine the situation in acarines, cDNAs encoding two RXR homologues, AamRXR1 and AamRXR2 have been isolated from A. americanum (Guo et al. 1998). The DNA-binding domains share ~95% and 87% identity, respectively, with such domains from insect USP and vertebrate RXR proteins. Surprisingly, ligand-binding domains of the AamRXRs are more similar to such domains from vertebrate RXRs than to insect USP ligand-binding domains (~71 vs. ~52%, respectively). The biological significance of this is unclear. Furthermore, Northern blot and RT–PCR analysis revealed both unique and overlapping patterns of AamRXR1 and AamRXR2 expression during development. Both AamRXR1 and AamRXR2 proteins, when paired with any of the three AamEcR isoforms, or the Drosophila EcRB1 isoform, can activate transcription of ecdysone response element-containing reporters. Thus, despite differences in the ligand-binding domains of vertebrate RXRs, AamRXRs, and insect USP proteins, all can pair with either insect or tick EcRs, yielding a functional ecdysteroid receptor in vitro in response to the ecdysteroid, muristerone A (Guo et al. 1998). The foregoing studies on A. americanum EcRs and RXRs have been reviewed (Palmer, Harmon & Laudet, 1999).

As we have seen, the DNA-binding domains of arthropod USPs and their vertebrate homologues, the RXRs, are highly conserved. However, interestingly the ligand-binding domain sequences divide into two district groups: (1) sequences from members of the holometabolous higher insect orders, the Diptera and Lepidoptera, and (2) sequences from vertebrates, a fiddler crab (Uca pugilator), a tick (A. americanum) and a hemimetabolous orthopteran insect, the locust, Locusta migratoria (Hayward et al. 1999). The reason for this evolutionarily sharp divergence of the lepidopteran and dipteran sequences is unknown.

Determination of total body ecdysteroid titres during development in A. americanum revealed that one ecdysteroid peak was observed following larval apolysis. However, two distinct ecdysteroid peaks occurred in the nymphal moulting cycle, the first following apolysis and the second occurring at about the time of ecdysis. Determination of the whole body profiles of the EcR and RXR mRNAs by RT–PCR showed that they were both correlated with the hormone titre (Palmer et al. 2002). However, using an electrophoretic gel mobility shift assay, it has been demonstrated that AamEcR-AamRXR1 (but not AamEcR-AamRXR2) exhibits broad DNA-binding specificity, suggesting that functional differences exists between the RXR1 and RXR2 proteins.

Ecdysteroid inactivation

Before considering ecdysteroid inactivation in ticks, it is germane to summarise such mechanisms in insects, since these are better understood. Ecdysteroid inactivation in insects may occur via several different routes, depending on species, stage in development and tissue (Lafont et al. 2004). The major ecdysteroid inactivation pathways include: (1) Conjugate (ester) formation, primarily polar phosphates or apolar fatty acyl derivatives. Such conjugates were originally identified as inactive maternal, storage ecdysteroid 22-esters in adult females/newly laid eggs for utilization in early stages of embryogenesis before de novo synthesis of hormone in embryos. However, analogous phosphate conjugates (at the C-2, C-3, or C-22 positions of the ecdysteroid) and fatty acyl esters (see Fig. 3) are formed in immature stages of insects; (2) An apparently fairly universal pathway of ecdysteroid inactivation involves ecdysteroid 26-oic acid formation via 26-hydroxyecdsyteroid (see Fig. 5); (3) In many insect systems, irreversible 3-epiecdysteroid formation is emphasised. This occurs via ecdysone oxidase-catalyzed 3-dehydroecdysteroid formation that then undergoes irreversible 3-dehydroecdysteroid 3α-reductase-catalyzed reduction to 3-epiecdysteroid (see Fig. 4). Analogous reactions occur with both ecdysone and 20-hydroxyecdysone.

Fig. 3. Formula of ecdysteroid 22-long chain fatty acyl esters.

Fig. 4. Conversion of ecdysone into 3-epiecdysone.

Fig. 5. Formula of ecdysteroid 26-oic acid.

Most of the work on ecdysteroid inactivation/metabolism in nymphs has been undertaken in O. moubata. In last-instar nymphs, injected [³H]ecdysone was efficiently converted into 20-hydroxyecdysone, the transformation having wide tissue distribution but which is particularly active in the midgut (Bouvier, Diehl & Morici, 1982; cited in Diehl et al. 1986). 20-Hydroxyecdysone is further transformed into putative 20,26-dihydroxyecdysone and all three ecdysteroids are subsequently converted into polar compounds of unknown structures. Significantly, this polar pathway was particularly active when the titre of endogenous ecdysteroids was decreasing, with accumulation of polar products in the digestive tract, thus suggesting its importance for inactivation of endogenous nymphal ecdysteroids. However, it was not operative with ingested ecdysteroids nor in adult females (see below).

However, apolar ecdysteroid 22-long-chain fatty acyl esters are also quickly formed from injected ecdysteroids, but even more rapidly produced from ingested hormones (Diehl et al. 1985; Connat, Diehl & Thompson, 1986a). Such apolar conjugates were then gradually converted into slightly more polar conjugates that are uncharacterized. Interestingly, most ingested ecdysteroid was retained in the digestive tract, where the midgut cells transformed it into apolar conjugates that accumulated in the midgut lumen (Diehl et al. 1986). It had been suggested that the apolar metabolic pathway may serve to inactivate ecdysteroids ingested in blood from hosts feeding on phytoecdysteroid-containing plants (Diehl et al. 1985). Such apolar esters are important and predominant endogenous components in all nymphal stages of O. moubata, particularly in the fourth and fifth stages (Connat et al. 1997). Interestingly, although 20-hydroxyecdysone predominated over ecdysone in all nymphal stages, the proportion of the latter was enhanced in the later three nymphal stages. The ecdysteroid metabolic pathways in fed nymphs of the ixodid, A. hebraeum, were similar to the ones in O. moubata (Diehl et al. 1985).

Undoubtedly, formation of ecdysteroid 22-fatty acyl esters is a prominent metabolic route in ticks. Formation of such esters is widespread in arthropods (Connat & Diehl, 1986; Kubo et al. 1987; Robinson et al. 1987).

Sperm maturation

In male ticks, sperm production and maturation has two phases (for review see Sonenshire, 1991), spermatogenesis (the development of haploid spermatids by meiotic and mitotic division) and spermiogenesis (the growth and maturation of spermatids). Spermatogenesis begins during the nymphal-adult moult and in a few exceptional species proceeds to completion during that moult so that unfed males have mature prospermia and can mate (see Lomas & Rees, 1998). However, in most species, spermatogenesis stops after the development of secondary spermatogonia during adult ecdysis and remains arrested until the adult male begins feeding.

Although the mechanism of reinitiation of sperm development is uncertain, ecdysteroids appear to be involved. Ecdysteroids occur in males (Oliver, 1986a; Oliver & Dotson, 1993) and injection of 20-hydroxyecdysone into unfed males strongly stimulated germ cell DNA synthesis; similarly, injection of a crude synganglial extract could mimic this effect (Oliver, l986a). It is significant that the accumulation of differentiated spermatocytes in D. variabilis nymphs is biphasic and parallels the ecdysteroid titres reported in A. hebraeum (Dumser & Oliver, 1981; Diehl et al. 1982). There is evidence for ecdysteroid synthesis in insect testis under the influence of a brain peptide (Loeb et al. 1987; Jarvis, Earley & Rees, 1994), but such steroid synthesis has not been demonstrated in testes of ticks.

The final growth and maturation of spermatids (spermiogenesis) can be divided further into a growth and elongation phase (resulting in prospermia) and a capacitation phase (sperm maturation; El-Said et al. 1981). Spermatogenesis continues with sperm growth and elongation, resulting in mature prospermia throughout the testis after five days of feeding (Khalil, 1970). The final phase of sperm development, capacitation, requires 24 hours and is triggered by a low molecular weight protein(s) from the male accessory gland (El-Said et al. 1981; Shepherd, Oliver & Hall, 1982).

For further discussion of various factors from male ticks involved in reproduction, see the article by Kaufman in this Supplement.

Regulation of salivary glands

In female ixodid ticks, salivary glands perform several important functions relating to tick feeding and maintaining osmoregulation (for reviews see Kaufman, 1989; Sauer et al. 1995; Sauer, Essenberg & Bowman, 2000). A major function is to secrete excess fluid from the blood meal back to the host, resulting in concentration of the blood meal and regulation of the haemolymph volume. A detailed account of tick salivary gland function and regulation is given in the chapter by Bowman & Sauer in this Supplement.

After the female tick has engorged, the salivary glands degenerate, an event characterized by the appearance of autophagic vacuoles (Harris & Kaufman, 1981). This autolytic degeneration of the salivary glands is triggered by 20-hydroxyecdysone (Harris & Kaufman, 1985; Kaufman, 1991). This hormone also triggers vitellogenesis (Sankhon et al. 1999) and, more recently, a role for ecdysteroid in inhibiting re-attachment to the host has been demonstrated (Weiss & Kaufman, 2001).

The molecular action of ecdysteroid, particularly the role of the receptors, EcR and USP, in salivary gland degeneration has been studied in Amblyomma hebraeum, where the glands degenerate within four days of engorgement. Extensive evidence has been furnished for the existence of a salivary gland EcR in A. hebraeum: (1) [³H]ponasterone A binds to a salivary gland protein (Mao, McBlain & Kaufman, 1995); (2) the specific ponasterone A-binding protein binds to a Drosophila ecdysone response element, hsp27 EcRE, in a sequence-specific manner, the binding being enhanced by biologically active ecdysteroids; (3) monoclonal antibodies against Drosophila EcR and ultraspiracle protein (USP) cross-react with counterparts in tick salivary gland extracts by Western blotting; and (4) the monoclonal antibody against USP retards the hsp27 EcRE-tick EcR complex in a gel mobility shift assay (Mao & Kaufman, 1998).

In A. hebraeum, the profile of the functional ecdysteroid receptor (EcR/USP) determined by [³H] ponasterone A binding, gel mobility shift assays and Western blotting, has been correlated with the haemolymph ecdysteroid titre during the feeding period and six days post-engorgement (Mao & Kaufman, 1999). EcR was undetectable in unfed ticks, but following onset of feeding, specific ponasterone A binding and two major EcR bands detected by Western blots appeared. Both measurements were sustained throughout the feeding period, but declined after detachment when the salivary glands were degenerating. A discrete DNA-binding band, shown by gel mobility shift assay using Drosophila hsp27 EcRE as probe, intensified when haemolymph ecdysteroid titre reached its peak during the rapid phase of feeding, but declined along with decreasing EcR/USP levels and with specific ligand binding activity following engorgement. These results, taken together, substantiate a physiological role for ecdysteroid, acting via nuclear receptors, in salivary gland degeneration. Furthermore, the latter study suggested a role for the small haemolymph ecdysteroid peak during the rapid phase of feeding in initiating salivary gland degeneration, that may constitute a signal somewhat analogous to the ‘commitment peak’ of ecdysteroid in larval Lepidoptera and Drosophila that triggers the behavioural and molecular changes leading to metamorphosis (Mao & Kaufman, 1999).

Regulation of sex pheromone production

The various types of pheromone systems in ticks are considered in the article by Sonenshine in this Supplement, with previous reviews also being available (Sonenshine 1986, 1991; Hamilton, 1992). This short section will consider the involvement of ecdysteroids, particularly in regulation of sex pheromone production.

Evidence has been furnished that ecdysteroids may regulate aspects of sex pheromone production in some species. Production of the attractant sex pheromone, 2,6-dichlorophenol, by the foveal glands begins shortly after the nymphal-adult moult. An increase in 2,6-dichlorophenol production in unfed Hyalomma dromedarii females has been demonstrated when they were stimulated by exogenous 20-hydroxyecdysone prior to moulting (Dees, Sonenshine & Breidling, 1984b, 1985; also see Sonenshine, 1991). Furthermore, a similar increase was observed in unfed females and males when treated with 22,25-dideoxyecdysone (Jaffe et al. 1986).

In addition, ecdysteroids may also be components of pheromone systems per se. In Dermacentor, the genital sex pheromone consists of long-chain saturated fatty acids together with ecdysone and 20-hydroxyecdysone (Allan et al. 1988; Taylor, Sonenshine & Phillips, 1991c).

Regulation of oogenesis and oviposition

In most ticks, feeding and mating are required for oogensis and oviposition to occur. Ixodid females must copulate in order to complete the blood meal, whereas argasid females generally feed to repletion whether or not they have copulated. Whereas ixodid ticks undergo one gonotrophic cycle and die, argasids have several such cycles. It is clear that mating triggers factors regulating oogenesis and oviposition, with involvement of interactions between nervous, endocrine and reproductive systems (for earlier reviews, see Connat et al. 1986b; Oliver, 1986b; Oliver & Dotson, 1993; Lomas & Rees, 1998).

Egg maturation in all arthropods involves synthesis of the major egg yolk protein, vitellogenin (Vg) and its uptake in modified form, vitellin (Vn) by oocytes. In ticks, Vg is synthesized outside the ovary, released into the haemolymph and transported to the ovary, where it is selectively taken up as Vn (Chinzei & Yano, 1985; Rosell-Davis & Coons, 1989), which is a haemoglycolipoprotein (Chinzei, Chino & Takahashi, 1983; Rosell & Coons, 1991; James & Oliver, 1997).

In argasids, there is evidence that the fat body is a site of Vg production (e.g. Chinzei & Yano, 1985; for review see Chinzei, 1986). Clearly, vitellogenesis is under hormonal control in ticks, but there are appreciable conflicting data. In addition to neuropeptides, ecdysteroids and juvenile hormones are the main candidate hormones (see Oliver & Dotson, 1993; Lomas & Rees, 1998). However, the status of the occurrence of juvenile hormones in ticks is uncertain (see below).

In the argasid, O. moubata, Vg production is stimulated by a Vitellogenesis Inducting Factor (VIF) released from the synganglion following the time of coxal fluid secretion during feeding and continues to be released for approximately one hour after feeding (Chinzei et al. 1992). Although the mechanism of action of VIF is uncertain, it has been suggested to trigger the release of ‘Fat body Stimulating Factor’ (FSF), which in turn, stimulates vitellogenesis in the fat body (Chinzei & Taylor, 1990). Although the identity of the FSF is unknown (see Oliver & Dotson, 1993), some evidence exists that it may be a juvenile hormone (Pound & Oliver, 1979; Connat, Ducommun & Diehl, 1983b) with stronger evidence that it may be an ecdysteroid (Sankhon et al. 1999; Taylor, Nakajima & Chinzei, 2000; Ogihara, 2003). Furthermore, evidence has been obtained in three other argasid species that an Egg Development Stimulating Factor (EDSF) from the synganglion may be involved in incorporation of Vg into oocytes (Argas arboreus, Shanbaky & Khalil, 1975; Argas hermanni, Shanbaky et al. 1990; O. parkeri, Oliver et al. 1992).

Administration of ecdysteroids to females of the argasid, O. moubata, not only induced supermoulting, but inhibited oogenesis and caused resorption of previously formed eggs (Diehl et al. 1986). Such action of ecdysteroids has been incorporated into a working hypothesis for possible control of the gonotrophic cycle in O. moubata (Connat, Dotson & Diehl, 1987a). Analogous inhibition of oogenesis by ecdysteroids has also been reported in ixodids, although there are conflicting results as well (see Diehl et al. 1986). Although such inhibitory effects of ecdysteroids on oogenesis might at first sight appear to be difficult to reconcile with the notion of ecdysteroid being the vitellogenic hormone (FSF), the situation is not so different from that in Drosophila where 20-hydroxyecdysone may trigger oocyte resorption or vitellogenesis at certain different precise stages of oogenesis (Soller, Bownes & Kubli, 1999).

In ixodids, there is evidence that the prime sites of Vg synthesis are the fat body and midgut (Rosell & Coons, 1992; see Oliver & Dotson, 1993). Evidence has been furnished in Hyalomma dromedarii for the involvement in Vg production of a synganglion factor similar to VIF in argasids (Shriefer, 1991). Furthermore, in Ixodes scapularis and D. variabilis, a synganglion factor, analogous to EDSF in argasids, appears to stimulate Vg uptake by oocytes (Oliver & Dotson, 1993). Since high doses of 20-hydroxyecdysone also stimulate Vg uptake in I. scapularis, the possibility exists that the synganglion factor might be similar to the ecdysteroidotropic neurohormone (EtNH) identified in A. hebraeum synganglion (Lomas, Turner & Rees, 1997; see ‘Neuroendocrine’ section below).

For correlation of ecdysteroid titres with oocyte maturation, vitellogenesis and some other events in various species, see Lomas & Rees (1998). Recently, good evidence has been furnished for the regulation of vitellogenesis by ecdysteroid in two ixodid species. Using fat body organ cultures and backless explants of unfed female D. variabilis, it has been shown that 20-hydroxyecdysone stimulates vitellogenin production in the fat body trophocytes, whereas the juvenile hormone analogue, methoprene, was without significant effect (Sankhon et al. 1999). Similarly, it has been demonstrated in A. hebraeum that the natural rise in haemolymph Vg concentration lagged slightly behind the rise in haemolymph ecdysteroid and that Vg synthesis was stimulated by injections of 20-hydroxyecdysone into non-vitellogenic females (Friesen & Kaufman, 2002). The pyrethroid insecticide, cypermethrin, stimulates vitellogenesis in O. moubata by stimulating release of the normal VIF from the synganglion, that subsequently triggers the release of FSF, which stimulates yolk synthesis in fat body (Taylor et al. 1991a). However, in A. hebraeum, cypermethrin inhibits vitellogenesis and egg development, possibly in part, due to inhibiting release of 20-hydroxyecdysone (Friesen & Kaufman, 2003). The reason for this discrepancy between these argasid and ixodid species is unclear.

Ecdysteroid inactivation

As alluded to above, ecdysteroid 22-fatty acyl esters are prominent metabolites in immature stages of ticks. The situation is similar in some cases in adult females. For example, in R. appendiculatus, apolar esters occur throughout oviposition and are incorporated, with free ecdysteroids, into the eggs (Magee, Jones & Rees, 1996). Incubation in vitro of ovaries of O. moubata with [³H]ecdysone yielded 20-hydroxyecdysone and apolar 22-fatty acyl esters (Connat, Diehl & Morici, 1984). Furthermore, such esters accumulated in eggs of female O. moubata injected with [³H]ecdysone or 20-hydroxyecdysone during vitellogenesis (Connat, Dotson & Diehl, 1988). The demonstration that such esters remained unchanged during embryonic development and during the moulting cycle of larvae led to the conclusion that in O. moubata, apolar ecdysteroid conjugates are inactivation metabolites that are not re-utilized during development. However, in the ixodid species, Boophilus microplus, the situation is apparently different (see Embryogenesis section below). In this species, the tissue distribution of the production of fatty acyl esters from [³H]ecdysone in vitro was widespread, including Malpighian tubules, ovaries, gut, and fat body (Wigglesworth, Lewis & Rees, 1985). These esters were definitively identified as ecdysteroid 22-fatty acyl esters in eggs of B. microplus (Crosby et al. 1986a). The fact that these esters are utilised in early embryogenesis (see below), suggests that they are within the egg as opposed to be merely components of the egg wax. Similarly, in A. hebraeum, putative ecdysone 22-fatty acyl ester formation occurred rapidly in all tissues incubated in vitro with [³H]ecdysone (Connat, Lafont & Diehl, 1986c). Malpighian tubules and gut had the highest 20-hydroxylase activity, although only low amounts of 20-hydroxyecdysone were formed. In this work, a massive conversion of ecdysone into 3-epiecdysone was observed in ovaries, together with detection of 3-dehydroecdysone the expected intermediate (Fig. 4). In view of the large accumulation in the ovaries of only free endogenous hormones (ecdysone and 20-hydroxyecdysone; Connat et al. 1985), and of incorporation of only free ecdysteroids into the newly laid eggs in in vivo labelling experiments (Connat et al. 1987), the extensive formation of apolar esters in these ovary incubations is an enigma. As suggested for R. appendiculatus (Whitehead et al. 1986), it was surmised (Connat et al. 1987) that in vivo, the ecdysteroids may be bound to vitellogenins/vitellins and thus be protected from metabolism. Surprisingly, 3-epiecdysteroids were also not detected during [³H]ecdysone metabolism in vivo (Connat et al. 1987). It is conceivable that the epimerization enzymes occur in the oocytes for utilisation in vivo during embryogenesis, when 3-epiecdysteroid conjugates are formed as hormone inactivation products (cited in Connat et al. 1986c). The foregoing results demonstrating apolar ester and 3-epiecdysteroid formation in vitro but not in vivo, demonstrate that experiments in vitro may not always accurately reflect the situation in vivo.

The foregoing results, taken together with a comparative study of ecdysteroids in newly laid eggs of various tick species (Connat & Dotson, 1988) indicate that ecdysteroids in eggs either occur in the free form (A. hebraeum and A. variegatum), or as apolar fatty acyl esters (O. moubata and B. microplus), or a mixture of both forms (R. appendiculatus and H. dromedarii). In Amblyomma species, the significance of incorporation of the free ecdysteroids into the eggs is unclear.

After the identification of ecdysteroid 22-fatty acyl esters in newly laid eggs of ticks, such conjugates were also characterized in eggs of some insect species (for review, see Isaac & Slinger, 1989). Such esters may possibly serve as storage forms of maternal ecdysteroids, releasing free hormone during embryogenesis (Slinger & Isaac, 1988; Dinan, 1997), analogous to the situation for 22-phosphate conjugates in locusts (Rees & Isaac, 1984).