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Ecdysteroid Titer and Metabolism and cuticle deposition during embryogenesis of the ixodid tick amblyomma-hebraeum (Koch)
Three embryonic cuticles are formed before larval cuticle deposition during embryonic development of Amblyomma hebraeum. The quantity of radioimmunoassay-positive material varied between 50 and 200 pg ecdysone equivalents per mg, but no significant peaks were detected. Maternally incorporated [H-3]-20-hydroxyecdysone and [H-3]-ecdysone contained in freshly laid eggs appear to be conjugated to C-22 fatty acid esters and 3 alpha epimers of those esters and, thus, appear doubly inactivated. In addition, ecdysone is converted to an unknown product called 2'. The role of these maternally derived ecdysteroids is unknown.
Possible inactivation of ingested ecdysteroids by conjugation with long-chain fatty acids in the female tick Ornithodoros moubata (acarina:argasidae)
Ornithodoros moubata females proved to be extremely sensitive to ingested 22,25-dideoxyecdysone; 15-20 ng provoked molting in all females and temporarily inhibited vitellogenesis. In contrast, this tick was very resistant to ingested ecdysteroids containing 22-OH groups, such as ecdysone, 20-hydroxyecdysone, ponasterone A, and makisterone A. Dosages about 500 times greater were necessary to produce supermolting and reduce fecundity [Connat et al: Z Ang Ent 96, 520 (1983)]. Ingested tritiated ecdysone, 20-hydroxyecdysone, 2-deoxyecdysone, and ponasterone A were rapidly converted to apolar esterase-labile metabolites having approximately the same retention time as the AP2 identified as esters of ecdysteroids at C-22 with long-chain fatty acids (C16:0, C18:0, C18:1, C18:2) [Diehl et al: Int J Invert Reprod Dev 8, 1 (1985)]. These products were then gradually transformed to the more polar apolar conjugates, AP1. A more detailed study with ingestion of large quantities of 20-hydroxyecdysone (10 μ/ml blood) demonstrated that only small amounts of free hormone were present in the hemolymph during the first day after the blood meal. The hormone was rapidly metabolized to AP2, then to AP1, in the intestinal cells and to a lesser extent in the peripheral tissues. Finally, AP1 accumulated in the intestinal cells and midgut content, probably because excretion outside the animal is impossible in this tick species. In contrast, ingested 22,25-dideoxyecdysone was not metabolized to apolar products. This could account for its high biological activity. This compound was converted to unidentified more polar products. Two of them comigrated with ecdysone and 20-hydroxyecdysone on RP-18 HPLC column, but not on silica column, and therefore cannot correspond to these compounds.
We hypothesize that esterification of ecdysteroids at the C-22 position with fatty acids represents a detoxification mechanism for ingested ecdysteroids that might be present in blood from herbivorous or parasite-infected hosts.
Some aspects of the control of the gonotrophic cycle in the tick Ornithodoros moubata (Ixodoieda, Argasidae)
Juvenile hormone-like substances can induce vitellogenesis in the tick Ortnithodoros moubata (Acarina : Argasidae)
Topical application of different juvenile hormone analogs (JHA) or of a mixture of stereoisomers of insect juvenile hormone (JH) 1 and 3 to fed virgin female Ornithodoros moubata immediately after feeding induced vitellogenesis and egg-laying in up to 70 of treated females. In controls only 13.7 oviposited. The eggs were sterile, with abnormal shape, but their number versus the weight of engorged females was normal or sometimes greater than in mated females. However, preoviposition period was longer than in mated females.
It was more difficult to induce egg-laying by similar topical applications 100 days after feeding of virgin females. A maximum of 58 of ovipositing females was obtained with a very high dosage of JH mixture (500 μg). Injection of this mixture into the females was more potent; 15 to 50 μg induced oviposition in about 60 of the females. The preoviposition period was also longer than in control females.
Our results suggest the presence of a JH-like substance which is involved in the hormonal control of vitellogenesis. However, since natural isomers of JH were much less efficient than isomeric mixtures or JHA, we suppose that the natural tick hormone does not correspond to JH, but rather to a JH-like substance.
Ecdysteroid titre and metabolism and cuticle deposition during embryogenesis of the ixodid tick Amblyomma hebraeum (Koch)
Three embryonic cuticles are formed before larval cuticle deposition during embryonic development of Amblyomma hebraeum. The quantity of radioimmunoassay-positive material varied between 50 and 200 pg ecdysone equivalents per mg, but no significant peaks were detected. Maternally incorporated [3H]-20-hydroxyecdysone and [3H]-ecdysone contained in freshly laid eggs appear to be conjugated to C-22 fatty acid esters and 3 α epimers of those esters and, thus, appear doubly inactivated. In addition, ecdysone is converted to an unknown product called 2′. The role of these maternally derived ecdysteroids is unknown.
A new class of apolar ecdysteroid conjugate: esters of 20-hydroxy-ecdysone with long-chain fatty acids in ticks
Nymphs of the argasid tick Ornithodoros moubata rapidly transform most of the ingested molting hormone 20-hydroxy-ecdysone (20-hydroxy-E) into a family of 4 major apolar conjugates which are hydrolyzable by carboxylic-ester hydrolase. These conjugates - new among zoo- or phytoecdysteroids - are composed of 20-hydroxy-E esterified at C22 with the common long-chain fatty acids C16:0, C18:0, Cig18:1, or C18:2;. The chemical structure of the purified metabolites was identified by fatty acid analysis, by CI/D mass spectrometry and by comparison with a chemically prepared 20-hydroxy-E-22-palmitate.
The significance of the apolar metabolic pathway is discussed. It may serve to inactivate molting hormones which could be ingested with blood from hosts that feed on plants containing phytoecdysteroids. In addition, the conjugates may act as a storage form of hormones for the developing embryo.
Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata
Fifth (last) instar nymphs of the tick Ornithodoros moubata convert ingested 20-hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to the more polar conjugates AP1. Only small quantities of free hormone were transferred to the hemolymph and the carcass within the first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [3H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of AP1 conjugates. AP1 obtained in second instar nymphs fed with [3H]ecdysone ([3H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of AP1 remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., AP1 mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle.
Apolar products (mainly AP2) that accumulated in eggs of females injected with [3H]E or [3H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to AP1, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs.
We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.
Probable occurrence of ecdysteroid fatty acid esters in different classes of arthropods
We investigated the fate of injected [3H]ecdysone or [3H]20-hydroxyecdysone in various species of ticks, spiders, scorpions, myriapods, crustaceans and insects. Most of these arthropods were able to convert the ecdysteroids to esterase-labile metabolites with a very apolar behaviour in reverse-phase HPLC. Some of them have retention times similar to the apolar conjugates AP2 of the tick, Ornithodoros moubata, which have been identified recently as ecdysteroids esterified as C22 with palmitic, stearic, oleic or linoleic acid [Diehl et al. (1985a) Int. J. invert. Reprod. Devl. 8, 1–13]. Others are less apolar and could correspond to the AP1 from O. moubata. The possible function of these metabolites remains to be established. They could represent inactivation products and/or a hormone storage-form for embryos.
Metabolism of ecdysteroids during the vitellogenesis of the tick Ornithodoros moubata (Ixodoidea, Argasidae): Accumulation of apolar metabolites in the eggs
The fate of injected [3H]ecdysone ([3H]E) and 20-hydroxy-[3H]ecdysone ([3H]20E) has been investigated in the female tick Ornithodoros moubata (Murray, 1877; sensu Walton, 1962). When injected into fed mated vitellogenic females, [3H]E is converted into [3H]20E and two apolar classes of metabolites, AP1 and AP2. Injected [3H]20E is directly converted into AP1 and AP2. AP2 is incorporated into the ovaries in a high proportion and at the end of the vitellogenic cycle represents about 25% of the total label recovered from the animal. The fate of labeled hormones injected into virgin females which perform an abortive vitellogenic cycle is quite similar. However, the ovaries incorporated less of the AP2 products. Ovaries of mated females cultured in vitro in the presence of [3H]E are able to produce [3H]20E and AP2. AP2 is incorporated, while [3H]20E is mainly found in the medium. Ovaries of virgin females presented a slower rate of transformation and of incorporation of the label. Labeled AP2 is recovered in freshly laid eggs and AP1 in the females after oviposition. AP1 and AP2 can produce [3H]20E, [3H]E, and other minor polar peaks when submitted to hydrolysis by esterase. It is concluded that the female O. moubata possesses a special enzymatic mechanism for transformation of ecdysteroids into apolar products and for selective incorporation of AP2 into the ovaries. These products are present in the freshly laid eggs and could play a role during embryogenesis.