Dual ecdysteroid action on the epitracheal glands and central nervous system preceding ecdysis of Manduca sexta
it
anová1,2
an
it
an2,3,*
1 Institute of Medical Chemistry and Biochemistry, School of Medicine, University of Komensk, Sasinkova 1, 81108 Bratislava, Slovakia,
2 Departments of Entomology and Neuroscience, 5419 Boyce Hall, University of California, Riverside, CA 92521, USA and
3 Institute of Zoology, Slovak Academy of Sciences, Dúbravská Cesta 9, 84206 Bratislava, Slovakia
*Author for correspondence (e-mail: uzaedus{at}savba.sk)
Accepted July 13, 2001
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Summary |
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Key words: Inka cell, pre-ecdysis-triggering hormone, ecdysis-triggering hormone, ecdysteroid, hydrazine, behaviour, Manduca sexta.
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Introduction |
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Materials and methods |
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Since it was difficult to dissect EGs from pupae and adults, we used sectioned tissue surrounding the spiracles from the following stages: pharate pupae 3 h before ecdysis and at the onset of ecdysis, pupae 515 min after ecdysis and on days 15, 10 and 15, pharate adults 1 day before ecdysis and adults 515 min after emergence. This tissue was fixed, embedded, sectioned and processed for immunohistochemical staining with the antisera to PETH or ETH, as described previously (it
an et al., 1999).
Enzyme immunoassays
To prepare samples for enzyme immunoassays, 34 sets of 2040 EGs were dissected under PBS each day (19) during the fifth instar, heated to 90°C for 5 min, homogenized in PBS and centrifuged at 10 000 g. Inka cell peptides and their precursors were fractionated by high-performance liquid chromatography (HPLC) using a Microsorb C4 column (Rainin Instruments, Woburn, MA, USA), as described previously (it
an et al., 1999). Peptide levels were quantified by enzyme immunoassays with antisera to PETH and ETH, as described previously (Kingan et al., 1997;
it
an et al., 1999).
To determine total ecdysteroid titres, blood was collected 23 times each day during the fifth instar (N=511 for each time point), heated to 90°C for 5 min and centrifuged. Supernatants were subjected to enzyme immunoassays with an antiserum to ecdysone, as described previously (Kingan, 1989). To measure the ecdysteroid content of the largest prepupal peak, individual ecdysteroids in extracted blood serum were separated by HPLC using a Vydac C4 column and 40 % methanol under isocratic solvent conditions. Ecdysteroid peaks were identified by enzyme immunoassays, and their elution times were compared with those of synthetic ecdysone and 20-hydroxyecdysone (20E; Sigma, St Louis, MO, USA).
Steroid treatment
The ecdysteroid agonist tebufenozide (RH-5992) was used to measure the effects of ecdysteroid on the production of Inka cell peptides. The agonist was dissolved in 96 % ethanol, and 0.2, 1 or 5 µg per 15 µl was injected into larvae 1030 min after ecdysis to the fifth instar. Control larvae were injected with the same amount of ethanol vehicle. After 2022 h, the Inka cells were dissected under PBS, heated to 90°C for 5 min, homogenized and centrifuged. Saline supernatants were used for enzyme immunoassay with the antiserum to ETH, as described previously (Kingan et al., 1997).
To study ecdysteroid-induced sensitivity to ETH, we injected 20E or tebufenozide into isolated abdomens or intact freshly ecdysed larvae. The abdomens of fifth-instar larvae 5 min to 3 h after ecdysis were ligated (between abdominal segments 12), and the head together with the thorax was cut off. These isolated abdomens were injected with 3050 µg of 20E followed by an injection of ETH (50100 pmol) 12 days later, or the injections of 20E and ETH were repeated several times in the same isolated abdomens. Alternatively, intact ecdysed larvae were injected with tebufenozide (0.55 µg per 15 µl) followed by ETH (500 pmol) treatment 2 days later. ETH-induced behaviour patterns were observed under the dissection microscope.
Electrophysiology
To demonstrate that ecdysteroids directly induce CNS sensitivity to ETH, nerve cords were dissected from feeding fifth-instar larvae on days 13 and incubated in Graces medium (Gibco BRL) with 20E (0.2 µg per 200 µl) for 2428 h. Control nerve cords were incubated in parallel in steroid-free Graces medium. The CNS was then placed in fresh Graces medium, and ETH (500 nmol l1) was added to the bath. Induced burst activity was recorded with suction electrodes from the dorsal nerves of abdominal ganglia (it
an et al., 1996;
it
an et al., 1999).
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Results |
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We also examined changes in the EGs during pupal and adult development. Approximately 3 h before ecdysis, the EGs of pharate pupae were large, and the Inka cells showed strong PETH and ETH immunoreactivity. The pattern of ETH immunoreactivity is shown in Fig. 3A. At the onset of pupal ecdysis, the EGs were reduced in size and peptide staining was largely depleted. Weaker immunoreactivity was restricted to the periphery of Inka cells (Fig. 3B) and disappeared completely 515 min after ecdysis. Weak PETH and ETH staining reappeared in Inka cells approximately 6 h after pupal ecdysis. During the following 13 days, the Inka cells showed increased immunoreactivity, while the other gland cells degenerated completely (Fig. 3C). Strong peptide staining was detected during adult development (Fig. 3D), but considerably decreased after adult emergence. However, residual immunoreactivity usually remained in the Inka cells 515 min after eclosion (Fig. 3E). These data suggest that the Inka cells produce and store PETH and ETH throughout the intermoult period but that their content is released at each ecdysis.
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Details of the levels of PETH, ETH and their precursors in Inka cells and of the concentrations of blood ecdysteroids during each day of the last larval instar are shown in Fig. 5. During the feeding stage on days 14, peptide and steroid levels were low (Fig. 5A,B). The difference between the production of PETH and ETH was very pronounced at this time, since ETH represented only 628 % of the total ETH-IR, while the remaining 7294 % was made up of incompletely processed precursor forms. Peptide expression increased markedly after the appearance of ecdysteroid peaks in the haemolymph (Fig. 5B,C). In particular, levels of PETH and ETH were much higher than during the feeding stage (ETH represented 5463 % of the total ETH-IR). Precursor levels showed a less dramatic increase, indicating that they were rapidly processed into active peptides (Fig. 5C).
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Steroid-induced peptide expression in Inka cells
To test whether ecdysteroids actually cause increased peptide expression in Inka cells, we used the ecdysteroid agonist tebufenozide, which mimics the action of 20E and is more stable and effective than natural ecdysteroids (Dhadialla at al., 1998). Freshly ecdysed larvae with depleted stores of PETH and ETH were injected with tebufenozide (0.2, 1 or 5 µg), and peptide production in Inka cells was determined 2022 h later by enzyme immunoassay with the antiserum to ETH. Tebufenozide treatment resulted in an approximately two- (0.2 µg) and threefold (15 µg) increase in ETH-IR compared with control animals (Fig. 6). These results support the evidence that ecdysteroids induce expression of Inka cell hormones.
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To test this, freshly ecdysed intact or abdomen-ligated fifth-instar larvae were injected with 3050 µg of 20E, and the induction of CNS sensitivity was checked by injection of ETH (50100 pmol) 12 days later. Intact larvae rapidly metabolised injected 20E (Koolman and Karlson, 1985), so this treatment did not induce CNS sensitivity to ETH (N=8). Since degradation of the injected steroid was reduced in isolated abdomens, they showed sensitivity to ETH within 12 days in 10 out of 11 individuals. In these steroid-treated isolated abdomens, injection of ETH induced strong pre-ecdysis behaviour, within 515 min, lasting for 3040 min (36±2 min, mean ± S.D., N=10). Further ETH treatment 12 days later had no effect or caused only weak and short pre-ecdysis behaviour (Fig. 8A). Another group of ecdysed and ligated larvae (N=12) was injected with 20E (50 µg) followed by ETH injection (100 pmol) 2 days later, which induced (in 515 min) strong pre-ecdysis contractions for 3550 min (44±3 min, mean ± S.D.). When contractions ceased, the steroid and peptide injections were repeated twice more, and these always led to pre-ecdysis contractions. All isolated abdomens (N=12) initiated strong pre-ecdysis behaviour within 818 min, and this lasted for 4060 min (52±2 min, mean ± S.D.) with occasional weaker contractions persisting for up to an additional 3060 min. Thus, repeated injections of 20E followed by ETH induced pre-ecdysis behaviour three times within 6 days in the same instar (Fig. 8B). Isolated abdomens failed to show ecdysis contractions since the brain and subesophageal ganglion are required for activation of this type of behaviour by ETH (it
an and Adams, 2000).
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Steroid-induced CNS sensitivity to ETH in vitro
To demonstrate that ecdysteroids induce CNS sensitivity to ETH in ecdysed or feeding larvae, isolated nerve cords from fifth-instar larvae on days 13 (N=12) were incubated individually for 2428 h with 0.2 µg of 20E in 100 µl of Graces medium. These nerve cords were then treated with ETH (300500 nmol l1), which induced pre-ecdysis or ecdysis bursts in 10 out of 12 preparations (Fig. 9). Interestingly, pre-ecdysis bursts were recorded in the dorsal and ventral nerves of only three nerve cords (Fig. 9A). Most nerve cords (N=7) did not show obvious pre-ecdysis burst patterns (Fig. 9B), but after incubation with ETH for 4055 min proceeded to show normal ecdysis bursts (Fig. 9C). Two remaining nerve cords failed to display any behavioural bursts. Treatment of five control nerve cords with ETH after incubation for 24 h in steroid-free Graces medium did not induce any pre-ecdysis or ecdysis bursts. These results showed that high ecdysteroid levels act directly on the CNS of feeding larvae to induce sensitivity to ETH.
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Discussion |
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The exocrine components of the epitracheal glands resemble paired Versons glands attached to the dorsal epidermis of each segment. These exocrine organs are composed of the secretory, saccule and duct cells, which remain relatively small and intact during the feeding stages but enlarge considerably when ecdysteroid levels increase in the pharate larvae and pharate pupae. Experiments in vivo and in vitro have shown that protein synthesis in these glands is controlled by ecdysteroids (Lane et al., 1986; Horwath and Riddiford, 1988). These glands secrete polypeptide products to coat the epicuticle during larval and pupal ecdysis, and this is associated with an apparent decrease in the size of the glands. The Versons glands degenerate several days after pupation (Lane et al., 1986), suggesting that the exocrine functions and ecdysteroid regulation of epitracheal and Versons glands may be similar.
Ecdysteroid regulation of the eth gene in Inka cells
Tebufenozide mimics the action of natural ecdysteroids in many bioassays (Dhadialla et al., 1998; Farka and Sláma, 1999). Indeed, we show here that both elevated ecdysteroid levels and injected tebufenozide stimulate the production of Inka cell peptides and their precursors in fifth-instar larvae. In a previous study, we demonstrated that ecdysteroids induce the expression of the ecdysone receptor EcR-B1 in Inka cells, and this may interact with the ecdysteroid receptor response element (direct repeat of AGGTCA) in the eth gene to induce its expression (
it
an et al., 1999). The natural ecdysteroid receptor is a heterodimer formed by ecdysone receptor (EcR) and ultraspiracle (USP) (Yao et al., 1992; Yao et al., 1993) gene products. Distinct EcR and USP isoforms bind to the response elements of specific genes and determine the fate of different cells and organs throughout insect development (Talbot et al., 1993; Thummel, 1995; Antoniewski et al., 1996). The presence of steroid response elements in peptide hormone genes is rather uncommon. So far, these elements have been identified only in the promoter regions of the Drosophila and Manduca eth genes (Park et al., 1999;
it
an et al., 1999) and in the oxytocin gene (Richard and Zingg, 1990; Mohr and Schmitz, 1991). We found that EcR and USP are expressed in the nuclei of Inka cells, and this receptor complex binds specifically to the direct repeat of the eth gene (V. Filipov, Y. Park, D.
it
an, M. E. Adams and S. S. Gill, unpublished results). These results indicate that eth gene expression may be under the direct control of high ecdysteroid levels. However, a decline in steroid levels is required for the release of PETH and ETH and for the consequent reduction in the size of the EGs (
it
an et al., 1999; Kingan and Adams, 2000). Released peptides activate different motor units in the CNS to induce the ecdysis behavioural sequence (
it
an and Adams, 2000).
Mechanisms of ecdysteroid action on the CNS
We have shown that ETH injection induces ecdysis behaviour in pharate larvae and pupae with high ecdysteroid levels. These animals were able to repeat this behaviour 1 day later in response to the natural release of PETH and ETH and to shed their cuticle. In contrast, animals treated with ETH after the ecdysteroid peak had declined displayed strong and long-lasting ecdysis behaviour, but failed to ecdyse as a result of insufficient thinning of the old cuticle at this time (6 to 10 h). In both cases, peptide injection probably causes inactivation and/or internalization of ETH receptors in the CNS, and these have to be expressed again in order for ecdysis to be completed. We suggest that the high ecdysteroid levels in the first experimental group are sufficient to induce the expression of these receptors and to recover the sensitivity of the CNS to ETH. Natural release of Inka cell peptides 1 day later therefore results in complete ecdysis. Our experiments in which 20E or tebufenozide was injected into freshly ecdysed larvae that were subsequently treated with ETH in vivo provide evidence that ecdysteroids are required for the induction of CNS sensitivity to ETH. In vitro experiments showed that this sensitivity results from a direct action of 20E on the CNS. These results indicate that a pulse of ecdysteroids before each ecdysis induces the expression of receptors for PETH and ETH, which enables the CNS to respond to Inka cell peptide hormones.
The CNS undergoes dramatic changes during metamorphosis (Truman, 1990; it
an et al., 1993). For example, several motoneurons controlling larval pre-ecdysis contractions show obvious regression of axonal and dendritic arborizations before pupal ecdysis and die shortly after pupation, while other motoneurons survive until adult emergence (Levine and Weeks, 1989). These changes result in suppressed pre-ecdysis behaviour, while a normal ecdysis motor pattern prevails during pupation (Miles and Weeks, 1991; Weeks and Truman, 1984). Experimental evidence shows that changing levels of ecdysteroids and juvenile hormone (JH) in the last larval instar control the regression of some pre-ecdysis-specific neurons. The absence of JH during the first ecdysteroid peak determines the fate of these neurons, while the second ecdysteroid peak initiates their degenerative changes (Weeks and Truman, 1985; Weeks and Truman, 1986). Expression of the ecdysteroid receptor isoform EcR-B1 in the CNS neurons coincides with these regressive responses (Truman et al., 1994; Schubiger et al., 1998). The attenuation of pre-ecdysis bursts in our isolated nerve cord preparations may indicate that 20E treatment induced this neuronal regression in the absence of JH in vitro. These data also suggest that the continued presence of JH is important for maintaining a functional pre-ecdysis circuitry in the larval stages.
Comparative aspects of steroid regulation of peptide genes
Vertebrate gonadal and adrenal steroid hormones control a wide variety of vital organ functions and metabolic pathways by regulating neuropeptide gene expression (Harlan, 1988; Crowley and Amico, 1993; Woods et al., 1998). For example, steroids regulate a number of brain functions, including the expression and release of neurotransmitters, neural development, cell death and various types of behaviour such as feeding, courtship and mating (Pfaff, 1980; Spindler, 1997). The specificity of steroid action is determined by the expression of a particular receptor for a given steroid in a subpopulation of similar neurons (Rainbow et al., 1982; Schumacher et al., 1990). Vertebrate steroids also influence endocrine/paracrine functions. For example, glucocorticoids have been implicated in the regulation of peptide gene expression in pancreatic islets, which is associated with intense proliferation of endocrine cells (Myrsen-Axcrona et al., 1997). Oestradiol and progesterone stimulate the secretion of luteinizing hormone and pro-opiomelanocortin peptides and increase the number of endocrine cells secreting these hormones (Kandeel and Swerloff, 1997). These observations show that the basic principles of the hormonal regulation of development and behaviour in vertebrates are in many respects similar to those described in insects (Spindler, 1997; Levine and Weeks, 1989; it
an et al., 1999).
Manduca sexta and Drosophila melanogaster are excellent models for investigating developmental changes induced by steroid and peptide hormones that result in the behavioural sequence leading to ecdysis (Weeks and Truman, 1986; Baker et al., 1999; it
an et al., 1999;
it
an and Adams, 2000). In this paper, we have shown that ecdysteroids are involved in the regulation of morphological changes in the EGs during larval development and metamorphosis. We also provide evidence that increased levels of ecdysteroids simultaneously control the expression of peptides in Inka cells and induce CNS sensitivity to ETH before larval and pupal ecdysis.
Other arthropods, such as crustaceans, periodically shed their hard exoskeleton. The identification and functional analysis of several neuropeptides produced by the nervous and endocrine organs of crabs and lobsters have provided new insights into the endocrine regulation of ecdysis in crustaceans and insects (Gammie and Truman, 1997; Chung et al., 1999; Phlippen et al., 2000). Such studies on crustacean models complement those carried out in insects and may reveal new aspects of arthropod endocrinology. Since the mechanisms of steroid and peptide action are similar in both vertebrates and arthropods, findings in relatively simple invertebrate organisms may be applicable to much more complex vertebrate systems.
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Acknowledgments |
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