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Dual ecdysteroid action on the epitracheal glands and central nervous system preceding ecdysis of Manduca sexta

Inka itanová^1,2, Michael E. Adams² and Duan itan^2,3,*

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

View larger version (12K): [in a new window]   Fig. 1. The developmental stages investigated in this study. Fourth-instar larvae that developed a new cuticle 30 h before ecdysis (pharate fifth-instar larvae), last larval stage (fifth instar) just after ecdysis (day 0) and during feeding on days 1–4, wandering on days 5–6 (larvae that stop feeding and crawl to find a place for pupation) and post-wandering on days 7–9 (pharate pupae), freshly ecdysed pupae and pupae on days 1–5, developing adults on days 10 and 15, pharate adults 1 day prior to emergence and freshly ecdysed adults. The relative ecdysteroid titres shown in the trace were taken from itan et al. (itan et al., 1999), for pharate fifth-instar larvae, from the present study for fifth-instar larvae and pharate pupae and from Bollenbacher et al. (Bollenbacher et al., 1981) for pupal–adult development.

View larger version (110K): [in a new window]   Fig. 2. Double wholemount immunohistochemical staining of the epitracheal gland during the fifth instar of Manduca sexta larvae. Only Inka cells (large arrow) were double-labelled by the pre-ecdysis-triggering hormone (PETH) and horseradish peroxidase (HRP) antisera (orange-red colour), while exocrine cells (large arrowhead) reacted just with the FITC-labelled HRP antiserum (yellowish-green colour). The ‘narrow’ (small arrow) and ‘canal’ (small arrowheads) cells plus tracheal cells were revealed by nuclear DAPI dye (blue colour). (A) Strong PETH-immunoreactivity (IR) was detected in Inka cells 8 h before ecdysis into the fifth instar; (B) this staining disappeared completely 5 min after ecdysis. (C,D) Weak PETH-IR was again detected in feeding larvae on day 1 and increased on day 3. (E,F) The Inka cells of wandering larvae (days 5, 6) subsequently showed stronger staining as the entire glands increased in size. (G–I). The epitracheal glands reached maximal size 1–2 days prior to pupal ecdysis (days 8–9). Note that the duct process was quite long and obvious in the feeding and wandering stages (C–F), but was reduced in size 1–2 days prior to ecdysis (A,G–I). (G) A rare case of two Inka cells with one set of gland cells attached to the same trachea. Another separated set of gland cells was attached to the opposite side of this trachea. Days 1–9 refer to days after ecdysis into the fifth instar. Scale bar, 100 µm.

View larger version (128K): [in a new window]   Fig. 3. Changes in ecdysis-triggering hormone (ETH)-immunoreactivity (IR) in sections of epitracheal glands during pupal and adult development. (A) The Inka cell showed very strong ETH staining (red colour) in pharate pupa approximately 3 h before ecdysis. (B) At the onset of pupal ecdysis, all gland cells decreased in size and most staining disappeared from the Inka cell. (C) Strong ETH-IR was again observed in the pupal Inka cell on day 3, but the other gland cells degenerated. (D) High peptide levels persisted in Inka cells throughout adult development, as indicated by strong ETH-IR in the pharate adult 1 day before ecdysis. (E) Adult ecdysis was associated with a marked decrease in ETH staining. Scale bar, 150 µm.

View larger version (20K): [in a new window]   Fig. 4. Correlation between natural ecdysteroid titres and peptide production in Inka cells. (A) Total pre-ecdysis-triggering hormone (PETH)-immunoreactivity and ecdysis-triggering hormone (ETH)-immunoreactivity (IR) were low following ecdysis into the fifth instar, but increased considerably after the two ecdysteroid peaks appeared in the haemolymph and reached their highest levels on day 9. Total ETH-IR represents the levels of active ETH plus all precursor forms containing this peptide. The ratio of ecdysone to 20-hydroxyecdysone (20E) in the second (prepupal) peak was 1:20. (B,C) Elevation of ecdysteroid levels in the haemolymph was associated with a marked increase of the levels of isolated active hormones (PETH and ETH), and their precursor forms (PE, EA, PEA) in Inka cells. (B) Levels of isolated PETH were higher than those of ETH because some of the ETH contained the precursor EA. Levels of the two precursors (PE and PEA) decreased before pupal ecdysis on days 8–9, indicating peptide processing (C). Each ecdysteroid and peptide determination represents the mean ± S.D. of 4–11 samples, but the standard deviations were too small to be included in the figure. Note the difference in scale in C.

View larger version (19K): [in a new window]   Fig. 5. Blood ecdysteroid levels and expression of pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and their precursors in Inka cells during the last larval instar. (A-C) Throughout the fifth instar, PETH levels were higher than those of ETH and their precursors. (A,B) Differences in levels between PETH and ETH were very obvious during the feeding stage on days 1–4, when only 6–28 % of the total ETH immunoreactivity (ETH-IR) was active ETH and the remaining 72–96 % was represented by different unprocessed precursor forms. (B) Levels of ETH and its precursors increased after the pre-wandering steroid peak. (C) Both active peptides and their precursors reached their highest levels after the prepupal peak, when 54–63 % of the total ETH-IR represented ETH. Each peptide determination represents the mean + S.D. of 3–4 sets of 20–40 epitracheal glands. Each ecdysteroid determination represents the mean ± S.D. of 5–11 haemolymph samples. Note the differences in scale between A, B and C.

View larger version (13K): [in a new window]   Fig. 6. Increased production of ecdysis-triggering hormone (ETH)-immunoreactive peptides (ETH-IR) induced by the ecdysteroid agonist tebufenozide. Injection of this compound (0.2 or 1–5 µg) into freshly ecdysed fifth-instar larvae increased the production of ETH and precursor peptides within 20–22 h compared with control larvae (C). Each histogram represents the amount of ETH-IR per Inka cell expressed as the mean + S.D. of 10 control larvae, plus seven (0.2 µg) and 21 (1–5 µg) tebufenozide-treated larvae, respectively.

View larger version (13K): [in a new window]   Fig. 7. The appearance of central nervous system (CNS) sensitivity to ecdysis-triggering hormone (ETH) associated with natural ecdysteroid pulses before larval and pupal ecdysis. Pharate fifth-instar larvae and pharate pupae become sensitive to ETH injection approximately 30 and 48 h before natural ecdysis, respectively (–30 and –48 h, arrowheads) when ecdysteroids reach peak levels. Grey areas depict the periods in pharate larvae (–28 to –20 h) and pharate pupae (–46 to –20 h) with increased ecdysteroid levels during which ETH injection induces the ecdysis behavioural sequence. Since the old cuticle is not sufficiently digested at this time, these animals fail to ecdyse. However, their ecdysteroid levels are sufficient to recover CNS sensitivity to ETH. After steroids levels decline 1 day later, the natural release of Inka cell peptides activates the entire behavioural sequence, and most animals ecdyse normally at the expected time (short arrow). Black areas depict the periods (pharate larvae at –10 to –6 h and pharate pupae at –12 to –4 h) during which ETH triggers strong and long-lasting ecdysis contractions, but these animals fail to ecdyse. Since, at these stages, ecdysteroid levels are too low to induce CNS sensitivity to Inka cell peptides, these animals never resume pre-ecdysis behaviour and remain trapped in the old cuticle. See text and Table 2 for details. Relative ecdysteroid levels are from itan et al. (itan et al., 1999) and the present study.

View larger version (19K): [in a new window]   Fig. 8. Effects of 20-hydroxyecdysone (20E) or the ecdysteroid agonist tebufenozide (RH-5992) on sensitivity to ecdysis-triggering hormone (ETH) in freshly ecdysed fifth-instar larvae or isolated abdomens. (A) Injection of 20E (arrowhead) induced sensitivity of isolated abdomens to ETH within 1–2 days. The initial ETH treatment (arrow) triggered pre-ecdysis (PE) behaviour, but subsequent peptide injections 1 and 2 days later failed to induce this behaviour (No PE). (B) Repeated injections of 20E into isolated abdomens followed by ETH treatment 2 days later always resulted in pre-ecdysis contractions (PE). (C) Injection of tebufenozide (5 µg) into intact ecdysed larvae induced the production of new cuticle and sensitivity to ETH within 1–2 days. These larvae responded to ETH injection with pre-ecdysis and ecdysis behaviour (PE+E). See text for details. Numbers refer to days after ecdysis into the fifth-instar larva.

View larger version (45K): [in a new window]   Fig. 9. In vitro treatment of the isolated central nervous system with 20-hydroxyecdysone followed by ETH application 24–28 h later. (A) This treatment induced strong pre-ecdysis burst patterns in three nerve cords. Note the synchronous bursts in dorsal nerves (D) which alternate with synchronous bursts in ventral nerves (V) of this nerve cord. (B) Most nerve cords showed attenuation of pre-ecdysis bursts, but these preparations displayed ecdysis bursts (C) after incubation with ETH for 40–55 min. Note that bursts from adjacent ganglia of this nerve cord are delayed, which is characteristic of peristaltic ecdysis movements. Dorsal (D) and ventral (V) nerves of abdominal ganglia 4, 5, 6 (AG4,5,6)