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Molecular cloning and function of ecdysis-triggering hormones in the silkworm Bombyx mori

Duan itan^1,*, Laura Hollar², Ivana Spalovská^1,4, Peter Taká¹, Inka itanová^1,5, Sarjeet S. Gill³ and Michael E. Adams^2,3

¹ Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 84206 Bratislava, Slovakia
² Department of Entomology, 5429 Boyce Hall, University of California, Riverside, CA 92521, USA
³ Department of Cell Biology and Neuroscience, 5429 Boyce Hall, University of California, Riverside, CA 92521, USA
⁴ Department of Zoology, Comenius University, Mlynská dolina B2, 84215 Bratislava, Slovakia
⁵ Institute of Medical Chemistry and Biochemistry, School of Medicine, Comenius University, Sasinkova 2, 81108 Bratislava, Slovakia

View larger version (114K): [in a new window]   Fig. 1. (A-H) Morphology of epitracheal glands in pharate 5th instar larvae, pharate pupae and pharate adults revealed with fluorescein-labelled antiserum to horseradish peroxidase and the nuclear dye 4',6'-diamidino-2-phenylindole (DAPI). Each epitracheal gland of pharate larvae (A-C) and pharate pupae (D-F) is composed of a prominent elongated or spherical Inka cell (arrows) and two small gland cells of unknown function (arrowheads). In pharate adults, 1-2 small cells observed in earlier stages disappeared, while Inka cells became rounded with a blebby surface (G-I). In some cases, two glands were attached to the same trachea (H). (I) Wholemount staining with FMRF amide antiserum in Inka cells of pharate adult. Note the cytoplasmic processes (black arrows) extending from the Inka cell to the surface of the small unstained gland cell. (A-H) Scale bar, 400 µm. (I) Scale bar, 200 µm. PG, prothoracic glands.

View larger version (134K): [in a new window]   Fig. 2. Pre-ecdysis-triggering hormone (PETH)- and ecdysis-triggering hormone (ETH)-immunoreactivity in Inka cells before and after pupal ecdysis and adult eclosion. Intense PETH-immunoreactivity in Inka cells of pharate pupa (A) was reduced 5 min after ecdysis (B, arrow). Strong staining with ETH antiserum in Inka cells of pharate adult (C) decreased considerably 5 min after eclosion (D, arrow). Scale bar, 200 µm.

View larger version (17K): [in a new window]   Fig. 3. Isolation of pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and ETH-associated peptide (ETH-AP) from epitracheal-gland extracts of pharate pupae. All three active peptides were isolated by a single reverse-phase liquid chromatography (RPLC) fractionation step using a Microsorb C4 column (4.6x250 mm, 5 µm) and a linear gradient of acetonitrile and 0.1% trifluoroacetic acid. Molecular mass values indicated above each peak were determined by electrospray mass spectrometry.

View larger version (20K): [in a new window]   Fig. 4. Amino acid sequences of pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and ETH-associated peptide (ETH-AP) identified in Bombyx mori (Bom) Inka cells compared with related peptides from Manduca sexta (Mas) and Drosophila melanogaster (Drm). In both moths, PETH is identical and ETHs are very similar, while ETH-APs are conserved at the amino termini. Drosophila ETHs show homology with moth peptides at the carboxyl termini. Light shading shows amino acid similarity; dark shading shows amino acid homology.

View larger version (21K): [in a new window]   Fig. 5. Identification of the Bombyx mori cDNA and deduced protein sequence containing three active peptides. A signal sequence is followed by pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and ETH-associated peptide (ETH-AP) (enclosed in boxes) and a stop codon. Amidation and processing sequences between each peptide are underlined. Arrows indicate sequences used for designing primers.

View larger version (39K): [in a new window]   Fig. 6. Natural and peptide-induced ecdysis behavioural sequences in intact and ligated pharate larvae. (A) Natural pre-ecdysis was initiated by weak pre-ecdysis I contractions (stippled lines), which developed into strong pre-ecdysis I and II. Animals switched to ecdysis movements 1 h later, which resulted in cuticle shedding within 10-12 min. Injection of epitracheal-gland extract or ecdysis-triggering hormone (ETH) induced both pre-ecdysis I and pre-ecdysis II behaviours for 30-40 min, followed by ecdysis movements for 10-30 min. (B) Injection of pre-ecdysis-triggering hormone (PETH) 20-24 h prior to ecdysis elicited only pre-ecdysis I, but subsequent ETH injection induced pre-ecdysis II and ecdysis behaviours. Injection of PETH 10-15 h prior to ecdysis elicited the entire behavioural sequence, and subsequent ETH injection caused only weak pre-ecdysis II and ecdysis (stippled lines). (C) Isolated abdomens initiated natural pre-ecdysis and ecdysis behaviours at the expected time. ETH injection also induced the entire behavioural sequence, but latency to the onset of ecdysis behaviour was much shorter. (D) Shaded areas and arrows depict asynchronous dorso-ventral and leg contractions in thorax and abdomen during PETH-induced pre-ecdysis I. (E) The subsequent ETH injection induced asynchronous ventral, posterio-lateral and proleg contractions (pre-ecdysis II; shaded areas and arrows). (F) Ecdysis movements were characterized by subsequent dorso-ventral contractions and proleg retractions (shaded areas and arrows, arrowheads) during which each segment was moved anteriorly (arrowhead). See text for details.

View larger version (53K): [in a new window]   Fig. 7. Natural and peptide-induced ecdysis behavioural sequences of pharate pupae. (A) Natural behaviour was initiated by weak and occasional pre-ecdysis contractions (stippled line), which became gradually stronger. After apparent pre-ecdysis contractions for 1 h, animals initiated strong ecdysis peristaltic movements to shed the old cuticle in 10-12 min. Injection of pre-ecdysis-triggering hormone (PETH) induced pre-ecdysis behaviour for 30-60 min, which was then followed by ecdysis movements. Injection of intact animals or isolated abdomens with ecdysis-triggering hormone (ETH) induced the same pre-ecdysis contractions, but latency to the onset of ecdysis was generally shorter (25-44 min). As injected animals could not shed the old cuticle, strong ecdysis contractions lasted for up to 1 h (stippled lines). (B) Shaded areas and arrows indicate dorsoventral, leg and proleg contractions during pre-ecdysis induced by PETH or ETH injection. (C) Following pre-ecdysis, both peptides induced strong anteriorly directed ecdysis peristaltic movements (black arrowheads), which caused rupture of the old cuticle behind the head and moved it posteriorly with attached larval spiracles and trachei (white arrowheads).

View larger version (56K): [in a new window]   Fig. 8. Natural and peptide-induced eclosion behavioural sequences of pharate adults. (A) Natural behaviour was initiated by abdominal rotations every 2-5 s for approximately 40 min, followed by a quiescent phase for approximately 50 min and eclosion peristaltic movements for 12-15 min. Injection of pre-ecdysis-triggering hormone (PETH) or ecdysis-triggering hormone (ETH) induced the entire behavioural sequence, but the onset of ecdysis was accelerated in ETH-treated animals. (B) Separate injections of each peptide caused 40-80 rotations of the abdomen of pharate adults, each lasting 1-2 s with intervals of 2-6 s. (C) After a quiescent phase, pharate adults initiated peristaltic movements of the abdominal segments (arrowhead).

View larger version (50K): [in a new window]   Fig. 9. Pre-ecdysis and ecdysis burst patterns in the isolated central nervous system (CNS) of pharate larvae in vitro. (A) Ecdysis-triggering hormone (ETH)-induced asynchronous pre-ecdysis II bursts in ventral nerves of abdominal ganglia 4-6 (AG4-6V). (B,C) Ecdysis bursts in dorsal nerves of abdominal ganglia 2-5 (AG2-5D). Note that ETH-induced ecdysis burst patterns (B) are very similar to natural ecdysis bursts (C). Calibration bars: horizontal, 5 s; vertical, 10 µV.

View larger version (46K): [in a new window]   Fig. 10. Ecdysis-triggering hormone (ETH)-induced pre-ecdysis and ecdysis bursts in the isolated central nervous system (CNS) of pharate pupae in vitro. (A) Asynchronous pre-ecdysis I bursts in dorsal nerves of AG5-8D of an isolated chain of abdominal ganglia (AG1-8). (B) Approximately 40 min later, this isolated chain of AG1-8 switched to ecdysis bursts. The ecdysis motor pattern recorded from isolated AG1-8 closely resembled that observed in the intact CNS. Calibration bars: horizontal, 5 s; vertical, 10 µV.

View larger version (35K): [in a new window]   Fig. 11. Pre-ecdysis-triggering hormone (PETH)-induced pre-ecdysis bursts in intact and individually isolated abdominal ganglia of pharate pupae in vitro. Asynchronous pre-ecdysis I bursts are very similar in (A) the intact chain of abdominal ganglia 1-8 (AG1-8) and (B) individually isolated ganglia following transection of connectives between each ganglion. Calibration bars: horizontal, 5 s; vertical, 10 µV.

View larger version (70K): [in a new window]   Fig. 12. The effect of ecdysis-triggering hormone (ETH) injection on eclosion hormone-immunoreactivity (EH-IR) in the central nervous system (CNS) of intact or ligated pharate larvae. (A) Strong EH-IR in four ventro-medial (VM) cells, axons and arborizations in the brain of control pharate larva 12 h prior to ecdysis. (B—D) Four varicose axons of VM cells showed strong EH-IR along all ventral ganglia (B,C), connectives (D) and neurohaemal proctodeal nerves (E). (F—I) ETH-induced ecdysis behaviour of pharate larva (ligated between abdominal segments 5 and 6) was associated with strong EH-IR in the brain VM cells and axons (F) but a considerable reduction (arrows) or depletion of EH staining in axons of ventral ganglia (G,H). Accumulation of EH-IR was only found in four axons anterior to the ligated connective (I). (J) Depletion of EH-IR in proctodeal nerves of intact pharate larva 15 min after ETH-induced ecdysis behaviour. (K—N) Strong EH-IR in axons of abdominal ganglia (K,L), terminal nerve (M) and branching proctodeal nerves (N) in an ETH-injected isolated abdomen showing ecdysis movements for 15 min. Abbreviations: AG, abdominal ganglion; Con, connectives between AG5 and AG6; PN, proctodeal nerve; TG, thoracic ganglion; TN, terminal nerve. Scale bars, 100 µm (upper three rows) and 50 µm (lower two rows).

View larger version (75K): [in a new window]   Fig. 13. Ecdysis-triggering hormone (ETH)-induced cyclic 3',5'-guanosine monophosphate (cGMP) immunoreactivity in the ventral nerve cord of intact or ligated pharate larvae 10-15 min after the initiation of ecdysis behaviour. (A—D) Ecdysing intact pharate larvae showed strong cGMP staining in 27/704 neurons of all ventral ganglia. (E—H) ETH-induced ecdysis behaviour of isolated abdomens was associated with a strong cGMP response in abdominal ganglia. Note that in most abdominal ganglia, only cells 27 show strong cGMP elevation. Abbreviations: SG, suboesophageal ganglion; TG2, thoracic ganglion 2; AG3-5, abdominal ganglia 3-5; TAG, terminal abdominal ganglion. Scale bar, 200 µm.