Identification of ecdysis-triggering hormone from an epitracheal endocrine system

Science

Volumn 271, Issue 5245, 1996, Pages 88-91

  Žitňan, Dušan ^a,d   Kingan, Timothy G ^b,c   Hermesman, John L ^a   Adams, Michael E ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b AGRICULTURAL RESEARCH SERVICE   (United States)

^c UNIVERSITY OF MARYLAND BALTIMORE COUNTY   (United States)

^d INSTITUTE OF CHEMISTRY   (Slovakia)

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACID; BRAIN PEPTIDE; HORMONE;

ANIMAL EXPERIMENT; ARTICLE; CENTRAL NERVOUS SYSTEM; CONTROLLED STUDY; CUTICLE; DEVELOPMENT; ENDOCRINE SYSTEM; HEMOLYMPH; HORMONE DETERMINATION; IMMUNOHISTOCHEMISTRY; INSECT; LARVA; LATENT PERIOD; MOLTING; MOTOR ACTIVITY; NONHUMAN; PRIORITY JOURNAL; STAINING; STEREOTYPY;

EID: 0030022627     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5245.88     Document Type: Article

References (39)

1. P. F Copenhaver and J. W. Truman, J. Insect Physiol 28, 695 (1982)
2. R S Hewes and J W Truman, J Comp Physiol A 168, 697 (1991).
3. J W Truman, Prog. Brain Res 92, 361 (1992)
4. _, R S Hewes, J. Ewer, in Insect Neuro-chemistry and Neurophysiology 1993, A B. Borkovec and M J Loeb, Eds (CRC Press, London, 1994), pp. 39-51; J Ewer, V J. De Vente, J W. Truman, J Neurosci 14, 7704 (1994).
5. _, R S Hewes, J. Ewer, in Insect Neuro-chemistry and Neurophysiology 1993, A B. Borkovec and M J Loeb, Eds (CRC Press, London, 1994), pp. 39-51; J Ewer, V J. De Vente, J W. Truman, J Neurosci 14, 7704 (1994).
6. Endocrine glands homologous to the EG were described previously in the waxmoth Galleria mellonella during a search for organs containing phenylalanine-methionine-arginine-phenylalanine (FMRF) amide-like peptides [D Žitňan, thesis, Slovak Academy of Sciences, Bratislava (1989)]. Longitudinal sections of whole pharate and freshly ecdysed larvae, pupae, and adults were stained with an antiserum to FMRF-amide Intense staining was observed in large, segmentally distributed cells located near each spiracle in pharate stages; staining was not detected in freshly ecdysed animals, which suggests that some components of these cells may control processes associated with ecdysis Žitňan originally referred to the G. mellonella structures as penspiracular glands. However, because Keilin [ D Keilin, Parasitology 36, 1 (1944)] previously used the term "perispiracular glands" to describe a different anatomical structure, we chose here the name "epitracheal glands" to emphasize their association with the tracheal system and for consistency with the terminology recently adopted for homologous glands in the silkworm, Bombyx mon [ H Akai, Cytologia 57, 195 (1992)]
7. Endocrine glands homologous to the EG were described previously in the waxmoth Galleria mellonella during a search for organs containing phenylalanine-methionine-arginine-phenylalanine (FMRF) amide-like peptides [D Žitňan, thesis, Slovak Academy of Sciences, Bratislava (1989)]. Longitudinal sections of whole pharate and freshly ecdysed larvae, pupae, and adults were stained with an antiserum to FMRF-amide Intense staining was observed in large, segmentally distributed cells located near each spiracle in pharate stages; staining was not detected in freshly ecdysed animals, which suggests that some components of these cells may control processes associated with ecdysis Žitňan originally referred to the G. mellonella structures as penspiracular glands. However, because Keilin [ D Keilin, Parasitology 36, 1 (1944)] previously used the term "perispiracular glands" to describe a different anatomical structure, we chose here the name "epitracheal glands" to emphasize their association with the tracheal system and for consistency with the terminology recently adopted for homologous glands in the silkworm, Bombyx mon [ H Akai, Cytologia 57, 195 (1992)]
8. Endocrine glands homologous to the EG were described previously in the waxmoth Galleria mellonella during a search for organs containing phenylalanine-methionine-arginine-phenylalanine (FMRF) amide-like peptides [D Žitňan, thesis, Slovak Academy of Sciences, Bratislava (1989)]. Longitudinal sections of whole pharate and freshly ecdysed larvae, pupae, and adults were stained with an antiserum to FMRF-amide Intense staining was observed in large, segmentally distributed cells located near each spiracle in pharate stages; staining was not detected in freshly ecdysed animals, which suggests that some components of these cells may control processes associated with ecdysis Žitňan originally referred to the G. mellonella structures as penspiracular glands. However, because Keilin [ D Keilin, Parasitology 36, 1 (1944)] previously used the term "perispiracular glands" to describe a different anatomical structure, we chose here the name "epitracheal glands" to emphasize their association with the tracheal system and for consistency with the terminology recently adopted for homologous glands in the silkworm, Bombyx mon [ H Akai, Cytologia 57, 195 (1992)]
9. The Inka cell is named here in honor of the beautiful fairy goddess of the Tatra mountains, who is a source of great inspiration for the first author.
10. B. rinsed, and incubated with a peroxidase-labeled antibody to mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) for 1 hour, and bound peroxidase was stained with 3-ammo-9-ethyl carbazole (Sigma)
11. B. rinsed, and incubated with a peroxidase-labeled antibody to mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) for 1 hour, and bound peroxidase was stained with 3-ammo-9-ethyl carbazole (Sigma)
12. B. rinsed, and incubated with a peroxidase-labeled antibody to mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) for 1 hour, and bound peroxidase was stained with 3-ammo-9-ethyl carbazole (Sigma)
13. B. rinsed, and incubated with a peroxidase-labeled antibody to mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) for 1 hour, and bound peroxidase was stained with 3-ammo-9-ethyl carbazole (Sigma)
14. Pharate is the term used to describe animals that have synthesized a new cuticular layer yet remain encased in the old cuticle; this stage ends after ecdysis is completed.
15. Epitracheal glands were dissected into a microtissue grinder and kept on dry ice until extracted into Weever's saline Extracted samples were heated in a 90°C water bath for 2 to 3 min, cooled on ice, and centrifuged at 10,000g, and the supernatant was injected directly into the hemocoel.
16. 2O.acetic acid, 90·9 1) and the supernatant was evaporated to dryness Crude extracts of EGs were fractionated on a Microsorb C4 column (wide pore 300 A, 4 6 mm × 25 cm) with a linearly increasing gradient of acetonitrile (3 to 53% in 90 mm) in constant O 1% tnfluoroacetic acid in water. The flow rate was 1 ml/min. Mas-ETH was 95% pure after a single fractionation step
17. + is 2940.45 amu for the amidated peptide and 2941 44 ± 0 98 amu for the free carboxyl)
18. Automated Edman degradation sequencing was performed with an Applied Biosystems 475A pulsed-liquid sequencer that was coupled on-line with an ABI 120A analyzer for identification of phenylthiohydantoin derivatized amino acids
19. Single-letter abbreviations for the amino acid residues are as follows A, Ala; C. Cys, D, Asp, E, Glu, F, Phe, G, Gly; H, His, I, Ile, K, Lys. L Leu, M. Met, N, Asn; P, Pro, Q, Gln; R, Arg, S, Ser, T, Thr; V, Val, W, Trp; and Y, Tyr.
20. Synthetic Mas-ETH was prepared by solid-state synthesis on an Applied Biosystems peptide synthesizer by the Peptide Synthesis Facility at the Sussex Center for Neuroscience. University of Sussex, Brighton, U.K.
21. To quantify amounts of Mas-ETH in EGs, peak integrations from RPLC were related to molar quantities obtained from quantitative analysis of amino acid composition. Peptide samples (1 nmol) were hydrolyzed with HCl vapor at 150°C for 90 min and analyzed with an Applied Biosystems model 420 microamino acid analyzer Molar quantities of PTC amino acids were determined by peak integration and corrected against a 500-pmol norleucine standard
22. Animals were staged 3 to 4 hours before natural ecdysis by observation of the morphological marker, anterior shrink, as defined in (24).
23. H R Morris, M. Panico, A. Karplus, P E Lloyd, B. Riniker, Nature 300, 643 (1982)
24. 18 cartridges before fractionation as described (10). For electrospray mass spectrometry analysis, fractions were evaporated in a vacuum and redissolved in a solution of methanol and water (50 50). Mass was analyzed with a Finnigan-MAT high-resolution mass spectrometer fitted with an electrospray interface that operated in positive ion mode
25. J. W Truman and J. C Weeks, In Neural Control of Rhythmic Movements, A. Roberts and B L. Roberts, Eds (Cambridge Univ Press, Cambridge, 1983), vol 37, PP 223-241; J W Truman and J.C Weeks, in Model Neural Networks and Behavior, A I Selverston, Ed (Plenum, New York, 1985), pp 381-399.
26. J. W Truman and J. C Weeks, In Neural Control of Rhythmic Movements, A. Roberts and B L. Roberts, Eds (Cambridge Univ Press, Cambridge, 1983), vol 37, PP 223-241; J W Truman and J.C Weeks, in Model Neural Networks and Behavior, A I Selverston, Ed (Plenum, New York, 1985), pp 381-399.
27. J C Weeks and J W Truman, J Comp Physiol. A 155, 407 (1984).
28. Internal hydrostatic pressure was measured in pharate fifth-instar larvae and pharate pupae Of M. sexta with a Gould-Statham P23-ID pressure transducer The transducer was connected to a saline-filled tube, which in turn was attached 10 the posterior dorsal horn of pharate fifth-instar larvae and pharate pupae by dental wax with a low melting point.
29. About 36 hours before natural ecdysis in pharate fifth-instar larvae, the first signs of apolysis can be observed as an accumulation of molting fluid in the prothorax. About 2 hours later, head capsule slip occurs See also A T Curtis et al., J. Insect Physiol 30, 597 (1984).
30. S. E. Reynolds, P. H. Taghert, J W. Truman, J. Exp. Biol. 78, 77 (1979).
31. J. W Truman, P. H Taghert, S E. Reynolds, J. Exp. Biol 88, 327 (1980)
32. The entire CNS or a chain of abdominal ganglia dissected from pharate fifth-instar larvae or pharate pupae was placed in a 300-μl Sylgard bath and bathed in modified Weaver's saline Motor output was recorded extracellularly from the dorsal roots of abdominal ganglia A5, A6, and A7 through the use of polyethylene suction electrodes. Potentials were amplified with Grass P-15 AC amplifiers, captured on videotape, and played back on a Gould Brush pen recorder To record natural preecdysis and ecdysis, the nervous system was removed after initiation of the behavior For experiments involving bath application of synthetic Mas-ETH, pharate fifth-instar nerve cords were dissected 8 hours before ecdysis arid liquid-filled head and brown mandibles were used as morphological markers. Pharate pupae were dissected 8 hours before natural ecdysis, with brown bars as the morphological marker. See (7, 24) for explanations of developmental staging in each life stage
33. To observe natural preecdysis and ecdysis motor patterns, we observed animals until the initiation of preecdysis, at which time the nerve cords were quickly removed and prepared for suction-electrode recordings.
34. D Žitňan and M E. Adams, unpublished data.
35. J W. Truman, J Exp Biol. 74, 151 (1978); J C Weeks and J W Truman, J Comp. Physiol A 155. 423 (1984), C. I Miles and J. C. Weeks, ibid., 168, 179 (1991)
36. J W. Truman, J Exp Biol. 74, 151 (1978); J C Weeks and J W Truman, J Comp. Physiol A 155. 423 (1984), C. I Miles and J. C. Weeks, ibid., 168, 179 (1991)
37. J W. Truman, J Exp Biol. 74, 151 (1978); J C Weeks and J W Truman, J Comp. Physiol A 155. 423 (1984), C. I Miles and J. C. Weeks, ibid., 168, 179 (1991)
38. Methods for preparation of natural eclosion hormone form corpora cardiaca followed those described in (20)
39. We gratefully acknowledge the assistance of the following contributors to these studies: Edman sequencing and amino acid composition analysis were performed by G Porter of the University of California (UC) Riverside Biotechnology Instrumentation Facility, Mas-ETH was synthesized by C Kowalczyk of the Sussex Center for Neuroscience, University of Sussex, Brighton, UK; LSIMS was performed by A G Craig of the Salk Institute, La Jolla, CA; and electrospray mass spectrometry was performed by R Kondrat, Southern California Mass Spectrometry Facility, UC Riverside. D. Eastmond provided access to fluorescence microscopy. We appreciate the helpful comments, advice, and support of B Gray, University of Utah, M. O'Shea, Sussex Center of Neuroscience, and D A Schooley, University of Nevada, Reno We are particularly indebted to M. K. Rust, L. J Lund, and the Agricultural Experiment Station at UC Riverside for financial support. Partially supported by USDA competitive grant 93-37302-8968 to T.G.K. and by funds to the INHL at USOA ARS, Beltsville, MD