Isolation, Structure, and Absolute Configuration of a New Natural Insect Juvenile Hormone from Manduca sexta

Abstract

Two juvenile hormones are isolated from organ cultures of corpora allata of the tobacco hornworm moth, Manduca sexta Johannson, and are purified by high-resolution liquid chromatography. These are identified as methyl (2E, 6E)-(IOR)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate, a new natural hormone, and methyl (2E,6E) -(IOR,11S) -10,11 -epoxy -3,7,11 -trimethyl- Continued improvements in insect tissue culture techniques have led to their increased use in studies of endocrine physiology (5). Indirect indications have been obtained that corpora allata can survive and function in vitro (6), and recently cecropia juvenile hormone has been recovered and identified from cultures of brain-corpora cardiaca-corpora allata complexes of H. cecropia (7).We report both an in vitro system that cleanly produces two hormones 2 and 3 from corpora allata of Manduca sexta, and the greatly simplified purification and chemical identification of the active hormones, one of them identified for the first time as a natural product of insects. Preparation of Cultures. Adult female moths, 0-48 hr after eclosion, were anesthetized with CO2 and surface sterilized by immersion in 70% ethanol; the cuticular scales and hairs over the head and thorax were rubbed off with paper towel. The dorsal body wall over the head and cervical membrane was cut away, taking care not to bring the external surface into contact with the underlying tissues. The corpus cardiacum-corpus allatum complex was located attached to either side of the aorta posterior to the brain, and was removed by cutting the aorta, nerves, and tracheae in front of and behind the glands. Groups of excised glands were transferred through four successive baths of culture medium, the first two containing 1 mM recrystallized phenylthiourea, and then into a sterile Leighton culture tube containing 0.5 ml of fresh culture medium.During incubation at 20-25', the culture medium was replaced at 2-to 8-day intervals with a total of three changes over a 15-day period. The medium removed from the cultures was either extracted immediately or stored for up to 2 weeks at -20°.Culture Medium. We used Grace's Insect T.C. Medium (GIBCO) without methionine, supplemented with 1% (w/v) bovine plasma albumin (fraction V, Metrix) and L-[methyl-14C]methionine (ICN, 48.4 Ci/mol) to a final concentration of 0.04 mM, containing 2,uCi/ml. Extraction Procedure. Culture media were extracted three times by vigorous shaking with equal volumes of highly purified ethyl acetate. The pooled organic phases were reduced to a small volume under N2, applied to washed (hexaneethyl acetate 1:1) thin-layer chromatography plates (silica gel GF-254, Brinkmann, 0.25 mm thickness), and developed in hexane-ethyl acetate 1:1 (v/v). Chromatoplates were radioscanned (Packard model 7201) and appropriate zones were eluted with pure ethyl acetate and stored in that solvent at -200.Chromatography and Spectroscopy. High-resolution liquid chromatography separations were performed on a homemade instrument incorporating a Haskel E...

CYP15A1, the cytochrome P450 that catalyzes epoxidation of methyl farnesoate to juvenile hormone III in cockroach corpora allata

“…Chiral HPLC separation of the 10R and 10S enantiomers of JH III conclusively showed that CYP15A1 has a very high degree of selectivity in the epoxidation of methyl farnesoate, producing the natural 10R enantiomer. The absolute configuration is known for the JH III produced by the CA of Manduca sexta and Tenebrio molitor (21)(22)(23), and for the JH III produced enzymatically with L. migratoria and B. giganteus CA microsomes (9,18).…”

The Secretion of α-Ecdysone by the Prothoracic Glands of Manduca sexta In Vitro

“…This has been accomplished by culturing active prothoracic glands of Manduca sexta in vitro and analyzing the incubation medium by a variety of chemical and biological techniques; a similar approach has proved successful in elucidating the structure of the M. sexta juvenile hormones (13). Since Fukuda's classical experiments (1) on the commercial silkworm, Bombyx mori, it has been generally accepted that the hormone responsible for initiating the moulting process in all insects is the secretory product of the prothoracic glands (or analogous structures).…”

Biosynthesis of the Juvenile Hormones of Manduca sexta: Labeling Pattern from Mevalonate, Propionate, and Acetate

“…In addition, good incorporation of the epoxy acid derived from JH I, has been witnessed in H. cecropia, although it may not be an obligatory intermediate (8). However, no information has been available on biosynthesis of the carbon skeletons of the juvenile hormones, except for a slight incorporation of [2-'4C]acetate (7), and a complete lack of incorporation of methionine in the chain of homoterpenoids JH I (7) and JH II (6,7).…”