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...

No abstract

Recent isolation3 of the sex attractant of the male boll weevil (Anthonomus granáis Boheman) followed a most difficult phase of insect pheromone research4 and allowed structure elucidation4 of the essential components as the monoterpenes 1, 2, and 3 or 4, which have recently been synthesized by nonselective routes.5

A brief survey of gas and liquid chromatographic methods for analysis, purification and quantitation of natural insect juvenile hormones and one commercially used analoh, altosid (methoprene) insect growth regulator, includes references to the most recent literature.

Residues of isopropyl (2E,4E)-ll-methoxy-3, 7,1 l-trimethyl-2,4-dodecadienoate (Altosid®) insect growth regulator are determined in waters, soils, plants, milk, eggs, fish, shellfish, poultry and cattle tissues, blood, urine, and feces. Acetonitrile is the primary extraction solvent for all samples. Residues are extracted by high-speed blending followed by vacuum filtration. Fatty extracts are subjected to cold-temperature precipitation and filtration. Samples are cleaned up by petroleum ether partitioning and Florisil and neutral alumina chromatography. The concentrated eluants are analyzed by gas-liquid chromatography (GLC) on columns of differing polarity, using hydrogen flame ionization detectors. The identity of suspected residues is confirmed by additional GLC and by mass fragmentography. The lower limits of detection were: water samples, 0.0004–0.001 ppm; soils, blood, and urine, 0.001 ppm; forage grasses, forage legumes, and rice foliage, 0.005 ppm; and milk, eggs, fish, shellfish, poultry and cattle tissues, and feces, 0.010 ppm.