The insect cell line MRRL-CH derived from embryos of the lepidopteranManduca sexta responded rapidly, within 15 min, to 20-hydroxyecdysone (20-OH-Ec) (1×10−8 M) by increasing in cell volume. The induction time for this response contrasts with that for the cell elongation response that appears only after 24 to 48 h. The increase in cell volume represents the most rapid 20-OH-Ec-induced change in cell morphology reported to date. It also has some steroid specificity because no volume change was induced by ecdysone (4×10−6 M) under the experimental conditions. The magnitude of the cell volume response to 20-OH-Ec varied with the population dynamics of the cell line and was associatated with cell size at the time of treatment.

Cells from the continuous MRRL-CH line derived from embryos of the tobacco hornworm synthesized chitin. Digestion of the washed pellet from [ 14 C]-N-acetylglucosamine-labeled cells by chitinase yielded a water-soluble labeled compound. The lyophilized residue from the supernatant of the chitin digestion was analyzed by gas-liquid chromatography as its trimethylsilyl derivative. The major component cochromatographed with derivitized chitobiose. The presence of chitobiose was confirmed by gas chro-matography-mass spectrometry. The synthesis of chitin by this cell line is inhibited by diflubenzuron.

Cell lines derived from embryos of the tobacco hornworm,Manduca sexta (L.), showed a marked morphological response to treatment with physiological doses of β-ecdysone. The response of these cell lines with α-ecdysone indicated that the penta-ol (α-ecdysone) must be converted to the hexa-ol (β-ecdysone) form before the morphological response can appear. Liquid chromatographic analysis of the spent medium confirmed that the cells converted the α-ecdysone to β-ecdysone in amounts that exceeded the threshold level for a biological response. Constant exposure to β-ecdysone produced a cell line that was resistant to the killing effect of β-ecdysone and failed entirely to respond to the presence of α-ecdysone.

Hydroxylation and conjugation were the principal pathways of metabolism of 22,25-dideoxyecdysone in cockroach fat body cultures. The major metabolite isolated and identified was the tetrahydroxy steroid 22-deoxyecdysone; other exdysteroids isolated, in order of decreasing quantities, were 22-deoxy-26-hydroxyecdysone, 22,25-dideoxy-26-hydroxyecdysone, and 22-deoxy-20-hydroxyecdysone. Cockroach fat body from late-instar nymphs appears to lack the mechanism for hydroxylating at C-22. Radioanalyses of the material obtained from enzymic hydrolysis of the conjugate fraction showed 65, 15 and 20% of tetraols, pentaols, and unhydrolyzed conjugates respectively, and no 22,25-dideoxyecdysone. An azasteroid and two nonsteroidal amines that effectively inhibit the activity of 22,25-dideoxyecdysone in the cockroach leg regenerate-fat body culture system enhanced the metabolism of 22,25-dideoxyecdysone, decreased the quantity of the pentaol fraction present, and caused an increase or accumulation of the tetraol and conjugate fractions in the fat body culture system.

An organ culture assay system using cockroach leg regenerates and fat body has been developed in which compounds that either inhibit molting hormone metabolism or act as molting hormone antagonists can be tested. Representatives of three classes of compounds were tested in the system: ecdysteroids, azasteroids, and nonsteroidal amines and amides. Inhibitory compounds were found in all three of the classes. Certain of these inhibitors represent a new class of insect hormonal compounds with a novel mode of action—the disruption of molting hormone metabolism.

Hemolymph β-ecdysone levels are high (∼1.6 μg/ml) in late last instar cockroach (Leucophaea maderae) nymphs; the level of α-ecdysone (∼0.1 μg/ml) is evidently subphysiological. Cultured leg regenerates, target organs of ecdysone, are capable of slowly converting α- to β-ecdysone. Cultured prothoracic glands secrete α-ecdysone, which was identified by complete mass spectrometry. These results are consistent with the view that α-ecdysone, secreted by the prothoracic gland, functions as a prohormone which is converted into the active moulting hormone, β-ecdysone, in other tissues.

A study of the effects of β-ecdysone on the initiation of cuticle deposition and seta formation by cockroach leg regenerates in vitro showed that both processes are ecdysone-dependent and are initiated by the same threshold dose, but the responses differ qualitatively and quantitatively. The initiation of cuticle deposition depends primarily on the accumulation of hormone-initiated events by the target tissue. The initiation of seta formation has an additional requirement for simultaneous protein synthesis.

A review of the literature of the action of hormones on insect tissues in vitro published between 1915 and 1970 indicates that research has been concerned with four main areas: studies of the development of imaginal discs, studies of morphogenesis and cuticle secretion in epidermal tissues, studies of the development of gonads, and studies of secretory activity in endocrine glands. The progress made in each field and the possibilities of further investigations are discussed.

An in vitro system was used to study the effects of diffusible substances from various endocrine tissues on the growth of nerve tissue from regenerating cockroach legs. Incubates prepared from endocrine glands and gland combinations were tested for their effect on regenerative growth from the stump of the fifth mesothoracic nerve of 8-day leg regenerates from late instar nymphs of Leucophaea maderae. Incubates prepared from the prothoracic ganglion produced a stimulatory effect, while the prothoracic gland, allatum-cardiacum complex, and brain showed no effect. When prothoracic gland and prothoracic ganglion incubates were combined, the results were the same as with ganglion incubate alone. When the glands were incubated together and allowed to interact, the nerve growth stimulating effect of the ganglion was reduced by 50%. To explain these findings, it is postulated that a diffusible substance is released by the prothoracic ganglion which stimulates regenerative nerve growth from the leg regenerate. The release of this nerve growth stimulating factor is inhibited by interaction with the prothoracic gland. The possible effect of this interaction on the process of leg regeneration as it occurs in vivo is discussed.