John D. O'Connor

Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta.

Abstract The titer of ecdysone in whole animal extracts of Manduca sexta was determined by radioimmunoassay during the fifth (last) larval instar, pharate pupal development and pupation. A subtle peak in ecdysone concentration was noted at day 4 (just prior to the onset of the wandering stage) and a second and greater peak at day 8.5 (coincident with pharate pupal development). The titer fluctuations during development were a result of changes in tissue ecdysone and not of alterations in the ecdysone content of the gut. When prothoracic gland secretory activity was analyzed in vitro at the same stages, the most rapid rate of α-ecdysone secretion was shown to occur on day 7 (one day prior to the peak in whole-animal ecdysone concentration). An earlier peak in prothoracic gland activity may occur at day 4–5. Thin layer and gas-liquid chromatographic analyses revealed developmental changes in the ratio of β:α-ecdysone in hemolymph and whole-animal extracts. It is suggested that the steroid-hydroxylating capacity of the insect increases during the instar.

Arthropod Molting Hormone: Radioimmune Assay

A radioimmune assay for the arthropod molting hormone, ecdysterone, has been developed. The sensitivity of the assay is 200 picograms or 25 times the maximum sensitivity of the bioassay. Closely related steroids also bind the anti-body, but with lower affinities.

Ecdysone titers during postembryonic development of Drosophila melanogaster

Abstract The level of ecdysone in Drosophila melanogaster was determined by a radioimmune assay in organisms selected between the second larval instar and maturity. Maxima in the titer of the hormone were observed at puparium formation and 38 hr later, just prior to the secretion of the adult cuticle. The level of ecdysone was very low in adults of either sex. However, adult females had significantly more ecdysone per organism than did males. The magnitude of this difference could be correlated with ovarian development, suggesting a possible role for ecdysone in ovarian maturation in this organism.

Trace ahalysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay

Abstract A radioimmunoassay was compared to a gas-liquid Chromatographic assay and a bioassay as a method for quantifying ecdysone titers in arthropods. In addition a continuous-development thin layer Chromatographic technique was evaluated as a means for separating various ecdysone analogues. The carboxyuethoxyamine derivative of β-ecdysone was synthesized and characterized by several spectral methods. This derivative was conjugated to bovine serum albumin to afford a suitable antigen and injected into rabbits. The antisera produced shows a moderate affinity for β-ecdysone and cross-reacts with many ecdysone analogues. Ecdysones were also quantified directly as trlmethylsilyl ethers using a 63Ni electron capture detector in a gas-liquid Chromatographic system. Less than 50 pg of material could be detected per injection. Both of these techniques and the Mosca domeetica bioassay were used for the comparative analysis of a number of biological samples. The advantages and disadvantages of the various methods are discussed.

A high affinity antiserum specific for the ecdysone nucleus

Abstract The 22-hemisuccinate ester of α-ecdysone was conjugated to bovine thyroglobulin and used to raise an antiserum which has a greater affinity for α-ecdysone and an overall higher sensitivity than those antisera previously reported. Modification of the tetracyclic nucleus characteristic of the ecdysones by either hydroxylation or dehydroxylation appreciably diminishes the ability of the ecdysone to compete for the antibody binding sites. Deviations from the side chain structure of α-ecdysone also reduce the affinity of such molecules (e.g. β-ecdysone, ponasterone A and inokosterone) but to a significantly lesser degree than alteration of the tetracyclic nucleus.

The qualitative and quantitative determinations of ecdysones in tissues of the crab, Pachygrapsus crassipes, following molt induction

On the basis of chromatographic evidence, it appears that the major circulating molting hormone is β-ecdysone in the crab, Pachygrapsus crassipes. This circulating molting hormone does not appear to be bound to any carrier protein in the hemolymph. The effects of eyestalk extirpation and the removal of the Y-organ upon the tissue distribution of ecdysone are documented using a radioimmunoassay. From in vitro experiments, it is demonstrated that crab tissues have the ability to convert radiolabeled α- to β-ecdysone. Of the tissues examined, the testis appears to have the greatest capacity for this conversion.

Repression of Y-organ secretory activity by molt inhibiting hormone in the crab Pachygrapsus crassipes

Abstract Sinus glands from the shore crab Pachygrapsus crassipes were incubated in saline to extract a Y-organ inhibiting factor. This conditioned saline inhibited ecdysone secretion by Y-organs maintained in vitro and decreased incorporation of [ 14 C]leucine into protein. Inhibition was dose dependent. Media conditioned by other tissues did not significantly affect secretion by Y-organs.

Characterization and quantification of a crustacean lipovitellin

Abstract 1. 1. Using immunodiffusion and immunoelectrophoresis the blood-borne female speific protein of Procambarus sp. was found to be immuno-chemically identical to the major high density oocyte lipoprotein. 2. 2. The lipovitellin, which is 35 per cent lipid, was shown to have a molecular weight of 500,000 and exhibit a reversible pH-dependent spectral shift. 3. 3. The lipid portion of the lipovitrellin consisted of a carotenoid, phosphatidyl choline and phosphatidyl ethanolamine, while an amino acid analysis of the lipovitellin demonstrated the sum of the non-polar residues to be 50.3 mole% and the sum of he charged residues to be 35.3 mole %. 4. 4. The quantity of lipopvsitellin per oocyte increased over 1000-fold from a 0.35 mm oocyte to a 2.0 mm oocyte.

In vitro secretion and hydroxylation of α-ecdysone as a function of the crustacean molt cycle

Abstract With the use of an ecdysteroid-specific antiserum, the alterations in the hemolymph titer of molting hormones have been determined during the course of the molt cycle in the crab, Pachygrapsus crassipes. In addition, the in vitro secretory rate of the Y-organ has been determined as a function of the molt cycle. The rate of Y-organ secretion describes a pattern qualitatively similar to that of the hemolymph titer. The ability to hydroxylate the Y-organ secretory product (α-ecdysone) to the predominant circulating form of the molting hormone (β-ecdysone) is concentrated in the mitochondria-enriched fraction of the testes of the crabs, Pachygrapsus crassipes and Cancer antennarius. This hydroxylase activity increases during an induced premolt.

An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells

The addition of physiological concentrations of the arthropod molting hormone 20-hydroxyecdysone results in the cessation of cell division in the Kc cell line of Drosophila melanogaster. Fluorometric mononitoring of the cell cycle reveals that treatment of the cells with hormone for 12 hr causes a G2 arrest. The dose-response curves are in agreement with those obtained for other hormonal effects in both the Kc line and the intact animal. In the continual presence of hormone, cells remain G2-arrested for approximately 100 hr, resuming division by 120 hr. Cells which have responded once to ecdysteroids and subsequently reentered the cell cycle are insensitive to hormonal restimulation. This lack of response has been correlated with, and is probably due to, the loss of ecdysteroid receptors in stimulated cells.