In vitro activation of insect prothoracic glands by the prothoracicotropic hormone

Abstract

An in vitro assay for the prothoracicotropic hormone has been developed that utilizes an ecdysone radioimmunoassay to quantify the increase in the rate of ecdysone synthesis elicited by the neurohormonal activation of the prothoracic glands. The rapidity, reproducibility, and accuracy of the assay were maximized by using one member of a gland pair as the control and the other as the test gland. This was possible because the basal rates of ecdysone synthesis by the members of a gland pair were equivalent. Activation was demonstrated to be dose dependent'and specific, with prothoracicotropic hormone activity present only in homogenates of brain. The in vitro activation of the prothoracic glands was verified with the Manduca bioassay for the prothoracicotropic hormone in which the morphological responses to the hormone were correlated with increased in vivo ecdysone titers. These results provide unequivocal evidence that the activation of the prothoracic glands by the prothoracicotropic hormone is direct and suggest that activation represents an increase in a basal rate of ecdysone synthesis. The endocrine function of the insect brain in the process of metamorphosis was first demonstrated in 1922 (1), but nearly 2 decades passed before it was demonstrated that this function involved the control of molting (2, 3). The initial observations on the role of a cerebral neurohormone in the molting process have since been confirmed by various implantation, extirpation, and ligation experiments (4-6). On the basis of these in vvo studies, it has been assumed that this brain hormone, now termed prothoracicotropic hormone (PTTH), acts directly on the prothoracic glands (PGs) to stimulate the synthesis of the steroid prohormone ecdysone (7). Hydroxylation of ecdysone at C-20 to form ecdysterone (20-hydroxyecdysone) then occurs in tissues peripheral to the PGs, and it is ecdysterone that initiates the molting process (7).Although PTTH was the first hormone to be discovered in insects, of the three major hormones involved in metamorphosis it alone remains to be chemically characterized. A necessary prerequisite to the chemical characterization of this putative peptide hormone is the unequivocal demonstration that PTTH directly activates the PGs. Recent attempts have not been successful due in part to the assay methods used to measure PTTH activity (8-10). Current PTTH assays are biological in nature and indirect by definition, in that they measure secondary morphological responses presumably elicited by a PTTH-stimulated increase in ecdysterone titer (10-12). However, with the. availability of a radioimmunoassay for ecdysteroids (13) and culture techniques for maintaining prothoracic glands in vitro (14), it is now possible to demonstrate the direct activation of the PGs by PTTH.This study details the development of an assay for PTTH that utilizes these two methods and proves that PTTH acts directly on the PGs to stimulate ecdysone synthesis.MATERIALS AND METHODS Animals. The tobacco hornworm (Manduca sexta) was used for...

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

“…PTTH is synthesized in the brain and released from the corpora allata. PTTH stimulates the synthesis and secretion of ecdysone (Bollenbacher et al, 1979;Bollenbacher et al, 1981). The release of PTTH is dependent upon the time of day (Truman, 1972;Truman and Riddiford, 1974).…”

Spontaneous and evoked release of prothoracicotropin from multiple neurohemal organs of the tobacco hornworm

“…Recently, an in vitro bioassay for the ready quantification of PTTH extracts was developed based on the tropic action of the neurohormone on cultured PG and measurement of the secreted ecdysone by a radioimmunoassay (RIA) (14).…”

Baculovirus replication alters hormone-regulated host development

“…At various times after infection prothoracic glands were dissected under lepidopteran saline (Weevers, 1966) and rinsed for t5 min in saline to remove contaminating haemolymph ecdysteroids. Individual glands were incubated in 25 I11 of Grace's medium (Gibco) for 12 h at 25 °C under a high humidity atmosphere (Bollenbacher et al, 1979;Kelly et al, 1992). After incubation, the culture medium was dried and re-extracted in 100 pl of 80 % methanol.…”

“…Based on the data from several classical studies (Chino et al, 1974;King et al, 1974;Bollenbacher et al, 1979), the prothoracic glands are accepted as the primary physiological source of ecdysone during most of postembryonic development. Considering the high levels of ecdysteroids in virus-infected larvae we have conducted research on the prothoracic glands to investigate their activity during virus infection, in this paper we report a significant influence of virus replication on insect hormonal synthesis by the prothoracic glands.…”