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

Abstract

The tobacco hornworm, Manduca sexta, has been a useful model for studying insect endocrinology. In M. sexta the majority of growth occurs during the fifth (final) larval stage. This growth is concurrent with endocrine changes that lead to a metamorphic molt. Past research has focused on the interplay of three growth hormones: juvenile hormone (JH), prothoracicotropic hormone (PTTH) and ecdysone, in regulating metamorphosis. The prothoracic glands, source of ecdysteroid molting hormones in insects, play a crucial role in coordinating growth. Insulin/insulin-like growth factor (IGF) signaling has been shown to regulate growth and secretion of steroidogenic tissues in both vertebrates and invertebrates, but it had yet to be well-characterized in M. sexta.In this dissertation I examined the role of insulin-directed growth in prothoracic glands during both normal larval development and in response to nutrient deprivation. In particular, I predicted that in response to a reduction in nutrients, the prothoracic glands would stop growing and ecdysone secretion would be reduced. Changes were assessed in actin abundance and in two nutritionally sensitive transcripts, insulin receptor and 4EBP.Actin abundance in prothoracic glands increased in the first four days of this stage, demo nstrating significant growth. Actin decreased in glands from starved larvae, indicating a cessation of growth. Both insulin receptor and 4EBP transcript were present during normal larval development. As expected, both insulin receptor and 4EBP transcript increased in response to starvation. This research was the first demonstration of insulin signaling in prothoracic glands of fifth instar M. sexta larvae, and revealed nutritional sensitivity of the prothoracic glands. 5Finally, ecdysone assays were conducted to determine the impact of nutrition on glandular secretory capacity. Starved larvae were unable to secrete as much ecdysone as fed controls. Injections of insulin were unable to rescue the ability of the glands to secrete high levels of ecdysone. This demonstrated that although insulin signaling was correlated with normal growth of the glands, insulin is not sufficient to impact the ability of the glands to secrete ecdysone in the absence of nutrie nts. 6 This thesis is dedicated to my parents,Robert and Angela Walsh, whose love and support made this dissertation possible.

TOR signaling is involved in PTTH-stimulated ecdysteroidogenesis by prothoracic glands in the silkworm, Bombyx mori

“…In addition, our previous study showed that insulin signaling is involved in growth and ecdysteroidogenesis of prothoracic glands (Gu et al, 2009). Insulin signaling in relation to the growth and secretory activity of Manduca prothoracic glands was also recently demonstrated (Walsh and Smith, 2011). Both bovine insulin and bombyxin (an insulin-like peptide in B. mori) also activate TOR signaling in silkworm prothoracic glands (Gu, unpublished data).…”

Direct effects of hypoxia and nitric oxide on ecdysone secretion by insect prothoracic glands

“…Hence the glands were not incubated with trachea in an air-filled condition. Similar in vitro protocols are routinely used for assays of ecdysone secretion by insect prothoracic glands, for incubation periods ranging in length from 2 to 24 hr (Bombyx mori (Gu et al, 2009;Mizoguchi and Kataoka, 2005), Pseudaletia separata (Komiya et al, 2001), Rhodnius prolixus (Vafopoulou and Steel, 1998), M. sexta (Bollenbacher et al, 1984;Walsh and Smith, 2011;Watson et al, 1987)). …”

“…10 ll of sample were analyzed by procedures as previously described (Walsh and Smith, 2011;Warren et al, 1984). The ecdysone antibody was produced in rabbits against an ecdysone-22-succinyl thyroglobulin synthesized by Dr. D.H.S.…”

A molt timer is involved in the metamorphic molt inManduca sextalarvae

“…Coordination of growth within and among tissues and organs then is mediated by the insulin/insulinlike growth factor (IGF) pathway. In Manduca the prothoracic glands grow during the final instar, and this growth is dependent on amino acids and TOR signaling (42,43). Accompanying the growth is an increased capacity to secrete ecdysone in response to PTTH, and starvation for the first 2 d of the fifth instar strongly suppresses this response and increases transcript levels of the insulin receptor and 4E-binding protein (4EBP) (a translation inhibitor) (42).…”

“…In Manduca the prothoracic glands grow during the final instar, and this growth is dependent on amino acids and TOR signaling (42,43). Accompanying the growth is an increased capacity to secrete ecdysone in response to PTTH, and starvation for the first 2 d of the fifth instar strongly suppresses this response and increases transcript levels of the insulin receptor and 4E-binding protein (4EBP) (a translation inhibitor) (42). Injection of insulin, however, was unable to rescue ecdysone production by the glands although it rescued the starvation-induced deficits in protein synthesis.…”