LaVern R. Whisenton

Brain-mediated 20-hydroxyecdysone regulation of juvenile hormone synthesis by the corpora allata of the tobacco hornworm Manduca sexta

In the latter part of the last larval instar (Days 6-7) of Manduca sexta an increase in the hemolymph titer of juvenile hormone (JH) occurs which is involved in directing the pharate pupal development elicited by the large increase in the ecdysteroid titer on Days 7-8 of the instar. A time course analysis on Days 3-4 of the instar of both the first hemolymph ecdysteroid titer peak, which is responsible for wandering behavior and pupal commitment, and of the capacity of the corpora allata (CA) to synthesize JH I and III in vitro revealed a temporal correlation between the increasing ecdysteroid titer and a following increase in CA activity. The temporal arrangement of these increases suggested that ecdysteroids, i.e., 20-hydroxyecdysone, may elicit the increase in CA activity which contributes to the second increase in the JH titer during the latter part of the instar. Regulation of the CA by 20-hydroxyecdysone was investigated in vitro by assessing the effect of this ecdysteroid on JH I and III synthesis by CA incubated either as a complex with the brain-corpora cardiaca or as isolated glands. Dose-response analyses revealed that 20-hydroxyecdysone indeed had a stimulatory effect on JH synthesis by the CA, but only when the glands were complexed with the brain-corpora cardiaca. The effective concentration of 20-hydroxyecdysone was the same as the hemolymph concentration of the hormone at this time.(ABSTRACT TRUNCATED AT 250 WORDS)

Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta

Abstract Regulation of the haemolymph titres of ecdysteroids and the juvenile hormones (JH) during larval-pupal development of the tobacco hornworm, Manduca sexta , involves the interendocrine control of the synthesis of each hormone by the other. Temporal relationships between the ecdysteroid titre peaks in the fourth and early fifth larval instar and the increases in corpora allata (CA) activity at these times suggests that ecdysteroids are evoking the increases. Incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes and isolated CA from these stages with 20-hydroxyecdysone (20-HE) revealed that 20-HE stimulates CA activity and that it does this indirectly via the Br-CC. The resulting increase in the JH titre after the commitment (first) peak in the fifth instar stimulates the fat body to secrete a factor which appears to be the same as a haemolymph stimulatory factor for the prothoracic glands. This moiety acts as a secondary effector that modulates the activity of the prothoracic glands and thus the ecdysteroid titre. These findings together have begun to elucidate the mechanisms by which the principal developmental hormones in the insect interact to regulate postembryonic development.

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

Abstract The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.

Interendocrine control by 20-hydroxyecdysone of the corpora allata of Manduca sexta

Abstract A positive interendocrine control by 20-hydroxyecdysone regulates the corpora allata (CA) and thus the hemolymph titers of the juvenile hormones (JHs) during the fourth and fifth larval stadia of the tobacco hornworm, Manduca sexta. The effect of 20-hydroxyecdysone is exerted via the brain-corpora cardiaca (Br-CC), and the kinetics of stimulation by the hormone suggest that its control of the CA is indirect, possibly involving neural effectors. Co-incubation of precommitment CA with Br-CC resulted in a stimulation of the synthesis of JH I acid equivalent to that achieved by the incubation of brain-corpora cardiaca-corpora allata (Br-CC-CA) with 20-hydroxyedcysone, suggesting that a diffusible regulatory factor may be involved in the stimulation. During pharate pupal development, a close temporal relationship exists between the drop in the ecdysteroid titer and declines in CA activity and the JH hemolymph titer, suggesting that 20-hydroxyecdysone may now inhbit the CA. Incubations of day 6 Br-CC-CA with physiological concentrations of 20-hydroxyecdysone resulted in an apparent dose-dependent inhibition of CA activity. Thus in Manduca, 20-hydroxyecdysone may exert varying stage-specific interendocrine effects on the CA which direct the postembryonic development of this species.

Ecdysteroid titres during pupal-adult development of Aedes aegypti: Basis for a sexual dimorphism in the rate of development

Abstract The ecdysteroids are steroid hormones primarily responsible for moulting and metamorphosis in insects. In mosquitoes, much is known about their role in the control of ovarian development in adults, however, essentially nothing is known about their control of this dipterans postembryonic development. In this study, quantitative and qualitative changes in the ecdysteroid titre during the pupal-adult development of the yellow fever mosquito, Aedes aegypti, were correlated with a sexual dimorphism in the time of adult eclosion. During development there was a single large peak in the ecdysteroid titre of male and female Aedes. The peak in males (∼220 pg ecdysone equivalents mg−1), was ∼1.5-fold greater than that in females (∼150 pg ecdysone equivalents mg−1), and occurred ∼4 h sooner. Subsequently, males eclosed ∼12 h sooner than females. The ecdysteroids present in developing males and females included, ecdysone, 20-hydroxyecdysone, 26-hydroxyecdysone, 20, 26-dihydroxyecdysone and polar metabolites. The relative amounts of these ecdysteroids and their conjugates fluctuated during development, and were essentially the same in males and females. When total ecdysteroid levels were calculated relative to the radioimmunassay cross-reactivities of the ecdysteroids present, a quantitative sexual difference was not evident. Therefore, it appears that the sexual dimorphism in the rate of Aedes development is probably a result of temporal differences in the respective ecdysteroid titres. A biological explanation for the earlier eclosion of male Aedes is presented.

A kinetics analysis of brain-mediated 20-hydroxyecdysone stimulation of the corpora allata of Manduca sexta

An in vitro method for investigating 20-hydroxyecdysone regulation of the corpora allata (CA) has been used to assess the kinetics of stimulation of precommitment day 3 fifth (V) larval instar Manduca sexta CA by 20-hydroxyecdysone. The synthesis of juvenile hormone (JH) I and III acids by 20-hydroxyecdysone-stimulated CA incubated as complexes with the brain-corpora cardiaca (Br-CC) increased similarly over time; the synthesis of JH III acid was greater than that of JH I acid. Maximal stimulation of the CA to synthesize both JH acids occurred when the Br-CC-CA were exposed to 20-hydroxyecdysone for 30-60 min. Following stimulation, the elevated rates of JH I and JH III acid synthesis remained unchanged over an 11 h incubation in the absence of the steroid hormone, suggesting that once stimulated by 20-hydroxyecdysone the CA biosynthetic response is persistent. These kinetics data provide insight into the means by which 20-hydroxyecdysone stimulates the CA via the Br-CC.

Multiple forms of cerebral peptides with steroidogenic functions in pupal and adult brains of the yellow fever mosquito, Aedes aegypti

In the adult mosquito, Aedes aegypti, the cerebral egg development neurosecretory hormone (EDNH) regulates reproduction by activating the ovaries to synthesize the steroid hormone ecdysone, while during postembryonic development the cerebral prothoracicotropic hormone (PTTH) activates the prothoracic glands to synthesize ecdysone. In each case, ecdysone than drives stage-specific physiological processes. Since EDNH and PTTH share a common steroidogenic function, it is conceivable that they are similar, if not the same, peptide(s). This investigation has begun to address this possibility by determining structural and functional relationships between these two families of neuropeptides. The peptides having EDNH activity in adult and pupal heads of Aedes aegypti were characterized on the basis of their molecular weights and their biological properties in in vitro and in vivo bioassays for EDNH. Gel filtration chromatography of extracts revealed fractions with EDNH activity having molecular weights of approximately 11 kDa and approximately 24 kDa. The similarities in the functional dynamics of these two activities in the different bioassays suggested that comparable moieties existed in pupal and adult brains. The sensitivity of these moieties to proteolytic hydrolysis indicated that they were proteins. Thus at least two EDNH-like peptides appear to be present in pupal and adult heads of Aedes aegypti. The apparent existence of more than one molecular weight form of EDNH has not been reported previously. Since these moieties are present in both pupal and adult heads, it is possible that they have the same steroidogenic function in the two different stages, i.e., to activate ecdysone synthesis, by prothoracic glands in larvae and pupae and by the ovaries in adults.

Life cycle expression of a bombyxin-like neuropeptide in the tobacco hornworm, Manduca sexta

Immunocytochemistry was used to investigate the developmental expression of the insulin-like neuropeptide bombyxin in the tobacco hornworm, Manduca sexta. A mouse monoclonal antibody raised against a synthetic peptide corresponding to bombyxins A-chain N-terminus was used to localize a bombyxin-like peptide to a group of cerebral medial neurosecretory cells, the M-NSC IIa(2). Immunostaining was first detected on day 0 of the second larval instar, localized in the M-NSC IIa(2) somata and in the neurohemal organ, the corpora allata (CA). By day 0 of the fourth larval instar, the peptide was present throughout the M-NSC IIa(2) somata, axons, dendritic fields and CA. Between days 7 and 9 of the fifth instar, a dramatic reduction in the dendritic fields and CA staining occurred, suggesting the peptide is released. After day 2 of the pupal period, only M-NSC IIa(2) somata immunostained, a pattern that persisted through day 2 of the adult stage. The specificity of immunostaining was demonstrated by using a synthetic bombyxin peptide to block staining. These developmental data reveal times of potential Manduca bombyxin-like peptide release which should provide insight into the peptides function.

Regulation of juvenile hormone biosynthesis by 20-hydroxyecdysone during the fourth larval instar of the tobacco hornworm, Manduca sexta

A previous study of the role of 20-hydroxyecdysone in the regulation of juvenile hormone (JH) biosynthesis during pupal commitment in the last (fifth, V) larval instar of Manduca sexta revealed that the increase in the hemolymph edysteroid titer that elicits commitment also stimulates the corpora allata (CA) to synthesize JHs I and III and/or their acids. This stimulation is exerted indirectly via the brain-corpora cardiaca and results in the postcommitment increase in the JH titer that is important for the molt to a pupa. The possibility that a similar form of interendocrine regulation of JH biosynthesis by 20-hydroxyecdysone is involved in the control of larval molting in Manduca was investigated for the molt from the fourth (IV) to the V larval instar. During the IV instar, the increase in the hemolymph ecdysteroid titer and the capacity of the CA to synthesize JHs I and III in vitro correlated temporally in a manner suggestive of CA regulation by 20-hydroxyecdysone. With an in vitro approach, physiological concentrations of 20-hydroxyecdysone (greater than or equal to 1 microgram ml-1 hemolymph) were shown to have a dose-dependent trophic effect on CA activity, specifically stimulating JH I synthesis. Since neither basally active nor stimulated CA contained significant amounts of JH I, it appears that 20-hydroxyecdysone acts at the level of biosynthesis rather than at the level of release. The specificity of 20-hydroxyecdysone stimulation of JH I synthesis was demonstrated by the inability of the biologically inactive ecdysteroids 22-isoecdysone and 5 alpha-ecdysone to activate the glands.(ABSTRACT TRUNCATED AT 250 WORDS)

Inter-species cross-reactivity of the prothoracicotropic hormone of Manduca sexta and egg-development neurosecretory hormone of Aedes aegypti

Abstract The cross-reactivities of the big and small forms of prothoracicotropic hormone (PTTH) from pupal brains of Manduca sexta and egg-development neurosecretory hormone (EDNH) from heads of adult Aedes aegypti were examined for PTTH by the in vitro Manduca prothoracic gland assay and for EDNH by the in vitro and in vivo Aedes ovary assays. The synthesis of ecdysone by both larval and pupal prothoracic glands of Manduca was increased in a dose-dependent manner by crude extracts of Aedes aegypti heads, reaching a maximum of approx. 3- and 2-fold, respectively. Gel filtration chromatography of the Aedes head extract revealed a peak of EDNH activity with an apparent mol. wt of 11 kD. This partially purified EDNH did not possess prothoracicotropic activity in the in vitro prothoracic gland assay, nor did any other fractions from the gel filtration column. Similarly, partially purified big and small PTTH did not activate Aedes atropalpus ovaries to synthesize ecdysone in vitro , nor did they cause ovarian maturation in vivo . Thus, it appears that the structural differences between PTTH and EDNH are sufficient enough to prevent functional cross-reactivity. The apparent discrepancy in the results obtained with the crude and partially purified EDNH and PTTHs raises questions about the reliability of bioassays for screening the presence and cross-reactivity of peptide neurohormones in crude extracts.