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 bombyxin's 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 peptide's function.

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.

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)

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.

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)