Molecular cloning and characterization of an invertebrate cellular retinoic acid binding protein.

We have cloned a cDNA and gene from the tobacco hornworm, Manduca sexta, which is related to the vertebrate cellular retinoic acid binding proteins (CRABPs). CRABPs are members of the superfamily of lipid binding proteins (LBPs) and are thought to mediate the effects of retinoic acid (RA) on morphogenesis, differentiation, and homeostasis. This discovery of a Manduca sexta CRABP (msCRABP) demonstrates the presence of a CRABP in invertebrates. Compared with bovine/murine CRABP I, the deduced amino acid sequence of msCRABP is 71% homologous overall and 88% homologous for the ligand binding pocket. The genomic organization of msCRABP is conserved with other CRABP family members and the larger LBP superfamily. Importantly, the promoter region contains a motif that resembles an RA response element characteristic of the promoter region of most CRABPs analyzed. Three-dimensional molecular modeling based on postulated structural homology with bovine/murine CRABP I shows msCRABP has a ligand binding pocket that can accommodate RA. The existence of an invertebrate CRABP has significant evolutionary implications, suggesting CRABPs appeared during the evolution of the LBP superfamily well before vertebrate/invertebrate divergence, instead of much later in evolution in selected vertebrates.

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.

Involvement of microtubules in prothoracicotropic hormone-stimulated ecdysteroidogenesis by insect (Manduca sexta) prothoracic glands.

Secretion of ecdysteroid molting hormones by insect prothoracic glands is stimulated by neuropeptide prothoracicotropic hormones (PTTH). Studies reported here were conducted to assess the effects of microfilament and microtubule inhibitors on in vitro ecdysteroidogenesis by prothoracic glands of Manduca sexta. Microfilament inhibitors (cytochalasins B and D) had no effect on basal or big PTTH-stimulated ecdysteroidogenesis. Microtubule inhibitors (colchicine, podophyllotoxin, nocodazole) had no effect on basal ecdysteroid secretion, but suppressed PTTH-stimulated secretion in a concentration-dependent manner. The effect of nocodazole was partially reversible, suggesting it was not due to nonspecific toxicity. Colchicine had no effect on glandular ecdysteroid levels, indicating that inhibition was not due solely to blockage of secretion. The combined results are consistent with the hypothesis that microtubule-mediated transport of ecdysteroid precursors plays a critical role in stimulation of ecdysteroidogenesis by PTTH.

Stimulation of ecdysteroidogenesis by small prothoracicotropic hormone: role of calcium.

Insect prothoracic glands are regulated by neuropeptide prothoracicotropic hormones (PTTH). In Manduca sexta PTTH exists as two size variants, big PTTH (approximately 25.5 kDa) and small PTTH (approximately 7 kDa). Previous studies indicate that both size variants employ cAMP as a second messenger and that stimulation of ecdysteroid secretion by big PTTH is Ca(2+)-dependent. In the present study, experiments were performed to assess the role of Ca2+ in small PTTH-stimulated ecdysteroid secretion by prothoracic glands from fifth instar larvae. Basal ecdysteroid secretion was not affected by Ca2+ channel blockers (verapamil or lanthanum) or by omission of Ca2+ from the incubation medium. Treatment of glands with a Ca2+ ionophore (A23187 or ionomycin) produced a concentration-dependent stimulation of ecdysteroid secretion. Stimulation of ecdysteroid secretion by small PTTH was suppressed (1) by Ca2+ channel blockers and (2) in Ca(2+)-free medium. A cAMP analog (Sp-cAMPS) stimulated ecdysteroid secretion in the presence of a Ca2+ channel blocker (verapamil) and in Ca(2+)-free incubation medium, and ionophore-induced ecdysteroid secretion appeared to be suppressed by a cAMP antagonist (Rp-cAMPS). The combined results indicate that basal ecdysteroid secretion is not dependent on external Ca2+, and suggest that small PTTH-stimulated ecdysteroid secretion is mediated by an influx of Ca2+ that precedes cAMP formation.

Electrical properties of the cerebral prothoracicotropic hormone cells in diapausing and non-diapausing pupae of the tobacco hornworm, Manduca sexta.

Prothoracicotropic hormone (PTTH) is an insect brain neuropeptide that is a primary factor regulating an insect development. Curtailment of its release is thought to be responsible for the pupal diapause of tobacco hornworm, Manduca sexta. The cell synthesizing and secreting the PTTH has been identified as a pair of neurosecretory cells in the pars lateralis on each brain hemisphere. Using intracellular recording techniques, we have demonstrated electrical properties of the PTTH cells in different physiological status, i.e., diapausing and developing pupae. In diapausing pupae, they showed threshold value increasing and input resistance decreasing with the progress of diapausing state, indicating that they were getting unexcitable. Spontaneous action potentials and excitatory postsynaptic potentials (EPSPs) were rarely observed in deeply diapausing state. Non-diapausing PTTH cells were almost silent except day-2, showing rather constant values of electrical properties. On day-2, a significant proportion of the cells had spontaneous action potentials, showing less negative membrane potential values than inactive cells. Exclusively inhibitory postsynaptic potentials (IPSPs) were observed in significant numbers of the cells during the period from day-2 to day-5. On the basis of the results obtained, we proposed a working hypothesis that electrical activities of the PTTH cell may be primarily regulated by its membrane properties which are further modulated by the synaptic mediation.

Diapause-dependent changes in prothoracicotropic hormone-producing neurons of the tobacco hornworm, Manduca sexta.

The prothoracicotropic hormone (PTTH), which stimulates ecdysteroid synthesis in the prothoracic glands, is produced, in the dorso-lateral protocerebrum of Manduca sexta, by paired peptidergic neurons, the lateral neurosecretory cell group III (L-NSC III). Our study revealed ultrastructural features of L-NSC III, identified by immunogold labeling, and compared developing and diapause states. In developing and early-diapause pupae, L-NSC III soma ultrastructure is similar and is characterized by numerous clusters of neurosecretory granules (NSG) and an extensive trophospongium formed by satellite-glial cells. However, as diapause progresses, the ultrastructure changes, with the NSG becoming concentrated into large clusters separated by highly organized rough endoplasmic reticulum. Most conspicuous is a substantial reduction in the number of Golgi complexes and the glial trophospongium, and the presence of stacked plasma membrane separating the glia and neuron somata. The deep-diapause soma also has abundant glycogen deposits and autophagic vacuoles. With diapause termination, this morphology reverts to the nondiapause ultrastructure within three days, i.e. just before PTTH release that evokes development to the adult. During PTTH release the abundance of NSG in the soma does not change, suggesting that NSG depletion in the perikarya is not a marker for neurosecretion by the L-NSC III.

Physical characteristics of the cerebral big prothoracicotropic hormone from Manduca sexta.

The prothoracicotropic hormones (PTTHs) are cerebral peptides that control insect postembryonic development by stimulating the prothoracic glands to synthesize ecdysteroids. In Manduca sexta, the tobacco hornworm, two classes of PTTH are distinguished by their M(r), small (ca. 7 kDa) and big PTTH (ca. 25-30 kDa). Little is known about the physical nature of the PTTHs and this study takes a first step towards defining characteristics of the Manduca big PTTH. The neurohormone has a Stokes radius of 2.59 nm and a sedimentation coefficient of 2.76 S. Based on these data, an M(r) of 29,443.7 and an f/fo of 1.27 were calculated. Combined, the physical data reveal Manduca big PTTH is an asymmetrical acidic homodimeric peptide with intra- and intermolecular disulfide bonds.

The prothoracicotropic hormone (PTTH) of the commercial silkmoth, Bombyx mori, in the CNS of the tobacco hornworm, Manduca sexta.

Immunocytochemistry revealed that a Bombyx mori prothoracicotropic hormone (PTTH)-like peptide is expressed by the Manduca sexta big PTTH-producing neurons, the lateral neurosecretory cell group III (L-NSC III). Independent PCR of genomic DNA and a L-NSC III cDNA library yielded products with 99% sequence similarity to the cDNA encoding Bombyx PTTH. This similarity necessitated evaluation of the relationship between Manduca big PTTH and Bombyx PTTH by 1) bioassay of IEF separated Manduca PTTH and 2) direct assessment of Bombyx PTTH biological activity with Manduca prothoracic glands. Together, these studies indicate that Bombyx PTTH and Manduca PTTH are different peptides expressed by the L-NSC III. The possible physiological significance of a Bombyx PTTH-like peptide in Manduca and its coexpression with Manduca big PTTH by the L-NSC III are discussed.

Immunoaffinity purification of the neuropeptide prothoracicotropic hormone from Manduca sexta.

The prothoracicotropic hormones (PTTH) are cerebral peptides that control insect postembryonic development by stimulating the prothoracic glands to synthesize ecdysteroids. Using immunoaffinity chromatography and SDS-PAGE, a 25.5 kDa big PTTH has been purified from Manduca sexta. Based upon SDS-PAGE and Western blot analysis, the native form of big PTTH appears to be a dimer with monomers of 16.5 kDa. Four HPLC-separated fragments of this acidic peptide were sequenced and exhibited no sequence similarity with Bombyx mori PTTH. In agreement with this finding, the basic Bombyx PTTH had no PTTH bioactivity in Manduca. One sequenced fragment of the Manduca PTTH is approximately 70% similar to the vertebrate cellular retinoid binding proteins, suggesting these binding proteins may be present in insects.

Stimulation of ecdysteroidogenesis by small prothoracicotropic hormone: role of cyclic AMP.

Prothoracicotropic hormones (PTTHs) stimulate synthesis and secretion of ecdysteroids by insect prothoracic glands. In Manduca sexta, PTTH exists as two size variants, small and big PTTH. Experiments were performed to assess the possible role of cyclic AMP in small PTTH signal transduction. cAMP analogs, or agents that increase intracellular cAMP, stimulated ecdysteroidogenesis. Small PTTH enhanced glandular cAMP levels; the rise in cAMP preceded an increase in ecdysteroid secretion. Prothoracic glands accumulated less cAMP when treated with small PTTH than when treated with big PTTH. A phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine) (MIX) increased the amount of cAMP in glands treated with small but not big PTTH, suggesting that glandular phosphodiesterase activity may be elevated in the presence of small PTTH. PTTH-stimulated ecdysteroid secretion was suppressed by a cAMP antagonist (Rp-cAMPS). The effects of small and big PTTH on ecdysteroidogenesis were non-additive. The combined results suggest that cAMP is employed as a second messenger by both prothoracicotropins, and that there may be subtle differences in their respective mechanisms of action.

Ultrastructure of prothoracic glands during larval-pupal development of the tobacco hornworm, Manduca sexta: a reappraisal.

The structure of Manduca sexta prothoracic glands was investigated using a protocol that preserves membranes. During the last larval stadium, prothoracic gland cells increase in diameter, volume, protein content, and perhaps number, enhancing their capacity to produce ecdysteroids. The glands' strand-of-cells morphology, their in situ location, the presence of gap junctions between cells, and junctional foot-like structures within cells support previous findings that prothoracicotropic hormone stimulates ecdysteroidogenesis via Ca(2+)-induced Ca2+ release. A different method of tissue fixation from that previously used to investigate the ultrastructure of Manduca sexta prothoracic glands has revealed a significantly different ultrastructure. These new findings begin to define roles for endoplasmic reticulum and mitochondria in ecdysteroid synthesis and support the hypothesis that the glands secrete the steroid hormone via exocytosis. The structural dynamics of the glands are discussed in the context of the glands' function during Manduca sexta larval-pupal development.

A 28-kDa cerebral neuropeptide from Manduca sexta: relationship to the insect prothoracicotropic hormone.

1. A 28-kDa peptide from the brain of the tobacco hornworm, Manduca sexta, was purified via HPLC. The peptide copurified with the insect neurohormone, prothoracicotropic hormone (PTTH), through two HPLC columns. 2. Immunocytochemistry using polyclonal antibodies against the 28-kDa peptide revealed that the peptide was produced in the same protocerebral neurons that produce PTTH. Western blot analysis demonstrated that the 28-kDa peptide and big PTTH are different molecules. 3. A PTTH in vitro bioassay indicated that despite having chromatographic properties similar to those of big PTTH and being produced by the same neurons, the 28-kDa peptide did not have PTTH activity. 4. Amino acid sequence analysis yielded a 27 N-terminal amino acid sequence that had no similarity with known peptides. 5. Immunocytochemical studies revealed that the 28-kDa peptide is present as early as 30% embryonic development and is absent by adult eclosion. This is in contrast to big PTTH, which is expressed throughout the Manduca life cycle. 6. These data suggest that the 28-kDa peptide is another secretory phenotype of the lateral neurosecretory cell group III (L-NSC III) which may have functions distinct from those for big PTTH or may act synergistically with big PTTH.

Developmental expression of the prothoracicotropic hormone in the CNS of the tobacco hornworm Manduca sexta.

The prothoracicotropic hormone is an insect neuropeptide released into the hemolymph to signal molting and metamorphosis through its stimulation of steroidogenesis. The only known source of the prothoracicotropic hormone in the tobacco hornworm, Manduca sexta, has been a group of lateral cerebral neurosecretory cells, the L-NSC III. In this study, the developmental and spatial distribution of the prothoracicotropic hormone was examined throughout the life cycle of Manduca. In common with many vertebrates and invertebrates in which neuropeptides are located in several regions within the central nervous system (CNS), the prothoracicotropic hormone phenotype in Manduca is expressed by CNS neurons in addition to the L-NSC III. These neurons are located in the brain, frontal ganglion, and subesophageal ganglion. One cerebral neurosecretory cell group, the ventromedial neurons, expresses the prothoracicotropic hormone phenotype and the behavioral neurohormone, eclosion hormone. Whereas the L-NSC III and the ventromedial neurons express the peptide phenotype throughout the life cycle, the other neurons express the peptide only during the embryonic and larval stages. This precise spatial and temporal expression of the prothoracicotropic hormone by different groups of neurosecretory cells raises the possibility that in Manduca the peptide may, in addition to its known neuroendocrine function, play other physiological roles in different ways at different stages of the life cycle.

Three-dimensional architecture of identified cerebral neurosecretory cells in an insect.

The organization of identified neurosecretory cell groups in the larval brain of the tobacco hornworm, Manduca sexta, was investigated immunocytologically. Computer-assisted three-dimensional reconstruction was used to examine the architecture of the neurosecretory cell groups. The group III lateral neurosecretory cells (L-NSC-III) which produce the prothoracicotropic hormone are located dorsolaterally in the protocerebrum and extend axons medially that decussate to the contralateral lobe prior to exiting the brain through the nervi corporis cardiaci I + II. The group IIa2 medial neurosecretory cells (M-NSC IIa2) are located anteriorly in the medial dorsal protocerebrum. The axons of these cells also exit the brain via the contralateral nervi corporis cardiaci I + II. However, their axons traverse a different pathway through the brain from that of the L-NSC III axons. Each of the cell groups possesses elaborate dendrites with terminal varicosities. The dendrites can be classified into specific fields based upon their location and projection pattern within the brain. The dendrites for these two neurosecretory cell groups overlap in specific regions of the protocerebral neuropil. After the axons of these neurosecretory cells exit the brain through the retrocerebral nerve, they innervate the corpus allatum where they arborize to form neurohemal terminals in strikingly different patterns. The L-NSC III penetrate throughout the glandular structure and the M-NSC IIa2 terminals are restricted to the external sheath. A third group of cerebral neurosecretory cells, the ventromedial neurons (VM) which stain with the monoclonal antibody to prothoracicotropic hormone in Manduca, are located anteriorly in the medial region of the brain. The axons of these cells do not exit the brain to the retrocerebral complex, but rather pass through the circumesophageal connectives and ventral nerve cord. These neurons appear to be the same VM neurons that produce eclosion hormone. One dendritic field of the L-NSC III terminates in close apposition to the VM neurons. The distinct morphologies of these neurosecretory cell groups in relation to other cell groups and the distribution of neuropeptides within the neurons suggest that insect neurosecretory cells, like their vertebrate counterparts, may have multiple regulatory roles.

The development of identified neurosecretory cells in the tobacco hornworm, Manduca sexta.

The prothoracicotropic hormone (PTTH) is a principal neuropeptide regulator of insect postembryonic molting and metamorphosis. In the tobacco hornworm, Manduca sexta, PTTH is produced by two neurosecretory cells (NSC) located in each protocerebral lobe of the brain. The development of these neurons, the L-NSC III, has been investigated immunocytologically to establish the time course of their morphological differentiation. PTTH may be one of the earliest neuropeptides expressed in insect embryos. PTTH-immunoreactivity was initially detected in the somata at 24 to 30% of embryonic development. Neurites sprouted shortly thereafter and began to grow medially through the brain anlage. By 42% embryonic development, the neurites had decussated to the contralateral brain lobe. As development progressed, the L-NSC III neurites grew along specific tracts through the contralateral brain lobe reaching the ventrolateral regions of the brain by approximately 60% development. The axons exited the brain through a retrocerebral nerve, the nervi corporis cardiaci I + II. At approximately 63% development, the axons innervated the corpus allatum and began branching to form neurohemal terminals for PTTH release. At 60% development, short collaterals began extending in the protocerebral neuropil. During the remainder of embryogenesis, both the dendritic collaterals and the terminal neurohemal varicosities continued to elongate and arborize. By 85% embryonic development, the basic architecture of the L-NSC III was established.

Regulation of ecdysteroidogenesis in prothoracic glands of the tobacco hornworm Manduca sexta.

Ecdysteroidogenesis in Manduca sexta prothoracic glands is regulated by a set of bioregulatory molecules, including prothoracicotropic hormone (PTTH) and a protein factor present in larval hemolymph, and by the competence of the glands to synthesize ecdysteroids in response to those molecules. A larval molting bioassay was used to assess the in vivo activity of Manduca PTTHs. Crude PTTH, big PTTH, and small PTTH each elicited a larval molt in head-ligated larvae. However, big PTTH was approximately 10-fold more potent than crude PTTH, which was, in turn, several orders of magnitude more potent than small PTTH. When big and small PTTH were combined, the molting response was similar to that elicited with crude PTTH. The chemical nature of the hemolymph protein factor was also investigated. Injection of [3H]cholesterol into last-instar larvae and fractionation of the radiolabeled hemolymph by gel filtration chromatography revealed three peaks of radioactivity. One peak eluted in fractions containing the hemolymph protein factor, a result consistent with the notion that the factor transports a sterol substrate. The possibility that the factor is a 3(2)-ketoreductase was investigated by assessing the effect of the factor on the accumulation of RIA-detectable ecdysteroids in prothoracic-gland-conditioned medium. Three of five preparations of the factor significantly enhanced the amount of RIA-detectable ecdysteroids in conditioned medium, indicating that at least some preparations of the factor may contain ketoreductase activity. The above findings are discussed in the context of current hypotheses of how bioregulatory molecules interact with the prothoracic glands to regulate ecdysteroidogenesis in Manduca.

Regeneration of the neurohemal terminals for identified cerebral neurosecretory cells in an insect.

The axons of specific neurosecretory cells, L-NSC III, in the brain of the tobacco hornworm, Manduca sexta, were transected during larval-pupal development to study the effects of this type of lesion on these peptidergic neurons and to begin to identify factors that may regulate their regeneration and growth. The two somata of these bilaterally paired neurons produce the prothoracicotropic hormone and are located in the pars intercerebralis. Their axons exit from the contralateral brain lobe via a retrocerebral nerve and pass through the corpus cardiacum before terminating at the glandular corpus allatum. At the corpus allatum, the L-NSC III axons arborize to form the terminal neurohemal organ for prothoracicotropic hormone release. The retrocerebral nerve was severed either in vitro followed by brain transplantation or in situ; in either protocol, the distal axon segments and corpus allatum were removed. The ability of the injured L-NSC III axons to regenerate was assessed immunocytologically by using a monoclonal antibody against the prothoracicotropic hormone. In both treatments, the proximal axon stumps exhibited regenerative growth as early as 1 day after axotomy, and, by the third day, neurites had extended. By the fifth day, the regenerating axons had branched to form terminal varicosities similar to those of a normal neurohemal organ. The regenerated neurohemal structure appeared to be functional, because larvae that had been bilaterally axotomized were able to metamorphose to pupae, a process requiring temporally precise periods of prothoracicotropic hormone release. In addition to the regeneration of the terminal axon structures, several other responses to axotomy and retrocerebral organ excision occurred. These included an apparent accumulation of prothoracicotropic hormone in the axons and regenerating neurohemal-like structure, sprouting of ectopic neurites from the axotomized somata, and a change in shape of the cell bodies from spherical to avoid.

A monoclonal antibody to the insect prothoracicotropic hormone.

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating molting and metamorphosis. "Big" PTTH, one of several molecular forms of the neurohormone, was isolated from brains of the tobacco hornworm Manduca sexta, and fractionated by high-pressure liquid chromatography (HPLC) for use in antibody production. A murine polyclonal antiserum and a monoclonal antibody (MAb) have been generated using this highly purified preparation of big PTTH. Antisera and hybridoma supernatants were screened with an indirect, brain whole-mount immunocytological assay, and antibody specificity was confirmed by immunocytological, ELISA, and functional criteria. In brain whole-mount preparations, the MAb (A2H5) and antiserum specifically immunostained the lateral protocerebral neurosecretory cells (L-NSC III), the prothoracicotropes, which produce PTTH. This immunostaining was blocked by preadsorbing the antibodies with big PTTH. Analysis of the elution of HPLC-fractionated big PTTH with an in vitro bioassay for the neurohormone and an ELISA employing the A2H5 MAb resulted in peaks of activity that were superimposable. Finally, the antiserum and A2H5 MAb inhibited big PTTH activation of the prothoracic glands to synthesize ecdysone in the in vitro bioassay for the neurohormone. With these specific antibodies, the organization of the PTTH neuroendocrine axis has been defined. It is now evident that both of the peptidergic neurons that comprise the L-NSC III are prothoracicotropes, and that the corpora allata are the neurohemal organs for the release of big PTTH into the hemolymph. This study indicates that these specific antibodies will be useful in investigations of numerous aspects of the biology of this cerebral neuroendocrine axis.

Juvenile hormone regulates the steroidogenic competence of Manduca sexta prothoracic glands.

The acquisition of steroidogenic competence by prothoracic glands of last instar Manduca sexta larvae is regulated by juvenile hormone (JH). Topical treatment of pre-commitment larvae with JH I or (7S)-hydroprene (a JH analog) delays development by increasing the time to pupal commitment and wandering. Prothoracic gland competence is suppressed in JH-treated larvae: Unstimulated and prothoracicotropic hormone (PTTH)-stimulated rates of in vitro ecdysone secretion are decreased relative to rates of secretion by competent glands. (7S)-Hydroprene also suppresses the competence of glands in head-ligated pre-commitment larvae, suggesting the hormone acts directly on the glands. Two results indicate PTTH plays a role in controlling competence, and that JH regulates competence indirectly by inhibiting PTTH release: (1) head-ligation prevents the acquisition of full competence, and (2) cAMP levels are elevated in glands from JH-treated larvae. Thus, the decrease in the JH titer that precedes pupal commitment in Manduca is permissive for the acquisition by prothoracic glands of steroidogenic competence.

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.