Intracellular stimulation of an identified neuron evokes cardioacceleratory peptide release.

The central nervous system of the tobacco hawkmoth, Manduca sexta, is known to contain two cardioacceleratory peptides (CAP's), both of which function in vivo as cardioregulatory neurohormones. Intracellular electrical stimulation of a single abdominal ganglion neuron evokes the release of CAP-like bioactivity. This stimulation-evoked bioactivity is destroyed by prior treatment with protease. The possibility that intracellular stimulation of a CAP-containing neuron synaptically activated additional spiking neurons is eliminated.

From behavior to molecules: an integrated approach to the study of neuropeptides.

Despite extensive information on many aspects of peptide neurobiology, the links between the behavioral effects of neuropeptides and their actions at the cellular and molecular levels are not fully understood. A pair of insect neuropeptides, the cardioacceleratory peptides (CAPs) of the tobacco hawkmoth Manduca sexta, provide an opportunity to elucidate these links. The CAPs are involved in the modulation of four distinct types of behavior during the life cycle of this moth. Functional differences at these four developmental periods can be explained by stage-specific changes in target sensitivity and the distribution of the CAP-containing neurons, including a set of peptidergic neurons that alter their transmitter phenotype postembryonically. Studies show that inositol 1, 4, 5-trisphosphate (IP3), linked to intracellular Ca2+, mediates the response of the cells to the CAPs. This preparation thus provides additional insights into the mechanisms underlying the action of multifunctional neuropeptides.

Primary structure of a cardioactive neuropeptide from the tobacco hawkmoth, Manduca sexta.

The amino acid sequence of the first of a family of insect cardioregulatory peptides from the tobacco hawkmoth, Manduca sexta, has been determined using a combination of Edman degradation microsequencing and mass spectroscopy. This peptide contains 9 amino acid residues and an observed mass for the monoisotopic protonated molecule of 956.4 Da. There are two cysteines at positions 3 and 9 forming a disulfide bridge and the carboxyl-terminus is amidated. The structure of this peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, is identical to a peptide recently isolated from crabs called crustacean cardioactive peptide (CCAP) and we propose that this peptide be named Manduca CCAP.

Amino acid sequence of CAP2b, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta.

The primary structure of a novel insect neuropeptide, Cardioacceleratory Peptide 2b (CAP2b), from the tobacco hawkmoth Manduca sexta has been established using a combination of mass spectroscopy, Edman degradation microsequencing, amino acid analysis, and biological assays. The sequence of CAP2b, pyroGlu-Leu-Tyr-Ala-Phe-Pro-Arg-Val-amide, has a molecular weight of 974.6 and is blocked at both the amino and carboxyl ends. Examination of several national computer protein data bases failed to reveal other peptides or proteins with any sequence homology to CAP2b indicating that this is likely to be a novel insect neuropeptide. This peptide may be a general activator of insect viscera since it causes an increase in heart rate in Manduca and in Drosophila, and has also been implicated in the regulation of fluid secretion by the Malphigian tubules of Drosophila.

Roles of glutamate and FMRFamide-related peptides at the chromatophore neuromuscular junction in the cuttlefish, Sepia officinalis.

Body patterning behavior, the expression of highly intricate patterns, is ubiquitous among all unshelled cephalopods. These body patterns are in part generated by the coordinated activity of millions of skin chromatophore organs, each of which is regulated by a set of chromatophore muscles directly innervated by centrally located chromatophore motoneurons. This study addresses the question of the identity and function of the transmitter(s) at the chromatophore neuromuscular junction (NMJ) in the European cuttlefish Sepia officinalis. Glutamate application causes a rapid contraction of the chromatophore muscles, resulting in chromatophore expansion. Pharmacological studies demonstrate that the chromatophore muscles contain receptors blocked by glutamate-specific antagonists. Glutamate-like immunoreactivity is also present in the somata of putative chromatophore motoneurons. These findings suggest that glutamate likely acts as a neurotransmitter at the chromatophore NMJ. Evidence is also presented suggesting that FMRFamide-related peptides (FaRPs) also function as neurotransmitters at the Sepia chromatophore NMJ. FMRFamide application causes contraction of chromatophore muscles; however, the FMRFamide effect is slower and longer lasting than that of glutamate. Pharmacological data show that FMRFamide acts directly on the chromatophore muscles. FMRFamide-immunopositive cells are present in the posterior chromatophore lobe, the putative location of the chromatophore motoneuron somata. A combination of immunocytochemistry and in situ hybridization shows that some putative chromatophore motoneurons express FaRP-like immunoreactivity and an FaRP-coding mRNA transcript. Many FMRFamide-immunopositive cells in the posterior chromatophore lobes also express glutamate-like immunoreactivity. We conclude that glutamate and FaRPs likely function as fast and slow transmitters, respectively, at the Sepia chromatophore NMJ.

Peptide detection in single cells using a dot immunoblot assay.

A highly sensitive dot immunoblot assay (DIA) for the detection and quantitative measurement of small peptides in single cells is presented. This DIA protocol is simple, rapid, and produces no radioactive waste. Its femtomole sensitivity is 100 fold greater than previously described DIAs. This DIA method is sufficiently sensitive to allow reliable peptide measurements to be obtained from a single cell in a manner than is faster and easier than other peptide detection procedures. This method can also be used for several other purposes, including assessing antibody specificity and peptide quantification.

Peptidergic regulation of chromatophore function in the European cuttlefish Sepia officinalis

Color patterning in cephalopod molluscs involves activation of a peripheral chromatophore system that is under neuromuscular control. The complex behavior of individual chromatophores is mediated by a specific set of muscles, the chromatophore muscles, that receive direct innervation from the central nervous system. To date, glutamate is the only excitatory transmitter that has been proposed to act at the chromatophore neuromuscular junction of cephalopods. We present data demonstrating that the chromatophore muscles in the European cuttlefish Sepia officinalis are also regulated by the FMRFamide family of neuropeptides. Using an in vitro chromatophore bioassay, it has been determined that several FMRFamide-related peptides (FaRPs) are potent excitors of the chromatophore muscles, causing chromatophore expansion. Immunocytochemical analyses of the central nervous system using an FMRFamide antibody revealed the presence of FMRFamide-like immunoreactive cell bodies in the posterior chromatophore lobes, the region of the brain containing the chromatophore motoneurons of the fin and mantle. FMRFamide-like immunoreactivity was also seen in the periphery, in the nerves around the chromatophores and in close apposition to the muscles in the chromatophore layer of the fin. HPLC analysis of the fin dermis isolated four bioactive peaks that were FMRFamide-immunoreactive when tested on an immunoblot assay. Two of these peaks co-eluted with known FaRPs, FMRFamide and ALSGDAFLRFamide, a decapeptide isolated from squid. Taken together, these data suggest that the FaRPs are likely to be endogenous excitors of the chromatophore muscles in cephalopods.

Insect cardioactive peptides: neurohormonal regulation of cardiac activity by two cardioacceleratory peptides during flight in the tobacco hawkmoth, Manduca sexta.

The relationship between two cardioactive neuropeptides, the cardioacceleratory peptides (CAPs), and changes in heart rate during flight was investigated in the tobacco hawkmoth, Manduca sexta. In vivo heart recordings from intact, tethered adults revealed a marked increase in heart rate associated with flying. Both anterior-to-posterior and posterior-to-anterior contraction waves showed a measurable elevation in contraction frequency. These changes in heart activity were noted in animals engaged in short (20 min) or long (60 min) bouts of continuous flight. Bioassay of blood taken from flying animals revealed the presence of an activity-dependent, blood-borne cardioacceleratory factor(s). Biochemical analyses of the blood of flying insects on HPLC identified two cardioacceleratory factors which co-eluted with the two CAPs. A depletion in the ventral nerve cord levels of both CAPs was observed during flight. In vivo injections of an anti-CAP monoclonal antibody blocked the increase in cardiac activity associated with flight. These results confirm the hypothesis that both CAPs act as cardioregulatory neurohormones during flight in Manduca sexta.

Insect cardioactive neuropeptides: peptidergic modulation of the intrinsic rhythm of an insect heart is mediated by inositol 1,4,5-trisphosphate.

The beat frequency of the myogenic heart of the tobacco hawkmoth, Manduca sexta, markedly increases at adult emergence in response to 2 blood-borne peptide neurohormones, known as the cardioacceleratory peptides (CAP1 and CAP2). Three independent lines of evidence are presented supporting the hypothesis that the CAPs exert their cardiostimulatory effects on the insect myocardium through a change in the intracellular levels of inositol 1,4,5-trisphosphate (InsP3). I show that (1) InsP3 levels increase in response to CAP2 in a timely fashion, (2) exogenous application of InsP3 mimics the effects of CAP2 application, and (3) a blocker of InsP3 metabolism inhibits the effect of CAP2. These results provide strong support for the hypothesis that InsP3 is likely to be the second messenger in the regulation of heart beat activity by CAP2. Besides establishing the nature of the signaling system between CAP2 and the heart, these data also identify a novel role for InsP3, namely, the control of contraction frequency in a myogenic muscle. Given the widespread distribution of cellular systems employing InsP3 as a second messenger, it is suggested that InsP3 may also be involved in the long-term regulation of rhythmic activity in other spontaneously contractile muscles and endogenously active cells.

Molecular analysis of FMRFamide- and FMRFamide-related peptides (FaRPS) in the cuttlefish Sepia officinalis.

The display of complex color patterns of the cuttlefish Sepia officinalis is under the regulation of the FMRFamide-related peptide (FaRP) family, but their exact identities are unknown. We report the isolation and characterization of a full-length FaRP cDNA from the brain of S. officinalis. This cDNA is 1850 base pairs long, including an open reading frame of 996 base pairs. The cDNA encodes a precursor protein containing four FaRPs: ALSGDAFLRF, FIRF, FLRF and FMRF. Each propeptide has a C-terminal glycine residue that is presumably converted post-translationally to an amide. Every FaRP propeptide is also flanked by basic amino acid residues at the amino and carboxy termini, indicative of putative cleavage sites during post-translational processing. Each of the four FaRPs encoded by this cDNA causes chromatophore expansion when assayed in an in vitro chromatophore bioassay. Thus, it is likely that one or more of the FaRPs identified in this study are involved in controlling chromatophore activity in cuttlefish.

Insect cardioactive peptides: cardioacceleratory peptide (CAP) activity is blocked in vivo and in vitro with a monoclonal antibody.

We demonstrate here that a specific monoclonal antibody can be utilized as a physiological tool to analyze neuropeptide function. Two cardioacceleratory peptides (CAPs) have been recently isolated from the CNS of the tobacco hawkmoth, Manduca sexta (Tublitz and Truman, 1985a), and it has been suggested that they act as cardioregulatory neurohormones during adult emergence and wing inflation (Tublitz and Truman, 1985b). Evidence is presented here indicating that a monoclonal antibody, 6C5, selectively and specifically precipitated the biological activities of both CAPs. In vivo injections of 6C5 markedly reduced CAP hemolymph titers in newly emerged adults. The 6C5 treatment also blocked the primary physiological effect of the CAPs, the increase in cardiac activity seen during adult wing expansion. In addition, removal of the postemergence CAP pulse with 6C5 prolonged the duration of wing-inflation behavior. Thus, by neutralizing CAP hemolymph activity with a CAP-specific antibody, we have shown that the CAPs are involved in cardioregulation in newly emerged moths.

Insect cardioactive peptides. I. Distribution and molecular characteristics of two cardioacceleratory peptides in the tobacco hawkmoth, Manduca sexta.

Using an in vitro heart bioassay, the pharmacological and biochemical properties of two cardioactive peptides derived from neural tissues of the moth, Manduca sexta, were analysed. Gel filtration of ventral nerve cords (VNC) from pharate adults identified two cardioacceleratory peptides (CAP1 and CAP2) with apparent molecular weights of 1000 and 500 Da, respectively. Both CAPs were localized to the abdominal perivisceral organs, the major neurohaemal release sites in the insect VNC. Pulse application of CAP1 or CAP2 on the in vitro Manduca heart produced a dose-dependent increase in rate but had no effect on beat amplitude. The threshold dose for the action of each peptide on the isolated heart bioassay was less than 0.05 abdominal nerve cord equivalents. Both CAPs were present in the pharate adult VNC of several other Lepidopteran species. Neither CAP1 nor CAP2 was detected in the prepupal VNC of Manduca sexta.

Postembryonic alteration of transmitter phenotype in individually identified peptidergic neurons.

Many neurons are now known to undergo dramatic morphological or biochemical changes long after they have completed differentiation and maturation. The ability of fully mature neurons to alter their transmitter phenotype has been amply demonstrated in culture, but direct in vivo data on single neurons have been difficult to obtain. Here we show that a set of 4 individually identified neurosecretory neurons in the moth Manduca sexta, previously demonstrated to contain the peptide hormone bursicon, also stain with a monoclonal antibody directed against 2 insect cardioacceleratory peptides (CAPs). These lateral cells exhibit CAP-like immunoreactivity in larvae but not in pupae or adults, in contrast to other CAP-containing neurons which are strongly immunoreactive in all postembryonic stages. Biochemical analyses using high-pressure liquid chromatography confirm that the lateral neurons in larvae contain CAP2, one of the CAPs. CAP measurements of cell clusters containing these cells indicate high levels only in caterpillars. When the same neurosecretory cells are individually dissected and assayed for CAP bioactivity, high CAP levels are again found in larvae, whereas the same neurons in pupae show no such CAP bioactivity. Simultaneous determinations of both bursicon and CAP levels in single lateral cells indicate that these cells express high levels of CAP activity and low amounts of bursicon in larvae yet are solely bursicon-containing in pupae and adults. Thus, by demonstrating that these cells alter their secretory profile in vivo during metamorphosis, our results confirm the notion that functionally mature neurons are capable of altering their transmitter expression after the completion of embryonic development.

Hormonal control of transmitter plasticity in insect peptidergic neurons. I. Steroid regulation of the decline in cardioacceleratory peptide 2 (CAP2) expression.

Transmitter plasticity, the ability to alter transmitter expression, has been documented in several different preparations both in vivo and in vitro. One of these is the tobacco hawkmoth, Manduca sexta, whose central nervous system contains four individually identified lateral neurosecretory cells (LNCs) that undergo a postembryonic transmitter switch in vivo. In larvae, the LNCs express high levels of a myoregulatory peptide, cardioacceleratory peptide 2 (CAP2). In contrast, the predominant LNC transmitter in adult moths in bursicon, a classic insect peptide hormone responsible for cuticular tanning. Here we show that the CAP2-to-bursicon conversion by the LNCs is a multi-step process beginning with a decline in CAP2 levels midway through the final larval stage. We provide several lines of evidence that this CAP2 drop is regulated by the insect steroid hormone 20-hydroxyecdysone (20-HE). The LNCs exhibit a fall in CAP2 levels at the beginning of metamorphosis, immediately after the commitment pulse of 20-HE when steroid levels are elevated. LNCs not exposed to this 20-HE rise do not exhibit a decline in CAP2 level. The transmitter switch can also be prevented by using an analog of juvenile hormone to create a larval hormonal environment during the commitment pulse of 20-HE. The CAP2 decline in the LNCs could be directly induced by exogenous steroid application, but only under conditions where the LNCs remained connected to the brain. Thus, the first step in the transmitter switch by the LNCs, the decline in CAP2 levels, is triggered by the commitment pulse of 20-HE, which may act indirectly through a set of steroid-sensitive cells in the brain.

Hormonal control of transmitter plasticity in insect peptidergic neurons. II. Steroid control of the up-regulation of bursicon expression.

Each abdominal ganglion of the central nervous system of the tobacco hawkmoth, Manduca sexta contains four individually identified lateral neurosecretory cells (LNCs) that undergo a postembryonic transmitter switch in vivo. In the embryonic and caterpillar stages, the primary LNC transmitter is cardioacceleratory peptide 2 (CAP2), a myoregulatory peptide. During metamorphosis, these cells stop expressing CAP2 and instead produce bursicon, a classic insect peptide hormone responsible for cuticular tanning. We have previously reported that this transmitter plasticity is under the control of the insect steroid hormone 20-hydroxyecdysone (20-HE), which surges twice during the last larval instar. In that report we showed that the CAP2 decline is indirectly regulated by the first 20-HE rise, the commitment pulse (CP). Here we provide evidence that the rise in bursicon levels in the LNCs is directly triggered by the second 20-HE surge, the prepupal peak (PP). We performed several experimental manipulations that exposed LNCs to the PP without the CP; cells treated in this manner exhibited a significant rise in bursicon content. In contrast, bursicon levels remained unchanged in those LNCs exposed only to the CP. Exposure to the PP triggered a precocious increase in bursicon expression in LNCs from the penultimate larval stage. Increased bursicon levels in the LNCs were also induced by direct infusion of 20-HE. Taken together, the results of these experiments suggest that the rise in bursicon in the LNCs during metamorphosis is due to the direct action of the PP on the LNCs. Thus, the two 20-HE surges combine to regulate the CAP2-to-bursicon switch in the LNCs, the first acting indirectly to cause a decline in CAP2 levels and the second triggering a rise in bursicon expression, possibly by a direct action on the LNCs.

Identification of neurones containing cardioacceleratory peptides (CAPs) in the ventral nerve cord of the tobacco hawkmoth, Manduca sexta.

The abdominal ganglion neurosecretory cells responsible for the synthesis and release of two insect neurohormones, cardioacceleratory peptides 1 and 2 (CAP1 and CAP2), from the perivisceral organs (PVOs) have been identified in the tobacco hawkmoth, Manduca sexta. Previous work established the existence of two groups of abdominal ganglion cell bodies with axons projecting to the PVO: four laterally-situated pairs and five pairs lying on the midline (Taghert & Truman, 1982b). Micro-dissection and bioassay of various parts of an abdominal ganglion revealed that CAP activity was greatest in the medial portion of the ganglion, the portion containing the 10 midline neurones. Six of the 10 midline neurosecretory cells, the new midline bilateral (MB) cells, appeared to differentiate post-embryonically, commencing differentiation late in the last larval instar and reaching maturity midway through adult development. The development of the new MB cells was mirrored by the accumulation of CAP activity in the abdominal nerve cord. Not present in measurable amounts in larvae, CAP activity was first detectable a few days after pupation and reached maximal levels midway through adult development. CAP-like bioactivity was collected from the PVO in response to antidromic stimulation of the nerve containing the new MB axons. No CAP-like bioactivity was detected in those preparations in which the new MB axons were severed or in which other nerves were stimulated. Intracellular stimulation of a new MB neurone evoked the release from the PVO of measurable levels of CAP bioactivity. It was shown that this stimulation-evoked, cardioacceleratory activity was sensitive to protease treatment, and was released only from the cell that was stimulated. On the basis of these experiments, it was concluded that the CAPs are synthesized and secreted from the new MB cells.

Insect cardioactive peptides. II. Neurohormonal control of heart activity by two cardioacceleratory peptides in the tobacco hawkmoth, Manduca sexta.

The physiological characteristics of two cardioacceleratory peptides (CAPs) were analysed in the tobacco hawkmoth, Manduca sexta, to determine if either CAP functioned as a cardioregulatory neurohormone. In vivo heart recordings from pharate and newly emerged adults revealed a dramatic increase in heart rate associated with wing-spreading behaviour. Bioassay of whole blood taken from wing-spreading (WS) animals indicated the presence of a stage-specific, blood-borne cardioacceleratory factor(s). Gel filtration of WS blood identified two cardioacceleratory factors which co-eluted with the two CAPs. A depletion of the ventral nerve cord levels of both CAPs was observed during WS behaviour. Measurements of blood CAP levels showed that the peak CAP titres were coincident with the initiation of WS behaviour. Experimental manipulations that delayed the onset of WS behaviour also prevented CAP release. High potassium incubation evoked the release of both CAPs in a calcium-dependent manner. In vivo injections of CAP1 or CAP2 caused a dose-dependent increase in heart rate. These results confirm the hypothesis that both CAPs function as cardioregulatory neurohormones during wing-spreading behaviour in Manduca sexta.

Modulating a modulator: biogenic amines at subthreshold levels potentiate peptide-mediated cardioexcitation of the heart of the tobacco hawkmoth Manduca sexta.

The central nervous system of the moth Manduca sexta contains a group of myoregulatory neuropeptides, the CAPs (Cardioacceleratory Peptides), which cause a physiologically important, dose-dependent increase in heart rate during wing inflation and flight in adult moths. We report here that the response of the adult heart to a subset of the CAPs, the CAP2S, is potentiated nearly twofold in the chronic presence of subthreshold levels of the biogenic amine octopamine or near-threshold levels of the biogenic amine serotonin. Subthreshold levels of the CAP2S fail to alter the response of the heart to octopamine. We have begun to investigate the molecular mechanisms underlying this potentiation. Previous work on the adult heart has shown that the CAP2s act through an inositol-1,4,5-trisphosphate second-messenger system. Here, we demonstrate that the cardioexcitatory effects of the two amines, in contrast to those of the CAP2S, are both mediated by cyclic AMP. Application to the heart of either 10(-5) moll-1 octopamine or 10(-6)moll-1 serotonin elicits a threefold increase in intracellular cyclic AMP levels. The CAP2S have no effect on cyclic AMP levels in the heart. These results illustrate a mechanism by which the effectiveness of a neurohormone can be increased with minimal cost to the animal. In Manduca sexta, subthreshold levels of octopamine are found in the haemolymph during wing inflation and flight. Thus, it is possible that octopamine up-regulates the effects of CAP2 via a cyclic-AMP-dependent mechanism during these activities.

Neurochemical fine tuning of a peripheral tissue: peptidergic and aminergic regulation of fluid secretion by Malpighian tubules in the tobacco hawkmoth M. sexta.

The actions of various peptides and other compounds on fluid secretion by Malpighian tubules in the tobacco hawkmoth Manduca sexta sexta are investigated in this study. Using a newly developed pharate adult Malpighian tubule bioassay, we show that three tachykinin-related peptides (TRPs), leucokinin I, serotonin (5-HT), octopamine, the cardioacceleratory peptides 1a, 1b and 2c, cGMP and cAMP each cause an increase in the rate of fluid secretion in pharate adult tubules. Whereas the possible hormonal sources of biogenic amines and some of the peptides are known, the distribution of TRPs has not been investigated previously in M. sexta. Thus we performed immunocytochemistry using an anti-TRP antiserum. We show the presence of TRP-like material in a small subset of cells in the M. sexta central nervous system (CNS). The larval brain contains approximately 60 TRP-immunopositive cells and there are approximately 100 such cells in the adult brain including the optic lobes. Every ganglion of the ventral nerve cord also contains TRP-like immunoreactive cells. No TRP-containing neurosecretory cells were seen in the CNS, but endocrine cells of the midgut reacted with the antiserum. We propose the hypothesis that the control in insects of physiological systems by hormones may not always involve tissue-specific hormones that force stereotypical responses in their target systems. Instead, there may exist in the extracellular fluid a continuous broadcast of information in the form of a chemical language to which some or all parts of the body continuously respond on a moment-to-moment basis, and which ensures a more effective and efficient coordination of function than could be achieved otherwise.

Immunological, biochemical and physiological analyses of cardioacceleratory peptide 2 (CAP2) activity in the embryo of the tobacco hawkmoth Manduca sexta.

The cells in the embryonic CNS of the tobacco hawkmoth, Manduca sexta, that synthesize a cardioacceleratory peptide 2 (CAP2)-like antigen were identified using immunohistochemical techniques. Two distinct neurosecretory cell types were present in the abdominal ventral nerve cord (VNC) that contain CAP2-like immunoreactivity during late embryogenesis: a pair of large (diameter range 15-20 microns) cells lying along the posterior, dorsal midline of abdominal ganglia A4-A8, and a bilateral set of four smaller (diameter range 6-11 microns) neurons which lie at the base of each ventral root in abdominal ganglia A2-A8. CAP2-like accumulation appeared to follow independent patterns in the two cell types. CAP2-like immunoreactivity began at 60% of embryo development (DT) in the medial cells, accumulated steadily throughout embryogenesis, and dropped markedly during hatching. Lateral cells synthesized the CAP2-like antigen later in development (70% DT) and showed a sharp drop in antigen levels between 75% and 80% of embryonic development. Extracts from developing M. sexta embryos were found to contain a cardioactive factor capable of accelerating the contraction frequency of the pharate adult moth heart in a fashion similar to CAP2. Immunoprecipitation with a monoclonal antibody that specifically recognizes the two endogenous Manduca cardioacceleratory peptides and purification using high pressure liquid chromatography identified this factor as cardioacceleratory peptide 2 (CAP2). Using an in vitro heart bioassay, the levels of this cardioactive neuropeptide were traced during the development of the M. sexta embryo. As with the immunohistochemical results, two periods during embryogenesis were identified in which the level of CAP2 dropped markedly: between 75% and 80% development, and at hatching. Embryo bioassays of CAP2 activity were used to identify possible target tissues for physiological activity during these two putative release times. CAP2 was found to accelerate contraction frequency in the embryonic heart and hindgut of Manduca in a dose-dependent fashion. Of these two possible targets, the hindgut proved to be more sensitive to CAP2, having a lower response threshold and a longer duration of response to a given concentration of the exogenously applied peptide. Based on these immunocytochemical, pharmacological and biochemical results, and on a previously published detailed analysis of Manduca embryogenesis, we conclude that CAP2 is probably released from a specific set of identified neurosecretory cells in the abdominal VNC to modulate embryonic gut activity at 75-80% of embryo development during ingestion of the extra-embryonic yolk.