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

Neuropeptides are universally recognized as an important class of intercellular messengers throughout the animal kingdom. In mature systems, neuropeptides have been demonstrated to act as neurotransmitters, neurohormones, and paracrine factors to regulate the function of most tissues including modulating CNS activity (Pickering et al. 1987; O’Shea and Schaffer, 1986; Mayeri et al. 1985; Jan and Jan, 1982; Guillemin, 1978). Despite this wealth of information in mature systems, the functional significance of neuropeptides during development has been largely ignored. In particular, little attention has been given to participation of peptides during embryogenesis. To date, efforts in this direction have been largely limited to tracing peptide levels and mapping peptide distribution using a variety of immunological and hydridization assay techniques (Pickering et al. 1987). In most cases, the functional role of these embryonic neuronal factors has proven very difficult to establish.

Of the few investigations concerning neuropeptide function in developing systems, most have utilized invertebrate preparations. In simple freshwater coelenterates, for example, Head/Foot Activating Peptide has been demonstrated to possess neurotransmitter and/or neuromodulatory properties that interact to establish determinate fate of regenerating and/or developing tissues (Schaller, 1979). In insects, the shedding of the embryonic cuticle is correlated to a drop in the storage levels of a neurosecretory peptide, eclosion hormone (Truman et al. 1981). Based on this evidence, eclosion hormone has been postulated to play a role in embryonic moults similar to its role during larval and adult development.

Given the diversity of neuropeptide roles post-embryonically, coupled with the few reports in the literature on their putative embryonic function, it seems plausible that peptides play a greater variety of roles in the developing embryo than previously thought. One approach to the study of peptides during development is to investigate the embryonic role of peptides that are present and functionally important in the mature nervous system. Such studies are best accomplished in a developing system that is relatively well characterized and experimentally tractable. One preparation that meets these criteria is the tobacco hawkmoth, Manduca sexta, whose CNS has been the subject of intensive study (Weeks, 1987; Levine, 1986; Tublitz et al. 1986; Truman, 1985).

Our focus in the present study is two cardioregulatory neuropeptides, cardioacceleratory peptide 1 and 2 (CAPj and CAP₂), that were originally isolated in the pharate adult stage of M. sexta (Tublitz and Truman, 1985a). Using in vitro and in vivo heart bioassays, these peptides were shown to be released into the haemolymph from individually-identified neurosecretory cells in the CNS, accelerating heart contraction frequency in a dose-dependent manner (Tublitz and Truman, 1985a, b). Biochemical and physiological studies demonstrated that both peptides play important physiological roles in the adult moth, acting as cardioexcit-atory hormones during wing-spreading behavior and flight (Tublitz and Truman, 1985b; Tublitz and Evans, 1986; Tublitz, 1989). Further work has recently shown that CAP₂ strongly excites the hindgut in fifth instar larvae, apparently to aid gut emptying during this developmental stage (N.J. Tublitz, unpublished observations). The CAPs thus have several different stagespecific functions.

Since the CAPs perform several different roles throughout post-embryonic life, we were interested in identifying other CAP roles even earlier in development, during embryogenesis. Results from preliminary immunocytochemical studies using an anti-CAP antibody identified several sets of CAP-immunopositive neurons in the latter stages of embryogenesis (Broadie et al. 1989b). In the present study, we use pharmacological, biochemical, and immunocytochemical techniques to trace the acquisition and distribution of the CAP peptides during embryo morphogenesis. Our results provide several lines of evidence strongly supporting a functional role for one of the CAPs during embryo formation.

Tobacco hawkmoths, M. sexta, were reared on an artificial diet (Bell and Joachim, 1978) in a controlled temperature (27°C [light], 25°C [dark]) room with a 17h photoperiod. Humidity was kept above 50% at all times. Under these standard conditions, the time duration from oviposition to hatching was 95±3h (mean±s.E.M.; N= 100). For this study, embryonic development is expressed as a percentage of the total development time (DT) using morphological development characteristics as observed in our laboratory (Broadie et al. 1989a) and elsewhere (Dorn et al. 1987).

A mouse monoclonal antibody, 6C5, shown to be highly specific for both CA?! and CAP₂ (Tublitz and Evans, 1986), was used in all trials. 6C5 isolation and preparation has been described previously (Taghert et al. 1983, 1984). In short, the crude extract of 3000 pharate adult Manduca perivisceral organs, the neurohaemal release site in the VNC for CAPs, was used as the inoculum. Antibody specificity was established by several independent criteria including morphological staining of known CAP-containing cells in pharate adult abdominal ganglia, in vivo immunoneutralization, in vitro ELISA assays and immunoprecipitation of both CAPs (Tublitz and Evans, 1986: Taghert et al. 1983, 1984). 6C5 was also shown to bind specifically both CAPi and CAP₂ that had been purified to homogeneity using high pressure liquid chromatography (HPLC; Tublitz and Evans, 1986).

To reconfirm the specificity of our anti-CAP antibody 6C5, a series of incubations was performed to establish the nature of the antigen(s) capable of immunoprecipitating the antibody. Both the suspect neuropeptide antigen, CAP₂, and a range of other known invertebrate cardioexcitatory neuropeptides were incubated with 6C5 (diluted 1:1000 in 0.4% saponin-PBS+1.0% BSA) for 30min prior to application onto a fixed embryo. Neuropeptides used were as follow: HPLC-purified CAP₂, peptide F (thr-asn-arg-asn-phe-leu-arg-phe-amide) kindly supplied by Dr B. A. Trimmer (Trimmer et al. 1987), and two molluscan cardioexcitatory peptides; FMRFamide (phe-met-arg-phe-NH₂; Greenberg and Price, 1979), and one of the small cardioactive peptides (SCP_D, met-asn-tyr-leu-ala-phe-pro-arg-met-NH₂; Lloyd, 1978). The staining protocol used with these putative 6C5-antigen complexes was in all other ways identical.

Embryos were dissected free of the chorion and yolk with fine forceps and the ventral CNS exposed. This dissection was accomplished by securely fastening the specimen in a Sylgard (Dow-Coming) dish containing Manduca saline, making an anterior-directed incision through the dorsal body wall from tail horn to head capsule, pinning open the body wall, and removing the embryonic gut to expose the ventral nerve cord (VNC). Dissected embryos were incubated at 4 °C with gentle agitation in a modified Bouin’s/glutaraldehyde fixative (2% glutaraldehyde, 25% saturated picric acid and 1% glacial acetic acid) for 1 h, washed three times in 0.4 % saponin-PBS for 30 min each, and taken through an ethanol dehydration series at 4°C. Fixed specimens were incubated in collagenase (1 mg ml^-1; Sigma, type XI) for Ih, followed by extinction of endogenous peroxidase activity with 0.75% hydrogen peroxide in methanol. Specimens were then blocked with goat serum (5mgml^-1; Sigma lot no. 28F-9401) and bovine serum albumin (1% w/v) in 0.4% saponin-PBS for2h.

A three-tier antibody system using the peroxidase-anti-peroxidase (PAP) method was employed to identify cells with CAP-like immunoreactivity. Primary antibody (6C5; dilution 1:1000), secondary antibody (whole molecule, goat antimouse IgG; dilution 1:100), and tertiary antibody (mouse peroxidase anti-peroxidase (PAP); dilution 1:100) were suspended with 1 % BSA in 0.4% saponin-PBS. Each antibody suspension was incubated for 24 h at 4°C. Specimens were washed five times in five hours with 0.4% saponin-PBS containing 1 % BSA between successive incubations. Following equilibration in 3,3′-diaminobenzidine (DAB), immunoreactivity was visualized by incubation in a solution of 0.8 % saponin-PBS containing DAB (0.5 mg ml^-1), hydrogen peroxide (0.003 % v/v) and NiCl₂ (0.001 % w/v) until complete, usually 10-15 min. Visualized embryos were taken through an ethanol dehydration series, equilibrated in xylene, and mounted in Permount for observation with Nomarski optics.

Developmentally staged embryos (Dorn et al. 1987; Broadie, 1989a) were heat-treated for 5 min at 80°C, ice-cooled, and homogenized in double-distilled H₂O (ddH₂O; 10μl/embryo) in a ground glass homogenizer. The homogenate was centrifuged (15 min, 12000g, at 4 °C) and the supernatant collected. The pellet was re-suspended in ddH₂O (20μl/embryo), reground, centrifuged, and the supernatants from both extractions pooled. The combined supernatant fraction was loaded onto a MeOH-activated, water-rinsed Waters C-18 Sep-Pak cartridge and washed in five times its volume with ddH₂O. This was followed by step-wise applications of 20 % and 80 % acetonitrile (HPLC grade; J.T. Baker no. 9017–2) in ddH₂O. From earlier studies (Tublitz and Truman, 1985c; Tublitz and Evans, 1986), it was known that both CAP] and CAP₂ elute in the 80% acetonitrile fraction. Accordingly, this fraction was collected, frozen in dry ice, and lyophilized to powder. Lyophilized samples were stored at −20°C for up to one month before use. Immediately prior to bioassay, samples were brought to room temperature and re-hydrated in normal Manduca saline.

An in vitro heart bioassay was used to quantify relative CAP levels in each fraction as described earlier (Tublitz and Truman, 1985c). In short, a portion of the abdominal heart was dissected from a pharate adult male immediately prior to eclosion. One end of the heart tissue was pinned in a small superfusion chamber; the other end was attached with fine suture thread (Ethicon, 6–0) to a Bionix F-200 isotonicdisplacement transducer powered by a Bionix ED1-1A Powerpack. The signal was amplified and displayed on a Hitachi VC-6026 oscilloscope. Concurrently, the signal from the force transducer was passed through a frequency converter with a window discriminator to determine instantaneous heart rate. Heart amplitude and contraction frequency were recorded continuously on a Gould 2200 chart recorder for later analysis.

Manduca saline of the following composition was used in all experiments: Pipes biological buffer (dipotassium salt; Sigma), 5mM; CaCl₂, 5.6ITIM; NaCl, 6.5 mM; KC1, 28.5HIM; MgCl₂, 16 mM; dextrose, 173 mM. The final pH was adjusted to 6.7±0.1 using a concentrated solution of HC1. During each bioassay. 80mlh⁻ saline flow rate was maintained at approximately through the open superfusion chamber containing the isolated heart. 100test samples were directly injected into the saline flow with a gas-tight Hamilton syringe.

Each injection of embryonic extract was bracketed with several graduated injections of known adult CAP activity. The resultant dose-response curves of adult CAP activity enabled a precise determination of an adult CAP activity equivalent for the embryonic extract. Thus, the CAP level in each embryo fraction was expressed as a percentage of the standard CAP levels in the abdominal nerve cord of the adult moth. Using these adult activity equivalents, measurements of the amount of CAP in embryo development stages could be quantitatively compared within the same bioassay, or between different heart preparations.

75 % DT Manduca embryos were extracted using the CAP extraction procedure as described above. The lyophilized sample was resuspended in Manduca saline and half of the sample incubated with the 6C5 antibody at a dilution of 1 part antibody: 10 parts saline. The other remaining aliquot was incubated with a non-reactive protein (1.0% BSA) as a control. Each aliquot was incubated at 4°C for 30min. The supernatant from each fraction was collected and bioassayed for cardioacceleratory activity on the isolated pharate adult Manduca heart as described above. Other controls included 6C5 alone, BSA alone, 6C5+serotonin (Sigma) and 6C5+peptide F (a crustacean cardioactive neuropeptide; Trimmer et al. 1987). Bioactivity of each treatment was compared to that of an untreated control.

Sep-Paked embryo extracts were chromatographed on a Brownlee Alltech C-18, reverse-phase HPLC column (4.6 ×250mm, 300 μm particle size). An acetonitrile-water solvent gradient with 0.1% trifluoroacetic acid (TFA) counter-ion was used in all trials. A linear acetonitrile-water gradient was used with the acetonitrile concentration increasing at 1.5% per min (Tublitz and Evans, 1986). For each chromatography run, 30 separate 1 ml fractions were collected at Imin intervals. Each fraction was lyophilized, stored at —20°C, and later re-suspended in Manduca saline for bioassay on the isolated heart.

Manduca embryos were dissected free of their chorion and yolk, and development stage was determined (Dorn et al. 1987; Broadie et al. 1989a). Specific target organs, either the embryonic hindgut or heart, were exposed by making incisions through the body wall and pinning back the epidermis with minute steel pins. When necessary, minimal surgery was performed to clearly expose the target organ. This semi-intact preparation was placed in a small superfusion chamber and perfused with Manduca saline at 60 ml h^-1.

HPLC-purified CAP₂, and a range of other known neurohormones and transmitters, were applied to the preparation and the effect on myogenic contraction frequency in the heart and gut quantified. The applied substances included: small cardioactive peptide_B (SCP_B; Lloyd, 1978), FMRFamide (Greenberg and Price, 1979), peptide F (Trimmer et al. 1987), proctolin (Sigma), serotonin (5-HT; Sigma), octopamine (Sigma), and acetylcholine. All substances were dissolved in 100 /d of Manduca saline and injected directly into the saline flow with a gas-tight Hamilton syringe.

The target tissue was observed under high magnification (×800) with a Wild binocular dissecting microscope, and myogenic contractions counted during 30 s intervals for up to 8 min following an injection. From these observations, contraction frequency over 30 s intervals was computed. A second injection was applied if and only if the organ returned rapidly to a basal contraction frequency.

Distinctive patterns of CAP-like immunoreactivity were observed repeatedly in the posterior abdominal ganglia during the late stages (70–100 % DT) of Manduca embryonic development. Cells showing CAP-like immunoreactivity fit into two spatially distinct groups. The first group consisted of a pair of large neurons that lie along the posterior midline of the caudal abdominal ganglia (A4-A8; Figs 1 and 2). The axons from these cells bifurcate and exit the ganglion via each ventral nerve, ultimately projecting posteriorly to both ipsilateral and contralateral transverse nerves (data not shown). The second group consisted of lateral neurosecretory cell clusters that lie at the base of each ventral root in the abdominal ganglia (A2-A8; Figs 1 and 2). These lateral cells also have processes in the ventral nerve leading to the transverse nerve. Midline staining was observed in an average of two medial neurons (range: 1-3 cells) in all four posterior abdominal ganglia. The mean number of CAP-like immunoreactive cell bodies in each lateral cluster varied greatly depending on position in the VNC (range: 2–8 cells/ cluster). In general, more caudal ganglia contained a higher number of immunoreactive somata in the lateral clusters as compared to the rostral ganglia (Fig. 2). The cellular morphology of both types of CAP-like immunoreactive cells was commensurate with observations of other insect neurosecretory cells (Rowell, 1976): large and clearly evident cell bodies displaying the Tyndall Blue effect characteristic of neurosecretory function.

CAP-like immunoreactivity appeared earliest in development and at the highest intensity in the fused terminal abdominal ganglion (A7/A8; Figs 3A and 4A). Both immunoreactive midline and lateral cells were observed in the terminal ganglion. The midline cells in A8 appeared anterior and dorsal, just posterior of the transverse nerve branching, whereas the midline cells in A7 were found very posteriorly, just anterior to the juncture between A7 and A8. These midline cells were very large relative to other neurosecretory cells in the ganglion, with cell body diameter ranging from 15 μm to 20 μm. The cell bodies of these cells displayed strong CAP-like immunoreactivity throughout late embryonic development (Figs 1, 3A and 4A).

The lateral cell clusters in the fused terminal ganglion also stained intensely throughout the last quarter of embryonic development. The lateral cells in A8 showed a distribution different from that of A7 and the anterior unfused ganglia, in that the more numerous cell bodies in A8 were less clustered and tended to be isolated into one or two cells lying at the bases of the major posterior ventral root branches (Figs 1 and 2C). In this ganglion (A8), an average of six cell bodies containing CAP-like immunoreactivity were observed, ranging from 2-10 cells per preparation. In contrast, the lateral cell clusters in A7 formed close-knit, bilaterally symmetrical clusters at the bases of the posterior ventral root (Figs 1 and 2C). Both groups of lateral cells in the terminal ganglion (A7/A8) had relatively small somata, with diameters in the range of 6/un to 11 pm each. In all preparations, only the cell bodies of the lateral cells were intensely immunoreactive.

In general, more CAP-like immunoreactive cell bodies were located in ganglion A8 than in the more anterior ganglion. We surmise that this is due to the fact that ganglion ‘A8’ is actually a condensation of multiple neuromeres found during the initial formation of the CNS (Jacobs and Murphey, 1987). This unique development explains the different distribution of CAP-like immunoreactive cells in A8 relative to A7 and the more anterior, unfused abdominal ganglia (Fig. 2).

Unfused abdominal ganglia showed immunoreactive patterns similar to A7 (Figs 1 and 2). An average of two midline cells per ganglion stained in ganglia A4 to A6 (range: 1–3 immunoreactive cells per ganglion). The cell bodies lay along the posterior, dorsal midline and were often positioned asymmetrically across the ganglion midline, with one cell body lying slightly anterior relative to the other. The morphology and size of these cells was comparable to the midline cells in the fused terminal ganglion (Figs 1 and 2). The midline cells in the unfused ganglia were less immunoreactive than in the fused terminal ganglia, with intensity of the DAB reaction product progressively decreasing from A6 to A4 (Fig. 4A). No midline cells showed CAP-like immunoreactivity in the first three abdominal ganglia during any stage of embryo development.

The lateral cell bodies in the unfused abdominal ganglia remained clustered at the base of the posterior ventral root in a pattern similar to A7 (Figs 1 and 2). The number of cells in the lateral clusters decreased in the more anterior unfused ganglia: an average of four cells per cluster in A6 (range: 4–5), three cells in A5 (range: 2–4), and two lateral cells, one cell at the base of each ventral root, in A2–A4 (range: 1–2). As with the midline cells, immunoreactive staining intensity progressively decreased in the lateral cells of the more anterior abdominal ganglia: A6 lateral cells were the most intensely stained, and A2 lateral cells were the least immunoreactive (Fig. 4B). Lateral cell body morphology and size were similar in all the abdominal ganglia. CAP-like immunoreactivity was not detectable in the lateral cells of ganglion Al.

In summary, CAP-like immunoreactivity during embryogenesis was restricted to midline and lateral neurosecretory cells in the abdominal ganglia, predominantly the posterior five ganglia (A4–A8). Immunoreactivity was limited to the cellular cytoplasm with no observed nuclear staining. In most preparations, CAP-like immunoreactivity was limited to the cell body; however, the base of the transverse nerve also demonstrated CAPlike immunoreactivity in approximately 10% of our trials. No CAP-like reactivity was observed in the thoracic ganglia or in the brain during embryonic development.

CAP-like immunoreactivity first appeared in the 60% developed embryo. The first cells to demonstrate immunoreactivity were the two pairs of large, dorsal midline cells in the terminal abdominal ganglion (A7/ A8; Figs 3A and 4A). Immunoreactive intensity was very weak in these cells in the 60 % developed embryos, and positively stained cells were only observed in a small fraction of our animals (12%; Figs 3A and 4A).

The number of immunoreactive midline cells and the immunoreactive intensity of these cells increased gradually during the later stages of embryonic development (70–100 % DT; Figs 3A and 4A). After the first appearance of CAP-like immunoreactivity in the midline cells of the fused, terminal ganglion, the homologous cells in an unfused abdominal ganglion (A6) first appeared in the 70% developed embryo. However, only a small fraction (10%) of the preparations exhibited positive staining. By 75 % DT, the percentage of preparations with immunoreactive midline cells in A6 had increased to 25 %, and the immunoreactive intensity of these cells had become more pronounced (Figs 3A and 4A). Midline cells in A5 first demonstrated immunoreactivity at 80% development. From 80 to 95% DT, no new midline cells with CAP-like immunoreactivity appeared (Fig. 3A). Immunoreactive intensity also remained fairly constant from 80 to 90% DT, but began to increase in the midline cells of the last four abdominal ganglia by 95% DT (Fig. 4A). Immunoreactive A4 midline cells, the most anterior midline cells to have CAP-like immunoreactivity during embryogenesis, were first detected in the 100%, fully developed embryo, just prior to hatching. In the fully developed embryo, a high percentage of preparations had immunoreactive midline cells in the last five abdominal ganglia, ranging from 75 % positive staining in A8 down to 10% staining in A4 (Fig. 3A). In addition, the intensity of CAP-like immunoreactivity peaked in the midline cells within the last two hours of embryonic development.

A marked decrease in midline cell CAP-like immunoreactivity was observed at hatching. During larval emergence, the number of immunoreactive midline cells and the intensity of their staining decreased (Figs 1E-H, 3A, and 4A). Specifically, midline cell CAP-like immunoreactivity in ganglia A4 and A5 disappeared completely and the midline cell immunoreactive intensity decreased markedly in A6 through A8 (Figs 3A and 4A).

By way of contrast, the lateral neurosecretory cells acquired CAP-like immunoreactivity in a developmentally unique pattern, distinct from the midline cells. First appearing at 70% DT, lateral cell immunoreactivity peaked in the 75 % developed embryo (Figs 1A,C and 4B). At this stage we saw the largest number of CAP-like immunoreactive lateral cells (Fig. 3B), the most intense CAP-like reactivity in these cells (Fig. 4B), and the greatest number of abdominal ganglia (A2-A8) possessing immunoreactive lateral cells (Fig. 3B). A sharp decline in lateral cell immunoreactivity was observed between 75% and 80% DT: lateral cell immunoreactivity in the more anterior two abdominal ganglia (A2-A3) disappeared completely, while the percentage of CAP-like reactive cells and the immunoreactive intensity of these cells declined markedly in all the remaining posterior abdominal ganglia (A4-A8; Figs 3B, 4B and 1A-D). By 85% DT, both immunoreactive intensity and the total percentage of CAP-like immunoreactive lateral cells began to recover in A5 through A8, whereas no recovery was detected in the lateral cells of ganglion A4 at this stage of development (85 % DT).

CAP-like immunoreactivity of the lateral neurosecretory cells continued to recover during the last 15 % of development. By 95 % DT, a large number (Fig. 3B) of intensely immunoreactive lateral cells were once again observed in A4-A8. Less intense staining was seen in ganglia A3. The immunoreactivity of the lateral cells plateaued at this level for the remainder of embryonic development (Figs 3B and 4B). Unlike the midline cells, lateral cell immunoreactivity remained constant during hatching behavior (Fig. 1E-H).

In summary, we found that levels of the CAP-like antigen were modulated independently in the two abdominal ganglia cell types. Expression of the CAPlike antigen in the midline cells was characterized by early development (60% DT), a gradual continuous increase in immunoreactivity during embryogenesis, and a marked drop in the levels of the immunoreactive antigen during hatching. In contrast, lateral cells expressed the CAP-like antigen later at 70 % DT, rapidly accumulated levels of the antigen during the next 5 % of development, and demonstrated a dramatic drop in CAP-like immunoreactivity between 75 % and 80 % of embryo development.

To reaffirm its specificity, our primary antibody (6C5) was pre-absorbed with a variety of antigens to help verify the nature of molecule responsible for the observed immunoreactivity patterns. All CAP-like immunoreactivity was completely abolished after pre-incubation of the primary antibody in CAP₂ (Fig. li). In contrast, pre-incubation of 6C5 with other invertebrate cardioactive peptides (peptide F, SCP_B and FMRF-amide) in no way altered the immunoreactivity patterns observed with the free antibody (data not shown).

The immunohistochemical observations described above clearly demonstrated the presence of a CAP-like antigen in a well defined subset of neurosecretory cells in the embryonic caterpillar. We next carried out a series of biochemical tests to establish the relationship between this antigen and the CAPs. Embryos at various developmental stages were extracted for cardioacceleratory activity as described in Materials and methods, with the crude homogenate partially purified through a C-18 Sep-Pak. The resultant fractions then were assayed for biological activity on the isolated Manduca heart. Using this procedure, we determined that all embryonic development stages possessed some degree of cardioacceleratory bioactivity. Even samples of unfertilized Manduca eggs, purified with the identical purification scheme, contained very low yet measurable levels of cardioacceleratory activity (0.9% of the activity in an adult abdominal nerve cord (ANC); n=10). Thus, all measurements of the embryonic cardioexcitatory levels found at later development stages were corrected for this background cardioacceleratory activity by subtracting the level of unfertilized egg bioactivity from the observed response of the pharate adult heart to a given sample.

Cardioacceleratory activity substantially above background levels was first detected in the 40 % developed embryo (Fig. 5). Extracts of this stage had 2.0±0.8% (mean±s.E.M.) of the cardioacceleratory activity in an adult ANC as determined by quantitative bioassay on the isolated pharate adult heart (Fig. 5). This activity increased rapidly between 40 % and 75 % DT, where it peaked at 14±3.8 % of the adult ANC levels. Cardioacceleratory bioactivity in the entire embryo decreased markedly between 75% and 80% DT, dropping to 4.5±1.2% of adult ANC cardioacceleratory activity (Fig. 5). A gradual increase in bioactivity was observed from 80 % to 100 % DT, with fully developed embryos containing 8.2±1.8% adult ANC units of CAP activity. A second, substantial drop in cardioacceleratory activity was recorded at hatching when bioactivity levels decreased by approximately 50 %, with newly-hatched first instar larvae having 4.3±0.9 % of adult ANC levels (Fig. 5).

As a first step in identifying the cardioactive factor found in the partially-purified embryonic extracts, a monoclonal anti-CAP antibody, 6C5, (Taghert et al. 1983, 1984) was tested for its ability to precipitate the bioactive factor. This antibody has been previously demonstrated to specifically recognize an epitope common to CAPx and CAP₂ (Tublitz and Evans, 1986). When extracts from 75 % developed embryos were incubated with the 6C5 antibody for 30 min prior to bioassay on the in vitro Manduca heart, bioactivity was reduced by 81 % when compared to untreated controls (Fig. 6; Table 1). The 6C5 antibody alone produced no detectable effect when applied to the isolated pharate adult heart. Furthermore, the 6C5 antibody had no effect on the bioactivity of a biogenic amine (serotonin) or on peptide F (Trimmer, 1987), another arthropod cardioactive peptide (Table 1).

To unequivocally ascertain the relationship between the CAPs and the cardioactivity in embryos, partially-purified material from embryos at various stages was chromatographed on a HPLC using a reverse phase C-18 column, and the resultant fractions bioassayed on the in vitro Manduca heart. Chromatographic profiles, such as the one shown in Fig. 7, indicated the presence of a single peak of cardioexcitatory activity during late embryonic development. This activity co-eluted with purified CAP₂ from the pharate adult moth. No detectable cardioregulatory bioactivity eluted in the fractions associated with CAPi, or elsewhere during the chromatographic run (Fig. 7). This sole activity peak accounted for all the cardioacceleratory activity present in the embryonic crude extracts. Cardioacceleratory activity could not be detected in HPLC samples from embryos less than 50% developed, even with sample sizes ten times larger than those assayed with older embryos.

Our biochemical results, combined with our previously described immunocytochemical observations (Figs 1, 3 and 4), clearly demonstrate that the level of CAP₂ fluctuates dramatically during embryogenesis, and that the observed drops in peptide levels may be related to CAP₂ release from individual neurosecretory cells. Hence, we were interested in the possible physiological role(s) of this neuropeptide in the embryonic system. As a first step in addressing this question, we analyzed the effect of exogenously applied CAP₂, as well as several other known invertebrate cardioregulatory factors, on the beat frequency of myogenically active embryonic tissues utilizing an in vitro preparation as described in Materials and methods. In particular, we were interested in the effects of CAP₂ on the heart and hindgut, as both tissues are known targets of CAP modulation during larval (N. Tublitz, unpublished observations) and adult life (Tublitz and Truman, 1985a,b,c; Tublitz and Evans, 1986; Tublitz, 1989).

The contraction frequency of both the embryonic heart and hindgut were found to be particularly sensitive to three known neuroactive substances: two biogenic amines, serotonin and octopamine, and peptide F, a small neuropeptide isolated from crustaceans (thr-asn-arg-asn-phe-leu-arg-phe-amide; Trimmer et al. 1987; Table 2). In both embryonic tissues, serotonin (5-HT) had the lowest threshold (10⁻⁹M), where threshold is defined as the lowest concentration required to evoke a measureable increase (5 %) in contraction frequency in 50% of the trials (Table 2). The thresholds for octopamine and peptide F were at least an order of magnitude higher for both assays. The heart was more sensitive to octopamine (threshold 10⁻⁸M) than to peptide F (threshold 10⁻⁷M). In contrast, the hindgut was more sensitive to peptide F (threshold 10~⁸M) than to octopamine (threshold 10⁻⁷M; Table 2).

The embryonic heart and hindgut were significantly less sensitive to the other substances applied during this study. These included three other invertebrate bioactive peptides, e.g. FMRFamide, small cardioactive peptide (SCPB), and proctolin, as well as acetylcholine, another putative insect cardioregulatory substance (Miller, 1979). The thresholds for the three peptides were of the order of 10⁻⁵M or greater when pulse-applied onto either in vitro preparation (Table 2). Acetylcholine had no detectable effect in either assay at concentrations up to 10⁻³M.

CAP₂ was found to increase contraction frequency in both the embryonic heart and hindgut. Of the two organs, the hindgut was more sensitive to CAP₂ application, with a lower threshold (0.05 adult ANC equivalents of HPLC-purified CAP₂ activity) and a more prolonged response (response to 1.0 ANC equivalent CAP₂: 5.0±0.3min; Fig. 8A). The dose-response relationship followed a sigmoidal curve with maximal response occurring at a pulse application of 0.24 ANC CAP equivalents (Fig. 8B).

In contrast, the embryonic heart had a threshold sensitivity of 0.2 ANC equivalents (Table 2). The response latency to exogenously applied CAP₂ (1.0 ANC) was similar in both organs, but the duration of the cardiac response was significantly reduced (3.4±0.4min) relative to that of the hindgut (Fig. 9A). A similar sigmoidal dose-response relationship was observed in both organs. However, the lower sensitivity of the embryonic heart resulted in a displacement of the curve to the right, with a maximal increase in cardiac contraction frequency observed to 0.52 ANC CAP equivalents (Fig. 9B).

The spontaneous activity of the embryonic gut was measured in vivo and in vitro during the last 50% of embryogenesis. In vivo observations of gut activity were possible prior to 80 % DT through a semi-translucent embryonic cuticle. However, the increase of cuticle pigmentation after this stage prevented further in vivo observations. Comparison of contraction rates between the in vivo gut and our semi-intact in vitro preparation indicated that contractile activity was not perturbed during our manipulations. Specifically, contraction rates of the two preparations remained in agreement during early development and gut activity did not change in response to direct physical stimulation. Consequently, both assays were used interchangeably to track embryonic gut activity.

The gut was quiescent in all embryos prior to 65 % DT (Fig. 10A). Spontaneous gut contractions began at 70% of embryonic development, increased dramatically during the next 10% DT and reached peak contraction rates by 80% of development (Fig. 10A). Gut contraction frequency remained relatively constant at this level for the remainder of embryogenesis. A slight increase in contraction rate was observed immediately following hatching in the first instar larvae (Fig. 10A).

HPLC-purified CAP₂ was applied to each of the developmentally staged hindgut preparations, and response in contraction frequency quantified. We found that 0.25 ANC units of exogenously applied CAP₂ had no discernible effect on the gut prior to 70 % development (Fig. 10B), stages when the gut is normally inactive (Fig. 10A). In contrast, the spontaneously active gut was found to be very responsive to pulse applications of CAP₂. Application of 0.25 ANC units of CAP₂ at 75 % DT increased contraction frequency by 40±4% (Fig. 10B). Sensitivity to CAP₂ did not change significantly during the remainder of embryonic development.

Taylor and Truman (1974) were the first to identify two distinct groups of neurosecretory cells in the abdominal ganglia of the adult moth. Their work demonstrated the existence of four pairs of large (25-30 gm) somata lying along the dorsal midline and a bilateral set of four smaller neurons that lay at the base of each ventral root. Later work (Taghert and Truman, 1982) showed that both sets of neurons projected to the transverse nerve, the major neurohaemal release site in the insect ventral nerve cord (Raabe, 1982). Additional investigations, using a variety of techniques including intracellular stimulation of single identified peptidergic neurons (Tublitz and Truman, 1985c) and immunocytochemistry (Taghert et al. 1984, 1985; Tublitz and Sylwester, 1988) revealed that the four pairs of segmentally-reiterated medial cells in the adult moth synthesized and released high levels of cardioacceleratory peptide (CAP) activity, whereas the lateral cells expressed another neuropeptide, bursicon (Taghert and Truman, 1982). In contrast, studies in larvae indicated that both cell types contained CAP activity including the lone pair of medial cells that arises embryonically and the four laterally-situated pairs lying at the base of each ventral root. Our results with the anti-CAP antibody in the Manduca embryo (Figs 1 and 2) closely resembled that observed during the initial stages of post-embryonic development. As in early larvae (Tublitz and Sylwester, 1988, 1989), staining was limited to a single pair of medial cells and the lateral clusters at the base of the ventral root. All these cells had projections out the ventral nerve, apparently extending at least to the base of the transverse nerve, which also expressed strong CAP immunoreactivity. Given the highly specific nature of the anti-CAP antibody (Fig. II; Tublitz and Evans, 1986; Taghert et al. 1983, 1984) and that this antibody stains a set of cells in the Manduca embryo that closely resemble the well-characterized CAP-containing neurons in larvae, we conclude that these embryonic cells are likely to express one or both of the CAPS.

Three lines of evidence strongly suggest that CAP₂ is present during embryonic development in M. sexta, and that the level of this neuropeptide changes in dramatic and potentially physiologically important ways. First, immunohistological work with an anti-CAP antibody demonstrated the presence of an immunoreactive antigen specifically localized in embryonic neurosecretory cells known to contain CAP₂ during larval and/or adult life (Tublitz and Truman, 1985c; Tublitz and Sylwester, 1988, 1989). This embryonic staining pattern was completely abolished after pre-incubation of the anti-CAP antibody with CAP₂ (Fig. II). In contrast, pre-incubation of the anti-CAP antibody with a range of similar invertebrate cardioactive peptides resulted in no change in the observed immunoreactivity pattern. Consequently, we were inclined to attribute the observed immunoreactivity patterns to the presence of CAP₂ in the Manduca embryo.

It is, however, very rarely possible to unequivocally identify an antigen based on immunoreactivity alone. Absolute identification of an antigen can be achieved only by separating it from tissue extracts, purifying it, and subjecting the pure sample to specific analysis using chemical and/or physiological assays. Consequently, as a second line of evidence, extracts of embryos were prepared according to the isolation procedure for the adult CAPs and quantitatively tested for CAP-like activity on a highly sensitive in vitro heart bioassay (Tublitz and Truman, 1985a,b). Our biochemical and pharmacological results showed that Manduca embryos contained substantial levels of CAP-like cardioacceleratory activity (Fig. 5) which changed during embryonic development in a manner consistent with the changes in immunohistological patterns and immunoreactivity intensities (Figs 3, 4 and 5).

In general, the fluctuations in activity from immunocytochemistry and the in vitro heart bioassay followed comparable time courses. Although the isolated heart bioassay indicated the appearance of CAP₂-like activity at an earlier development time (40%) than the immunocytochemical results (60%), it is apparent that the biological cardioactivity of this factor was very low, below 60% DT (Fig. 5). This discrepancy may be due to the appearance of another cardioexcitatory substance at stages before 60 % DT which is not recognized by the 6C5 antibody. Alternatively, endogenous CAP₂ levels before 60% DT may be below the level of resolution by our immunocytochemical techniques. It is clear, however, that the first significant increase in cardioacceleratory activity (60%) precisely correlates with the appearance of the first anti-CAP immunoreactive cells (Figs 3 and 5). Between 60 and 75% of embryo development, both CAP-like immunoreactivity and cardioacceleratory activity levels increased sharply (Figs 3, 4 and 5). Even more compelling, the radical decline in cardioacceleratory activity between 75 % and 80% development (Fig. 5) was paralleled by a similar drop in immunoreactivity of the lateral cell clusters during this developmental span (Fig. 1). Similarly, immunocytochemical (Figs 3 and 4) and biochemical evidence (Fig. 5) both indicated a gradual increase in CAP-like activity during the remainder of embryonic development, culminating in a second drop in CAP-like cardioactivity and midline cell immunoreactivity during hatching.

As the last line of evidence, the embryonic cardioacceleratory activity was demonstrated to be attributable to CAP₂ using two independent tests. In the first, it was shown that over 80 % of the embryonic bioactivity was immunoprecipitated upon incubation with the antiCAP antibody 6C5 (Fig. 6). Additionally, CAP₂ was shown to specifically block the CAP-like immunocytochemical staining patterns in the Manduca embryo (Fig. II). In the second, the embryonic cardioacceleratory activity was shown to precisely co-elute with CAP₂ purified from pharate adult moths using high pressure liquid chromatography (Fig. 7). Moreover, all cardioacceleratory activity present in the late embryo stages eluted in a single peak, with no other cardioexcitatory factors detected in our tissue extracts at any embryonic stage (Fig. 7B).

The above data, taken together, strongly support the notion that the observed biological activities are due to CAP₂. Furthermore, the close correlation of the embryonic CAP₂ activity profile and the observed immunocytochemical staining patterns, as well as the absence of any other cardioexcitatory factors in the tissue extracts, strongly argues that the CAP-like immunoreactivity in the identified neurosecretory cells is directly attributable to the expression of CAP₂.

Biochemical, pharmacological, and immunohistochemical evidence all argue that a large drop in the storage levels of CAP₂ occurs twice during embryo development: between 75% and 80% development, and during hatching (Figs 1, 3, 4 and 5). These findings imply that CAP₂ may play an important physiological role(s) during the course of embryogenesis. In an attempt to define the role(s) of CAP₂ in the embryo, experiments were initiated to identify possible target tissues that CAP₂ may modulate during embryo development. Our results demonstrate that the contractile activity of the embryonic heart and gut musculature is pharmacologically modulated by exogenous application of several myoactive factors (Table 2). In particular, HPLC-purified CAP₂, when applied to the in vitro embryo, accelerated contractile rates of both the heart and hindgut (Figs 8 and 9).

Defining the precise physiological role of a neurochemical in vivo is always a difficult task. In this instance, given the scale of the Manduca embryo, direct, unequivocal identification of a physiological role for this peptide during embryogenesis may prove unfeasible. Nevertheless, several lines of indirect evidence lead us to propose a possible role for CAP₂ during its apparent release between 75% and 80% of embryo development. First, our work on Manduca development demonstrated that the hindgut initially becomes myogenically active at 70% DT (Broadie et al. 1989,a), and that the rate of gut contraction increases radically (3–4 fold) between 70% and 80% DT (Fig. 10A). Second, ingestion of the extra-embryonic yolk commences approximately at 75% DT (Broadie, unpublished observations). At this stage, the gut is empty of yolk and, by 85 % DT, ingestion of the extra-embryonic yolk is complete, resulting in a yolk-filled gut (Broadie, unpublished observations). Third, the embryonic gut is very sensitive to CAP₂ (Fig. 8), which, at physiological concentrations, accelerates contraction frequency in a fashion similar to the endogenous modulation of the gut observed between 70 % and 80 % DT in vivo (Fig. 10A). Fourth, the pharmacological sensitivity of the embryonic gut to exogenously applied CAP₂ increases markedly between 70 % and 80 % DT (Fig. 10B). It is particularly striking that the inactive gut at stages before 70 % DT is not responsive to exogenous application of the peptide and only becomes sensitive to CAP₂ immediately prior to the acceleration of gut contractions seen at 75% DT (Fig. 10). Fifth, our biochemical studies of CAP₂ levels in the embryo indicated a sharp drop in stored CAP₂ levels between 75 % and 80 % development (Fig. 5). Lastly, our immunocytochemical studies (Figs 1, 3 and 4), showed a marked decrease in the CAP₂-like immunoreactivity of identified lateral neurosecretory cells in the posterior abdominal VNC between 75 % and 80% development, confirming our biochemical results. Interestingly, a large proportion of this CAP₂-like immunoreactivity was localized in the terminal abdominal ganglia, which other studies have indicated is the primary control site of the digestive hindgut (Raabe, 1982). Taken together, these data provide strong circumstantial evidence that CAP₂ may be released from identified lateral neurosecretory cells in the abdominal VNC between 75 % and 80% of embryonic development, facilitating ingestion of the extra-embryonic yolk by directly accelerating the frequency of hindgut contractions.

The role of CAP₂ secretion during hatching behavior is less clear. Our biochemical studies have shown that a similar, if somewhat smaller, decline in CAP₂ levels in the embryo occurs at hatching (Fig. 5). Interestingly, the drop in CAP-like immunoreactivity at hatching occurs exclusively in the large medial neurosecretory cells of the abdominal VNC as opposed to the decrease in lateral cell immunoreactivity observed at 75 % DT (Figs 1, 3 and 4). This raises the tantalizing possibility that the two cell types may be independently regulated, playing distinctive roles at different stages in the Manduca embryo. CAP₂ secretion at hatching can be hypothesized to increase heart contraction providing the necessary hydrostatic force to aid hatching and/or it may increase gut contraction to facilitate the embryo’s ability to eat its way clear of the egg shell and ingest part of the shell immediately after emergence. However, it is obvious that additional work must be done to better understand the exact physiological role of CAP₂ during either embryonic period.

We would like to thank Dr J. C. Weeks for her comments on earlier versions of this manuscript and Dr B. Trimmer for his gift of peptide F used in this study. We also thank Mr T. Schilling for his input and advice during the preliminary stages of the immunocytochemical work. This work was supported by NIH grant no. NS 24613, a NIH Research Development Award no. NS 01258, and a Sloan Fellowship to N.J.T.