ABSTRACT
Allatotropin is a neuropeptide that was originally isolated from the sphinx moth Manduca sexta, in which it stimulates the synthesis of juvenile hormone in adults (Kataoka et al. 1989). An antiserum raised against this peptide was characterized, shown to be specific for allatotropin and used in a competitive enzyme-linked immunosorbent assay (ELISA) to demonstrate that allatotropin is present not only in the brain and retrocerebral complex but also in the ventral nerve cord of Manduca sexta (Veenstra and Hagedorn, 1993). In the present study, allatotropin-immunoreactive neurons were located using the allatotropin antiserum in a whole-mount immunofluorescence method adapted from Davis et al. (1989). Several cell types were found to be immunoreactive, including three pairs of intensely staining median neurons in abdominal ganglia 3–6 of the pharate adult. The location of these median pairs of cells in each ganglion is anterior, mid-dorsal and posterio-ventral (Fig. 1A). The cells project bilaterally via the ventral nerves of each ganglion to the next transverse nerve (Fig. 2), where their axons terminate in numerous superficial varicosities (Fig. 1D). It is well established that the transverse nerve is a neurohemal organ in Lepidoptera (Provansal, 1972; Taghert and Truman, 1982; Tublitz and Truman, 1985a–d). The location and projection pattern of these three pairs of median neurons indicate that they belong to a group of abdominal median neurosecretory cells identified previously (Taghert and Truman, 1982; Tublitz and Truman, 1985a). Using the nomenclature of Davis et al. (1993), these are the M1, M2 and M5 neurosecretory cells. The M1 and M2 cells mature during adult development, but the M5 cells are already differentiated in the larval stages (Taghert and Truman, 1982; Tublitz and Truman, 1985a). The M5 cells of larvae were not stained by the allatotropin antiserum and only became immunoreactive, along with the M1 and M2 cells, during adult metamorphosis. Two pairs of the abdominal median neurosecretory cells were not allatotropin-immunoreactive: (1) the M3 cells, which are ventral and mature during adult metamorphosis (Taghert and Truman, 1982); and (2) the M4 cells, which are already differentiated in the larval stages (Tublitz and Truman, 1985a).
Previously, the abdominal median neurosecretory cells of adult M. sexta were reported to contain two unidentified cardioactive peptides, CAP1 and CAP2 (Tublitz and Truman, 1985a,b); subsequently CAP1 has been shown to consist of several peptides and CAP2 of three peptides (Loi et al. 1992). One of the CAP2 peptides has been identified as crustacean cardioactive peptide (CCAP), a peptide first identified from a shore crab by Stangier et al. (1987). This peptide has now been isolated and sequenced from M. sexta (Hildebrand et al. 1990; Cheung et al. 1992; Lehman et al. 1993). Moreover, CCAP has been shown to be cardioactive in M. sexta (Davis et al. 1990; Lehman et al. 1993). Using an antiserum that has been shown to be specific to CCAP (Dircksen and Keller, 1988; Stangier et al. 1988; Lehman et al. 1993), a pair of abdominal median neurosecretory cells, the M4 cells, has been found to be CCAP-immunoreactive in ganglia of adults (Davis et al. 1993). These cells are not the same as the four pairs of so-called CAP cells identified by Tublitz and Sylwester (1990), using a monoclonal antibody (anti-CAP; Taghert et al. 1983, 1984) that has been reported to recognize an epitope common to CAP1 and CAP2.
For direct comparisons between CCAP-and allatotropin-immunoreactive neurosecretory cells in the same ganglia, double-fluorescence labeling was performed. Immunoreactivity to CCAP was visualized with a Rhodamine-labeled secondary antiserum (Fig. 1B), and the ganglia were subsequently incubated with purified allatotropin IgG, which had been labeled with FITC (Fig. 1A) using a kit from Boehringer Mannheim Biochemicals. The results clearly show that the two types of immunoreactivities are present in different cells (Fig. 1C). Moreover, the staining in the CCAP-and allatotropin-immunoreactive processes in the transverse nerve does not overlap (Fig. 1D–F).
Other than CCAP, the identities of the cardioactive peptides of M. sexta remain unknown, but the staining of median neurosecretory cells by allatotropin antiserum leads to the hypothesis that one of these peptides is allatotropin. When allatotropin was tested on the heart of pharate adult (stage 18) M. sexta, the peptide was strongly excitatory (Fig. 3). To provide further evidence in support of our hypothesis, an extract of abdominal nerve cords was separated on HPLC, and the fractions containing allatotropin-immunoreactivity, CCAP-immunoreactivity and those that had an excitatory effect on heart preparations were identified.
Two regions eliciting cardioexcitatory activity were found: these were in fractions 14–18 and in fractions 20–21. Allatotropin-immunoreactivity was identified in fractions 15 and 16, while fractions 16 and 17 exhibited CCAP-immunoreactivity (Fig. 4). Preincubations of the cardioactive fractions with allatotropin antiserum (1:20) abolished all of the cardioacceleratory activity present in fraction 15 and some of the effect of fraction 16, but such treatment was without effect on the cardioacceleratory activity of fractions 14, 17, 18, 20 and 21 (Fig. 5). The presence of both allatotropin and CCAP-immunoreactivity in fraction 16 suggested that the cardioactivity remaining in fraction 16 after preadsorption with allatotropin antiserum might be due to CCAP. Preincubation of fraction 16 with both anti-allatotropin and anti-CCAP abolished all cardioactive effect, but preincubation with anti-CCAP alone only reduced the cardioactive effect of this fraction. Preincubation with CCAP antiserum abolished the cardioacceleratory activity of fraction 17, but was without effect on fractions 14, 15, 18, 20 or 21.
Previously, the anti-CAP antibody has been shown to block the cardioacceleratory effects of CAP1 and CAP2 (Tublitz and Evans, 1986). It is now known that the so-called CAPs comprise at least five cardioactive peptides (Loi et al. 1992), and this conclusion is supported by the present study. Therefore, the anti-allatotropin and anti-CCAP antisera are apparently more specific in their blocking of the cardioacceleratory effects of the CAPs than is the anti-CAP monoclonal antibody. This greater specificity is also seen in the use of these antisera in immunocytochemistry; the CCAP (Davis et al. 1993) and allatotropin antisera recognize subpopulations of the abdominal neurosecretory cells (Fig. 1C), while the anti-CAP monoclonal antibody recognizes all but one pair of these cells (Tublitz and Sylwester, 1990).
The apparently different separation characteristics of our C-18 column, compared with the one used by Tublitz (e.g. Tublitz, 1989), make it difficult to determine whether any of the previously isolated CAP fractions may be the same as allatotropin. However, because the relative molecular mass of CAP1 was estimated to be about 1000, and that of CAP2 about 500 (Tublitz and Truman, 1985a), it seems more likely that one of the peptides in the CAP1 fraction is allatotropin (Mr 1487). This conclusion is supported by the report that CAP1 was not found in larval abdominal ganglia (Tublitz and Sylwester, 1990), and, correspondingly, we found no allatotropin-immunoreactive neurosecretory cells in larval ganglia.
Another characteristic of CAP1 is that it does not stimulate the heart rate in larvae (Tublitz et al. 1992). Therefore, we tested the effects of allatotropin and CCAP on the larval heart. The effects of CCAP were in marked contrast to those of allatotropin. Whereas 50 pmol of allatotropin failed to produce an observable change in heart rate, an equal amount of CCAP produced a maximal response on the larval heart (N=4). This amount of CCAP or allatotropin was sufficient to produce maximal responses in the pharate adult heart (Figs 1, 5). These findings, taken together, suggest that one of the peptides present in CAP1 is indeed allatotropin.
It has been shown before that both CAP1 and CAP2 are released at the time of eclosion and later, during flight (Tublitz and Truman, 1985c; Tublitz and Evans, 1986; Tublitz, 1989). Therefore, it seems likely that allatotropin is also released at these times, but, because there is more than one peptide in the CAP1 fraction (Loi et al. 1992), it cannot be assumed that allatotropin is released. We were able to measure allatotropin-immunoreactive material in Sep-Pak prepurified hemolymph samples obtained at eclosion, but the immunoreactive material did not co-elute with authentic allatotropin on HPLC. Hence, the release of allatotropin after eclosion still remains to be demonstrated.
ACKNOWLEDGEMENTS
We thank Drs H. Dircksen and R. Keller for the generous gift of the anti-CCAP antiserum and Drs H. H. Hagedorn and J. G. Hildebrand for encouragement, comments on the manuscript and use of their laboratory facilities. This work was supported by NIH grant AI 33429 to J.A.V., NSF grant IBN 9209780 to H.K.L. and a Whitehall Foundation grant to N.T.D.
