neuroanatomy and immunocytochemistry of the median neuroendocrine cells of the subesophageal...

MICROSCOPY RESEARCH AND TECHNIQUE 35:201-229 (1996)

Neuroanatomy and lmmunocytochemistry of the Median Neuroendocrine Cells of the Subesophageal Ganglion of the Tobacco H aw kmot h , Manduca Sexta ; I m m u nor e ac t ivi t i es to PBAN and Other Neuropeptides NORMAN T. DAVIS, UWE HOMBERG, PETER E.A. TEAL, MIRIAM ALTSTEIN, HANS-J. AGRICOLA, AND JOHN G. HILDEBRAND ARL Division of Neurobiology and Center for Insect Science, University of Arizona, Tucson, Arizona 85721 (N.T.D., J.G.H.), University of Regensburg, Institute for Zoology, 0-93040 Regensburg (U.H.), and Institute for General Zoology and Animal Physiology, Friedrich Schiller University, Jena (H.JA.) , Germany; Insect Attractants, Behavior, and Basic Biology Research Laboratory, USDA, ARS, Gainsville, Florida, 32604 (P.EA .T.); Department of Entomology, Institute of Plant Protection, ARO, The Volcani Center, Bet Dagan, Israel ( M A . )

KEY WORDS Insects, Neurosecretory cells, Axonal backfills, Sex pheromone, Proctolin, SCPB, FMRFamide

ABSTRACT The median neuroendocrine cells of the subesophageal ganglion, important components of the neuroendocrine system of the tobacco hawkmoth, Manduca sexta, have not been well investigated. Therefore, we studied the anatomy of these cells by axonal backfills and characterized their peptide immunoreactivities. Both larvae and adults were examined, and developmental changes in these neuroendocrine cells were followed. Processes of the median neuroendocrine cells project to terminations in the corpora cardiaca via the third and the ventral nerves of this neurohemal organ, but the ventral nerve of the corpus cardiacum is the principal neurohemal surface for this system. Cobalt backfills of the third cardiacal nerves revealed lateral cells in the maxillary neuromere and a ventro-median pair in the labial neuromere. Backfills of the ventral cardiacal nerves revealed two ventro-median pairs of cells in the mandibular neuromere and two ventro- median triplets in the maxillary neuromere. The efferent projections of these cells are contralateral. The anatomy of the system is basically the same in larvae and adults. The three sets of median neuroendocrine cells are PBAN- and FMRFamide-immunoreactive, but only the mandibular and maxillary cells are proctolin-immunoreactive. During metamorphosis, the mandibular and maxillary cells also acquire CCK-like immunoreactivity and the labial cells become SCP- and sulfakinin- immunoreactive. Characteristics of FMRFamide-like immunostaining suggest that the median neuroendocrine cells may contain one or more of the FLRFamides that have been identified in M . sextu. The mandibular and maxillary neuroendocrine cells appear to produce the same set of hormones, and a somewhat different set of hormones is produced by the labial neuroendocrine cells. Two pairs of interneurons immunologically related to the neurosecretory cells are associated with the median maxillary neuroendocrine cells. These cells are PBAN-, FMRFamide-, SCP-, and sulfakinin-immunoreactive and project to arborizations in the brain and all ventral ganglia. These interneurons appear to have extensive modulatory functions in the CNS. o 1996 Wiley-Liss, Inc.

INTRODUCTION Neuroendocrine cells in the brain of the tobacco

hawkmoth, Manduca sexta, and other insects are the principal sources of an array of peptide neurohormones released by the corpora cardiaca (CC) and corpora allata (CA). These hormones are involved in the regulation of various vital processes such as eclosion, metamorphosis, diuresis, and reproduction (Homberg, 1994; Kelly et al., 1994; Nassel, 1993; Raabe, 1989). The neuroanatomy and peptide immunocytochemistry of the neuroendocrine cells of the brain of M. sexta have been studied extensively (Copenhaver and Truman, 1986; Homberg et al., 1991; O’Brien et al., 1988; Truman and Copen- haver, 1989; Veenstra and Hagedorn, 1991; gitiian et al., 1995), and the chemical identities of several neu-

rohormones from the brain of M. sexta have been determined [e.g., eclosion hormone (Kataoka et al., 1987; Marti et al., 1987), diuretic hormone (Kataoka et al., 1989a) allatotropin (Kataoka et al., 1989b), corazonin (Veenstra, 1991), diuretic peptide (Blackburn et al., 19911, allatostatin (Kramer et al., 199113.

Neuroendocrine cells in the subesophageal ganglion (SeG) are also an important source of neurohormones released by the CC and CA of insects, but the anatomy of these cells and the hormones that they produce have

Received October 1, 1994; accepted in revised form December 1, 1994. Address reprint requests to N.T. Davis, ARL Division of Neurobiology, US-

versity of Arizona, PO BOX 210077 Tucson, AZ 85721-007.

0 1996 WILEY-LISS, INC.

202 N.T. DAVIS ET AL.

not been studied as well as have those of the brain. In various species of Lepidoptera, however, the SeG has been shown to be a source of a diapause hormone (Fukuda and Takeuchi, 1967), melanization, and reddish coloration hormone (Morita et al., 1988; Ogura, 1975), and pheromone biosynthesis-activating neuropeptide (PBAN; Kitamura et al., 1989; Masler et al., 1994; Raina et al., 1989). These hormones are all members of a family of structurally related neuropeptides, in that their C-termini end in FXPRL-NH2 (Nachman et al., 1993). Because members of the FXPRLamide family have not yet been demonstrated in M . sextu, a principal objective of the present study was to identify the PBAN-immunoreactive (PBAN-ir) neurons in this insect.

There remain uncertainties regarding the role of PBAN in the control of the pheromone gland of female Lepidoptera (Christensen and Hildebrand, 1995). In addition, PBAN is known to occur in larvae and in adult male Lepidoptera, but its function in these insects is unknown. Therefore, we wished to obtain immunocytochemical evidence that might be related to the questions of PBAN function. In addition to the median neuroendocrine cells, we found certain other neurons that are PBAN-ir, and because these neurons may have some bearing on the question of PBAN function, they have been included in our study.

The anatomy of some of the neuroendocrine cells of the SeG of M . sextu has been described previously; these are the serotonin-ir lateral neuroendocrine cells of the mandibular neuromere (Griss, 1989; Homberg and Hildebrand, 19891, the lateral neuroendocrine cells in the maxillary neuromere, and the medio-ventral neuroendocrine cells in the labial neuromere (Copen- haver and Truman, 1986). Immunocytochemical studies of the lateral neuroendocrine cells of the maxillary and labial neuromeres indicate that these cells are recognized by an antiserum raised to crustacean cardioactive peptide (Davis et al., 1993; Klukas et al., 1996). In addition to those in the labial neuromere, median neuroendocrine cells are known to occur in the mandibular and maxillary neuromeres of the SeG (Davis et al., 1989; Homberg et al., 1990), but knowledge of the anatomy of these cells and of their secretions is very incomplete.

Our earlier immunocytochemical investigation of the neuroendocrine cells of the brain of M . sextu demonstrated that most of these cells have immunoreactivity to several neuropeptides (Homberg et al., 1991). Therefore, it was of interest in the present study to determine if the neuroendocrine cells of the SeG of M . sextu also are characterized by colocalized neuropeptide immunoreactivities.

MATERIALS AND METHODS Rearing of Insects

Larvae of Munducu sextu (Lepidoptera: Sphingidae) were reared at 25"C, at 50-60% relative humidity, un- der a long-day photoperiod regimen (17/7 hours), and on an artificial diet adapted from that of Bell and Joachim (1976). Our study was based primarily on third-instar larvae, pharate adult stages 9-12 and 18, and mature adults 24 hours post-eclosion. The 18

stages of pharate adult developmental were determined by external features as described by Kent (1985).

Axonal Backfilling Ganglia were bathed in a Weever's-type saline solu-

tion (Davis, 1985) containing 33.5 g glucose/l. A 3% solution of cobalt-lysine was used for backfilling (Lazar, 1978), and the cobalt ions were precipitated in 3 ml of normal saline solution containing 100 p1 (NHJ2S. The tissues were fixed overnight in alcoholic Bouin solution at 4°C. The backfills were silver-inten- sified by a method adapted from that of Davis (1982).

Immunocytochemistry Seventeen primary antisera were screened for immu-

nostaining of SeG median neuroendocrine cells. No staining of these cells was observed with use of the following antisera: anti-allatotropin and anti-diuretic hormone I of M . sextu; anti-leucokinin IV (gifts of J.A. Veenstra, University of Arizona); anti-lys-vasopressin (IncStar Inc., Stillwater, MN); anti-Arg-vasopressin (Peninsula Lab. Inc., Belmont, CA); anti-crustacean cardioactive peptide (gift of H. Dircksen, Rheinische Friedrich Wilhelms University, Germany), anti-locus- tachykinin (provided by H . J . Agricola, Friedrich Schiller University, Germany), and anti-PBAN K92 (gift of T.G. Kingan and A. Raina, Insect Neurobiology and Hormone Lab, USDA, ARS, Beltsville, MD).

The following antisera showed promise in staining the median neuroendocrine cells and were selected for use: anti-crustacean P-pigment dispersing hormone (anti-PDH); anti-proctolin; anti-molluscan cardioactive peptide (anti-FMRFamide); anti-cholecystokinin 1-26 (anti-CCK); anti-perisulfakinin (anti-PSK); and four antisera raised to pheromone biosynthesis-activating neuropeptide (PBAN) of the corn eanvorm, Heli- couerpu zeu (anti-PBAN PT2, anti-PBAN YG16, and anti-PBAN YG17). In addition, we used a mouse monoclonal antibody to molluscan small cardioactive peptide B (anti-SCP,). The sources, references to characterization, working dilutions, and preadsorption controls for these antisera and the monoclonal antibody are given in Table 1.

Liquid-phase preadsorption controls were done at 4°C for 12 hours with the antigen of each antiserum. In addition, the C-terminal five amino acid fragment of leucopyrokinin (Sigma, St. Louis, MO) was used to preadsorb the anti-PBAN antisera (Table 1); four of the five amino acids of this fragment are identical to those of the C-terminus of PBAN. Because a SCP-like peptide (CAP,,) has been identified in M . sextu (Huesmann et al., 1995), synthetic CAP2,, rather than SCP, was used for the preadsorption control of SCP immunoreactivity.

Wholemount preparations were studied by an immu- nofluorescence method as described by Davis et al. (1993). Goat anti-rabbit IgGs conjugated to fluorescein, rhodamine, Texas Red, Cy3, or Cy5 (Jackson Immu- noResearch Laboratories, West Grove, PA) were used to label the primary antisera. Controls in which the primary antisera were omitted from the procedure

NEUROENDOCRINE CELLS OF THE SEG OF M. SEXTA 203

TABLE 1 . Primary antisera

Antiserudmonoclonal antibody Source and first characterization Working dilution Preadsorption control Anti-PBAN (PT2)

Anti-PBAN (YG16)

Anti-PBAN (YG17)

Anti-proctolin (A3) Davis et al. (1989) 1: 1,000 10 pM prodolin Anti-PSK Veenstra et al. (1995) 1:2,000 10 nM PSK Anti-FMRFamide (232) Watson 111 (O’Donahue et al., 1984) 1:2,500 10 pM FMRFamide Anti-CCK 1-26 (1199) Polak (Homberg et al., 1991) 1500 200 nM CCK Anti-PPDH Dircksen et al. (1987) 1:2,000 100 pM PPDH Anti-SCP Monoclonal Lab, U. of Washington (Masinovsky et al., 1988) 150 (supernatant) 250 p M CAP,,

1:2,000 10 p M PBAN

1:1,000 100 p M PBAN

1:1,000 100 pM PBAN

Teal et al. (1997)

Altstein (Gazit et al., 1992)

Altstein (Gazit et al., 1992)

100 pM 4-8 leucopyrokinin



indicated that there was no specific staining by the secondary antisera.

Immunostaining by the primary antisera also was investigated by means of the peroxidase-antiperoxi- dase (PAP) technique of Sternberger (1986) on Vi- bratome sections of pharate adults fixed within 24 hours prior to eclosion. Fixation, sectioning, and immunostaining was carried out as described by Homberg et al. (1991).

Colocalization of immunoreactivities was investigated by staining alternate paraffin sections of the SeG with two antisera by means of the PAP technique. Gan- glia from pharate adults within 24 hours of eclosion were fixed at 4°C for 2-4 hours in 1 part 25% glutaral- dehydel3 parts saturated picric acid/l% acetic acid (GPA; Boer et al., 1979) and embedded in Paraplast (Monoject Scientific, St. Louis, MO) as described by Homberg et al. (1987). Alternate frontal sections (6-8 pM) were mounted on separate slides and treated with two different primary antisera and visualized by the PAP-technique (Homberg et al., 1990). Various combi- nations of primary antisera to FMRFamide, CCK, PBAN, PDH, and proctolin were tested.

In addition, double-staining was performed on wholemounts as follows. Rhodamine-conjugated goat anti-mouse IgG (Jackson Laboratories) was used to label tissue treated with mouse monoclonal antibody to SCP, and this treatment was followed by use of fluorescein-conjugated goat anti-rabbit IgG to label this tissue treated with one of the various rabbit primary antisera.

Fluorescent-immunostaining was examined with a Nikon Optiphot epifluorescence microscope and photo- graphed with Kodak Technical Pan film. In addition, images of optical sections of fluorescent-immunostaining were made using a Bio-Rad (Richmond, CAI MRC 600 laser-scanning confocal microscope equipped with a YHS filter block and an argodkrypton laser. Slides stained by the PAP method were examined with a Zeiss (Thornwood, NY) compound microscope and photomi- crographs were made with Agfapan APX 25 film. Cam- era lucida drawings were made by use of a drawing tube mounted on a Nikon Optiphot microscope.

RESULTS Background Anatomy of the

Subesophageal Ganglion Although some descriptions of the anatomy of the

SeG are available (Copenhaver and Truman, 1986;

Eaton, 1974; Eaton and Dickens, 1974; Griss, 1990), further studies were undertaken to provide the additional details and depictions essential to the present study.

AMMC A0 Br CA Cb CB cc CCAP

E2 CN EF FMR-

FrC Famide

IN, IN,, -ir Lb-Hp LbN Lc LMX M4 MdN MLb Mhld MM, MXN MXP NCC-3 NCC-V OL OP P9-12 P Pb PBAN PDH PG PMM,

PSK RfaRP SCP SeG SGD Tc TN Tn TnA TT VMP WM,

Abbreviations antenna1 mechanosensory and motor center aorta brain corpus allatum cibarial pump central body corpus cardiacum crustacean cardioactive peptide cardioactive peptide 2b circumesophageal connective cardine branch of maxillary nerve esophageal foramen

molluscan cardioactive peptide frontal connective PBAN-ir interneurons of maxillary neuromere PBAN-ir interneurons of the thoracic ganglia -immunoreactive labio-hypopharyngeal complex labial nerve lacina lateral neuroendocrine cell of maxillary neuromere median neuroendocrine cell 4 of abdominal ganglia mandibular nerve median neuroendocrine cell of labial neuromere median neuroendocrine cell of mandibular neuromere median neuroendocrine cell of maxillary neuromere maxillary nerve maxillary palp third nerve of corpus cardiacum ventral nerve of corpus cardiacum optic lobe ocular plate stages 9-12 of pharate adult pedunculus proboscis pheromone biosynthesis-activating neuropeptide pigment dispersing hormone peripheral ganglion paramedian neurosecretory cells of mandibular neuromere perisulfakinin RFamide-related peptide molluscan small cardioactive peptide subesophageal ganglion salivary gland duct tritocerebrum transverse nerve tentorium tentorial arm trachea ventro-median protocerebrum ventral unpaired median neuron of maxillary neuromere


Larval SeG. As in other insects, the SeG of M. sexta is formed by a fusion of the neuromeres of the mandibular, maxillary, and labial segments. In the larval stages the SeG is joined to the brain by relatively long circumesophageal connectives (Fig. 1; SeG, CeC). Trunks of four major pairs of nerves arise from the larval SeG; these are the mandibular, maxillary, and two pairs of labial nerves. The mandibular nerve trunk and some of its branches are shown in Figure 1 (MdN), and this nerve serves as a neurohemal surface for the serotonin-ir, paramedian neuroendocrine cells (see Fig. 3a; PMMd) of the mandibular neuromere of the subesophageal ganglion (Griss, 1989).

Each maxillary nerve extends rostrally, medial to musculature of the maxilla (Fig. 1; MxN); successive branches of this nerve extend to muscles of the cardo and stipes (Fig. 1; b,c). Distally a sensory branch extends to the lacina and maxillary palp (d); beyond this sensory branch the maxillary nerve passes in front of the stipital muscles and extends posteriorly to a major bifurcation. The outer branch of this bifurcation is a nerve which innervates an abductor muscle of the maxillary lobe (e), and the inner branch (f) is the origin of the ventral nerve (NCC-V) of the corpus cardiacum (CC). The NCC-V continues posteriorly in a sheet of connective tissue (g) which is attached between various muscles of the maxilla, and then is joined to the cardine branch of the maxillary nerve (b). Thus, the maxillary nerve and NCC-V form a loop that completely encircles the stipital muscles (Fig. 1). Near the attachment of NCC-V to the maxillary cardine nerve there is a peripheral ganglion in NCC-V, and this ganglion contains a pair of neurons (see Fig. 4e; PG). Caudal to this peripheral ganglion, the NCC-V leaves the connective tissue sheath and extends dorso-laterally to the posterior end of the arm of the tentorium (Fig. 1; NCC-V, TnA); from there the nerve extends mesally to enter the CC (Fig. 1; CC). The labial nerves (Fig. 1; LbN-1, LbN-2) are not directly involved in the neuroendocrine system of the SeG.

Adult SeG. During metamorphosis, the circumesophageal connectives shorten, and the SeG becomes incorporated onto the underside of the brain (Fig. 2; SeG, Br). The mouthparts of the adult consist princi- pally of large labial palps and elements of the maxillae. As in many other adult lepidopterans, the two elon- gated galeae are interlocked and form the proboscis (Fig. 2; Pb), and the cibarium is modified to form a large cibarial pump (Cb). The mandibles and their muscles are eliminated, and the branches mandibular nerve that innervate these structures are lost. Conse- quently, in the adult only two components of the larval mandibular nerve persist, the hypopharyngeal motor nerve and the postgenal sensory nerve (Fig. 2; a,b). These nerves serve as the neurohemal surface for the serotonin-ir neurohemal system of the adult (N.T. Davis, unpublished results).

As in the larva, the main trunk of each maxillary nerve (Fig. 2; MxN) extends rostrally, and its branches extend to muscles of the cardo (c), and stipes (d). Near the base of each galea, two slender branches extend laterally from the maxillary nerve (f, g), and the main trunk of the maxillary nerve then extends into the lu-

men of the galea (e). The most rostra1 of these lateral branches (f) extends to sensory structures located at the base of the proboscis, and the other branch (g) is the origin of NCC-V. The NCC-V, along with the postgenal sensory nerve, extends laterally in front of the stipital muscles and then caudally between the stipital muscles and ocular plate (OP). Unlike the NCC-V of larvae, that nerve of adults is not embedded in a sheet of connective tissue. A short nerve-like process, NCC-V,, extends from NCC-V to the base of the maxillary cardine nerve (h), and caudal to this process, there is a small peripheral ganglion in the NCC-V; as in the larva, the ganglion contains the somata of two neurons (i). [The structures labeled h and i have been noted previously by Eaton and Dickens (1974) and Copenhaver and Tru- man (1986)l. From the peripheral ganglion, NCC-V extends dorso-laterally over the posterior end of the tentorial arm (Fig. 2; NCC-V, TnA), and from there continues dorsally to enter the CC.

As shown in Figure 2, the SeG of the adult has three pairs of labial nerve trunks (LbN-1, LbN-2, LbN-3). These labial nerves are not involved in the neuroendocrine system of the SeG. Nomenclature of the SeG Neuroendocrine Cells In earlier studies of the SeG neuroendocrine cells of

M . sexta (Copenhaver and Truman, 1986; Davis et al., 1993; Griss, 1989; Homberg and Hildebrand, 1989; Homberg et al., 1990), disparate terms were used in naming these cells. Consequently, there is a need to establish a uniform and comprehensive terminology comparable to that used for neuroendocrine cells in the thoracic and abdominal ganglia (Davis et al., 1993). We will describe sets of neuroendocrine cells located near the mid-line (M) of the mandibular (Md), maxillary (Mx), and labial (Lb) neuromeres of the SeG. In addition, there is a set of serotoninergic, paramedian (PM) neuroendocrine cells in the mandibular neuromere (Griss, 1989; Homberg and Hildebrand, 1989) and lateral (L) neuroendocrine cells are found in the maxillary and labial neuromeres (Davis et al., 1993; Klukas et al., 1996). Therefore, these sets of cells are designated the MMd, PMMd, MMx, MLb, LMx, and LLb neuroendocrine cells; their somata and projections are shown di- agrammatically in Figures 3a and b.

Axonal Backfills Backfills Through NCC-V. In larvae and adults,

bilateral backfills of NCC-V resulted in staining of two pairs of somata near the ventral mid-line of the mandibular neuromere and two triplets near the ventral mid-line of the maxillary neuromere (Fig. 4a,c; MMd, MMJ. Efferent processes of these cells extend anteriorly through the maxillary nerve (Fig. 4a, arrow; Figs. 5b, 6b, MxN), distally enter NCC-V, and then extend caudally to arborize in the CC (see Figs. 1 and 2 for details of these nerves). We were unable to fill somata in the SeG by backfills through the so-called NCC-Vb (see Fig. 2; h).

Unilateral backfills of NCC-V resulted in staining of a contralateral set of somata in the mandibular and maxillary neuromeres and never resulted in staining their ipsilateral homologues (Figs. 4b, 5b, 6b; MMd,


Fig. 1. Depiction of the larval SeG, its nerves, and related structures as viewed from the dorsal aspect (anterior is up). For abbreviations in this and all other figures, see Abbreviations. a, hypopharyngeal nerve; b-e, branches of the maxillary nerve (see text); f, origin of

NCC-V; g, sheet-like portion of NCC-V; h, sensory nerve of the pre- mentum; i, motor nerve of labial retractor muscle; j, nerve of salivary syringe.

MM,). Consequently, we conclude that these cells have unilateral, rather than bilateral, projections to the CC. Because the mandibular and maxillary cells project to neurohemal endings in the CC, they can be identified as neuroendocrine cells.

The two pairs of somata in the median anterior cortex of the mandibular neuromere are the M,, (median mandibular) neuroendocrine cells (Figs. 4a,b, 5a,b, 6a,b; MMMd). "he paired, median ventral triplets of cells in the maxillary neuromere are the MM, (median max-


Fig. 2. Depiction of the adult SeG, its nerves, and related structures of the head as viewed from the ventral aspect; anterior is up. a, hypopharyngeal nerve; b, postgenal sensory nerve; c-f, h, branches of

illary) neuroendocrine cells (Figs. 3a,b 4a,b; M M J . The locations of the mandibular and maxillary somata are essentially the same in larvae and adults, but during metamorphosis the M M , cells shift to a more rostra1 position (compare Figs. 5a, 6a; MM,), and the pairs of M M d cells shift to a position that is more paramedian (compare Figs. 5b and 6b; MMMd).

In both larvae and adults the neurites of the MMd and M M x cells are individually distinguishable at their origin, but as they extend from each cluster of somata, they fasciculate, cross over the tract of their contralat-

the maxillary nerve (see text); g, origin of NCC-V; i, peripheral ganglion; j, labial palp nerve; k, salivary syringe nerve; 1, hypostomal sensory nerve.

era1 homologues, and project upward to arborization in the median, dorsal neuropil (Figs. 5a, 6a; arrowhead, arrow). The pattern of these projections and arborizations is basically similar in larvae and adults. The ar- rangement of the arborizations appears somewhat dis- ordered and extends rostrally into the mandibular neuropil and caudally into the labial neuropil (Figs. 5b, 6b).

Poorly defined, paired regions of arborization were demonstrated by unilateral fills of the M M d and M M x cells, and there appeared to be somewhat fewer


I Fig. 3. Depiction of the larval (a) and (b) adult SeG showing the locations of the neuroendocrine cells

and their axonal projections (dorsal view, anterior is up). The stippled areas of 3b represent the surfaces which result from cutting the adult SeG from the brain.

branches in the band of arborization contralateral to the somata (Figs. 5b, 6b). Because the arborization of these cells is bilateral, there is considerable overlap of their respective dendritic fields. The dendrites of the M, cells probably contribute most to this overlap, because in backfills in which by chance only the MM,j cells were stained, the arborization of these cells was found to be unpaired and ipsilateral.

Just beyond the origin of the dendritic branches of the MMx cells, a bundle of axons projects contralaterally in a dorso-lateral arc and then descends anteriorly in a lateral tract that extends into the maxillary nerve (Figs. 4b, 5b; arrow). The bundle of axons from the MM, cells forms a similar transverse, arcuate tract that laterally becomes confluent with that of the MMx cells and extends with them into the maxillary nerve (Figs. 4b, 5b; arrowhead).

Backfills Through NCC-3. In both larvae and adults, unilateral backfills of the third nerve of the CC adults resulted in the staining of a contralateral neuroendocrine cell in the SeG (Fig. 7a). The pathway of fills of NCC-3 is through the tritocerebrum to the ipsi-

lateral circumesophageal connective, and then to contralateral cells in the maxillary and labial neuromeres. (Our results confirm the earlier demonstration of these neuroendocrine cells by Copenhaver and Truman, 1986). The lateral cell stained in the maxillary neuromere is the LMx (lateral maxillary) neuroendocrine cell (Fig. 7a; LMx), and the posterior median soma in the ventral cortex of the labial neuromere is the MLb (median labial) neuroendocrine cell (Figs. 4d, 7a; MLb). Backfills of the L,, and MLb cells indicated that their projections to the CC are unilateral. The initial seg- ment of the MLb. cell projects upward through the central neuropil (Fig. 7b, arrow) and gives rise to paired neurites which project diagonally upward to the dorsal maxillary neuropil (Fig. 7a,b; arrowhead). Anteriorly these neurites diverge from one another, each extending in a lateral arc of dense arborizations in the central neuropil of the mandibular neuromere (Figs. 4c, 7a); the arborizations ipsilateral to the soma are somewhat more abundant than those on the contralateral side. Beyond the origin of these paired neurites, the axon of the M, cell extends contralaterally in a dorso-lateral

208 N.T. DAVIS E T AL.

Fig. 4. Neuroendocrine cells of the SeG stained by cobalt backfilling (a-d), and peripheral neuroendocrine cells stained with methyl- ene blue (e); ganglia are viewed from the dorsal (a,b,d) and lateral (c) aspect (anterior is up). a: Bilateral backfills through the NCC-V/MxN to the SeG of a fifth-instar larva; two pairs of somata (MMJ are stained in the mandibular neuromere and paired triplets (M+) are stained in the maxillary neuromere (arrow points to axons in the maxillary nerve backfilled from NCC-V). b Unilateral backfills through NCC-V/MxN to the SeG of a stage-17 pharate adult; a contralateral pair of somata (MMJ is stained in the mandibular neuromere, and a contralateral triplet of somata (MMJ is stained in the

maxillary neuromere. c: Unilateral backfill through NCC-ViMxN to the SeG of a fifth-instar larva; neurites of somata on ventral surface (right) project upward to dendritic arborizations in the dorsal neuropil. d Unilateral backfill through NCC-3KeC to the SeG of a fifth- instar larva; a contralateral soma (MLJ and its dendritic arborization are stained. (In this preparation, another neuroendocrine cell [L,] in the maxillary neuromere, which also projects through NCC-3, by chance failed to stain). e: Staining of two neuroendocrine cells in a peripheral ganglion (PG) associated with NCC-V of a fifth-instar larva. Scale bars: 200 +m (a-d) and 100 Fm (e).

arc, then descends and projects rostrally in a lateral continues into the tritocerebrum and thence into tract that extends into the circumesophageal connective (Fig. 7a,b; double-headed arrow, CeC). This tract The neurite of the LMx cell projects transversely into

NCC-3.


Fig. 5. Camera lucida drawings of cobalt backfills through NCC- ViMxN to the SeG of a fifth-instar larva (anterior is to the left). a: Lateral view of paired M,, cells, a triplet of M,, cells, and the neurites of these cells (arrowhead, arrow) projecting to arborizations in dorsal neuropil. b: Dorsal view of the M,, and M, cells, their

arborizations, and the contralateral projections of their axons (arrowhead, arrow). Laterally the bundles of axons of two groups of cells become confluent in a tract that projects anteriorly into the maxillary nerve (MxN).


Fig. 6. Camera lucida drawings of cobalt backfills of NCC-VMxN to the SeG of a stage-16 pharate adult (anterior is to the left). a: Lateral view of M,, and M, cells, showing the projection of their neurites (arrowhead, arrow) to arborizations in the dorsal neuropil.

b: Dorsal view of the M,, and M, cells, their arborizations, and the contralateral projections of their axons (arrowheads, arrows). Later- ally these two groups of axons become confluent to form a tract that projects anteriorly into the maxillary nerve (MxN).


/

a

Fig. 7. Camera lucida drawing of cobalt backfills through NCC-3/ CeC to the SeG of a fiRh instar larva (anterior is to the leff). a: Dorsal view showing staining of a contralateral soma (&) in the maxillary neuropil and a median soma (M,) in the labial neuropil. Paired neurites project anteriorly (arrowhead) from the primary neurite of the labial cell to arching, bilateral arborizations deep in the neuropil of the maxillary neuromere. The L, cell projects contralaterally (ar-

rows), and very little of its arborization has stained. Laterally the axon of L,, becomes confluent with the laterally projecting axon (double-headed arrow) of the M,, cell to form a tract that projects anteriorly into the circumesophageal connective (CeC). b Lateral view showing the projections of the primary neurite (arrow), paired neurites (arrowhead), and the axon (double-headed arrow) from the ventral soma of the M,, cell.

the center of the dorsal neuropil of the maxillary neuromere, and then the projection continues to the contralateral side of the neuropil, where the axon extends anteriorly and fasciculates with the axon of the MLb cell (Fig. 7a; arrows). Together the axons extend ros-

trally into the circumesophageal connective (Fig. 7a; CeC), tritocerebrum, and then into NCC-3.

The anatomy of the M,, cell of adults is similar to that of larvae, but in larvae the somata are in a paramedian position and in adults they are median.

Fig. 8. Fluorescent-staining of PBAN-ir neurons in wholemounts of the SeG (ventral view, anterior is up). a: The SeG of a third-instar larva, showing the groups’ median neuroendocrine cells (M,,, M,,, and MLJ and PBAN-IR interneurons (INMx). b SeG of a stage-10 pharate adult, showing intense PBAN-immunostaining of the three groups of median neuroendocrine cells and neurohemal processes (arrow). The projections of the M,, cells are labeled A, and those of the M,, cells are labeled B. Note that these two bundles of axons become confluent to form a tract extending into the maxillary nerve. c: SeG of a stage-16 pharate adult. At this stage the immunostaining of the

somata of the median neuroendocrine cells is weak, but staining of their neurohemal processes associated with NCC-V (arrow) remains strong. d SeG of a stage-18 pharate adult. At this stage immunostaining of the median neuroendocrine cells is weak and their processes cannot be distinguished due to the opacity of the ganglion. e: SeG of an adult female 24 h after eclosion. The PBAN-immunostaining of the median neuroendocrine cells and their processes (arrow) has become somewhat stronger. f: SeG of a second-instar larva; at this stage PBAN-ir somata (arrow) adjacent to the M,, cells are stained. Scale bars: 100 pm.


Fig. 9. Fluorescent-staining of PBAN-ir wholemounts. a: PBAN- immunostaining showing somata of the IN,, and M,, cells and two of the four M,, cells in a fourth-instar larva; the IN,, somata are characteristically smaller and stain less intensely than the adjacent M,, somata. b: PBAN-ir axons in the maxillary nerve (MxN) and neurohemal processes associated with NCC-V (stage-10 pharate adult). The neurohemal processes extend to an adjacent nerve (CN) via NCC-V,. c: SeG of a stage-I2 pharate adult, showing the exten-

sion of neurohemal processes onto the surface of one side of the ganglion (arrows). d CA and CC of a fourth-instar larva, showing immunoreactive processes extending in NCC-V to the CC. e: CC-CA complex of a stage-12 pharate adult, showing immunoreactive processes extending to the CC via NCC-3 and NCC-V; some processes also extend onto the adjacent wall ofthe aorta (anterior is to the right). Scale bars: 100 pm.

PBAN-Like Immunostaining

PBAN antisera (pT2, YG16, and YG17) was very similar, and this staining was blocked by preadsorption of the antisera with H . zea PBAN and by the C-terminal fragment of leucopyrokinin (Table 1). Our initial studies were undertaken with the PT2 anti-PBAN, and,

therefore, this antiserum was used for most of the re- The array of neurons stained by the three anti- sults reported here. However, the PT2 antiserum

stained a slightly broader range of cells than did the YG16 and YG17 antisera and, thus, is probably somewhat less specific.

PBAN-ir processes were observed in the cervical and circumesophageal connectives, and it was of interest to


know the origin of these processes. Therefore, the investigation of PBAN immunoreactivity was extended beyond the SeG to the brain and to the thoracic and abdominal ganglia.

PBAN-ir Neuroendocrine Cells of the SeG. In the larval and adult SeG, three groups of cells were strongly PBAN-ir. The size, number, location, and projection of sets of neurons in the mandibular and maxillary neuromeres indicate that they are the MMd and MM, neuroendocrine cells (Fig. 8a,b; see also Fig. 10a; MM,, MM,). Similarly, the size, number, location, and projections of a pair of PBAN-ir cells in the labial neuromere indicate that these cells are the MLb neuroendocrine cells (Figs. 8a,b; see also Fig. 10a; MLb). An additional set of PBAN-ir interneurons was found closely associated with the MM, somata (Figs. 8a,e 9a; INM,), and these interneurons will be discussed below.

The arborizations of the PBAN-ir neuroendocrine cells are seen in Figure 10a (unlabeled) and in Figure l l a (arrowheads, arrows). As in backfilled cells, the lateral, arcuate efferent processes of the MMd and MM, cells can be seen, and these projections extend into the maxillary nerve (Figs. 8b, 10a, l l a ; A,B). We were able to follow these PBAN-ir processes through the maxillary nerve and NCC-V to terminal branches in the CC (Fig. 9b, MxN, NCC-V; Figs. 9d,e, NCC-V, CC). How- ever, the processes form a very extensive neurohemal- like meshwork on NCC-V (Fig. 9b; NCC-V), and, consequently, this nerve, rather than the CC, appears to be the principal neurohemal release site of the median mandibular and maxillary neuroendocrine cells. As is characteristic of neurohemal projections, the extent of this system was quite variable and extended via NCC-Vb to form a neurohemal meshwork on the cardine branch of the maxillary nerve (Fig. 9b; NCC-Vb, CN), on the trunk of the maxillary nerve, and, some- times, on the dorso-anterior surface of the SeG (Fig. 9c, arrows).

We also traced the immunostained efferent processes of each MLb cell through the contralateral circumesophageal connective, into the tritocerebrum, and then via NCC-3 to the CC (Fig. 9e, NCC-3; Fig. 10a, MLb, C, parallel arrows; Fig. lOc, parallel arrows, Tc, NCC-3). The CC, rather than the efferent pathway of the MLb cells, serves as the principal site of neurohemal release of these cells. In the adult a few immunoreactive processes extend onto the CA and onto the adjacent wall of the aorta (Figs. 9e, CC, CA, Ao). In larvae the somata of MLb were found to be distinctly smaller than the somata of the MMd and M M x cells, but in adults the labial cells had become the same size or larger than the mandibular maxillary neurosecretory cells (compare MLb to MMd and M M x in Fig. 8a,b,c).

Interneurons of the SeG With PBAN-Like Immu- rwreactivity. Two pairs of maxillary interneurons, the INMx cells (Fig. 8a,e,9a; INMx), are PBAN-ir. These cells are very closely associated with the MM, neuroendocrine cells, but the INM, somata can be distinguished by their slightly smaller size and weaker immunostaining (Fig. 9a; MMx, INMx). In backfills to the M M x cells, the IN,, somata were never stained, indicating that they do not project into NCC-V. The PBAN- immunostaining of the IN,, interneurons usually was

most evident in the larval stages and in mature adults (Fig. 8a,e; INM,) and usually could not be distinguished in pharate adults (Fig. 8b,d). The projections of each pair of INM, cells are in the same transverse tract that contains the projections of the M M x cells (Figs. 10a, l l a ; B). Usually we could not distinguish between the various processes in this tract, but in early larval stages, when fasciculation of processes tends to be incomplete, the projections of the INM, cells could be distinguished (Fig. 10a; curved arrow).

At the lateral margin of the neuropil, the processes of the INM, cells bifurcate into anterior and posterior projections; the anterior branches run next to the axon of the MLb cell, extend through the circumesophageal connective, and enter the tritocerebrum (Fig. lOc, single arrow). In the tritocerebrum the INM, processes turn mesally to arborize in a PBAN-ir neuropil described below (Fig. 1Oc; open arrow).

The paired posterior branches of the INMx cell descend in a dorso-lateral tract and extend the entire length of the ventral nerve cord (Fig. lOa,d,f; arrow). In the SeG and each of the ventral ganglia, the descending projections of the IN,, cells give rise to a series of short collaterals that extend into the dorso-lateral neuropil (Figs. lOa,d,e,f: double-headed arrow), and in the ninth neuromere of the terminal abdominal ganglion, the descending projections form a fine, extensive arborization (Fig. 1Of: arrowhead). The INMx collaterals

Fig. 10. Confocal microscope images of PBAN-immunostaining of cells and processes in wholemounts of the brain and various ventral ganglia (frontal view of the brain; dorsal view of the ganglia, anterior is up). a: SeG of a third-instar larva showing the projections of the three groups of median neuroendocrine cells. The projections of the M, cells (A) and M,, cells (B) extend laterally and converge to form a tract that extends anteriorly into the maxillary nerve. The projection of each M,, cell (C) extends contralaterally into a lateral tract that extends anteriorly in to the circumesophageal connective (parallel arrows). The projections of the IN,, cells extend laterally (curved arrow) and bifurcate; the anterior branches extend in a tract with the axons of M, (parallel arrows), and the posterior branches (arrow) extend posteriorly the length of the ventral nerve cord. These descending processes of IN,, give rise to varicose collaterals along the lateral margins of the neuropil (double-headed arrow). Immunoreac- tive processes apparently ascending from the thoracic ganglia (arrowhead) enter the SeG and extend into the circumesophageal connective (open arrow). b: Frontal view of the brain of a third-instar larva. Weakly immunoreactive somata are seen in the protocerebrum (small arrowhead), and processes ascending in each circumesophageal connective (large arrowhead) give rise to arborizations in the tritocerebrum (arrow) and extend in the median tract into the protocerebrum. c: The circumesophageal connective (CeC) and base of the tritocerebrum (Tc), showing the axon of M, (parallel arrows) extending through the tritocerebrum into NCC-3. Adjacent to the axon of M, is an axon of IN,, (arrow), which turns medially in the tritocerebrum to enter the immunoreactive neuropil (open arrow). Additional axons (arrowheads) also enter this neuropil; these axons may originate from PBAN-ir interneurons in the thorax. d Prothoracic ganglion of a third-instar larva, showing the somata of the ascending interneurons (INTG) and the ascending tract of these neurons (arrowhead). The paired axons of the IN, cells (arrow) and their varicose collaterals (double-headed arrow) are shown. e: Fifth abdominal ganglion of a third-instar larva, showing the M, neuroendocrine cells and their projections (arrowhead) to the transverse nerve (TN). In addition, the tracts and collaterals of the IN,, cells are seen (double-headed arrow). f: Terminal abdominal ganglion of a third-instar larva, showing the descending tract of the IN,, cells (arrow), varicose collaterals (double-headed arrow), and terminal arborizations in the end of the ganglion (arrowhead). Scale bars: 100 pm.

Fig. 10. Legend on facing page.


Fig. 11. Frontal Vibratome sections showing PBAN-immunostaining ( a x ) and FMRFamide-immunostaining (d) in the brain and SeG of a stage-18 pharate adult. a: Section through the SeG, showing that PBAN immunostained arborizations from the median neuroendocrine cells (arrowheads) are concentrated below the esophageal foramen (EF). Axons of the IN,,/M, neurons (A) and the M,, neurons (B) take arcuate lateral paths. In addition, the neuropil contains a meshwork of fine, varicose fibers (arrows) of unknown origin. b PBAN-immunostaining in the protocerebrum. Fine, varicose fibers

in the ventral ganglia are characteristically thick, ir- regular, and strongly varicose, and these features give these collaterals the appearance of neurosecretory-type endings (Figs. lOa,d,e,f; double-headed arrow). Com- pared to these branches, the arborizations of the INM, cells in the ninth neuromere are more extensive, finer, less varicose, and deeper in the neuropil (Fig. 1Of; arrowhead).

PBAN-Like Immutwreactivity in the Brain. In the larval brain, a weakly PBAN-ir interneuron was stained in each protocerebral hemisphere, but the projections of these cells could not be distinguished (Fig. lob; small arrowhead). These PBAN-ir neurons were not seen in the pharate adult brain.

Also, in larvae there was PBAN-immunostaining of an area of arborizations in the inner, ventral neuropil

are concentrated in the ventro-median protocerebrum (VMP) and continue along the inner edge of the pedunculus of the mushroom body (Pd) to the superior protocerebrum. c , d Lateral areas (arrows) of the antenna1 mechanosensory and motor center (AMMC) exhibit similar dense patterns of varicose fibers immunostained by PBAN antiserum (c) and FMRFamide antiserum (d). At least some of these fibers appear to be side branches of an ascending axon (arrowhead) of the IN, interneurons, which are stained by both antisera. Scale bar: 100 pm.

of the tritocerebrum (Fig. lob; arrow). This area of arborization appears to originate from the INM, cells described above. In addition, there is a paramedian band of arborization that extends from the tritocerebrum into the median protocerebrum via the median bundles of the brain (Fig. lob). This band of PBAN-ir neuropil appears to arise in part from the IN,, cells. In addition, the arborizations of the median protocerebrum appear to arise from several processes that ascend on the inner side of each circumesophageal connective (Fig. 1Oc; CeC, arrowheads). These processes can be traced down the circumesophageal connective and into a dorsal diagonal tract in the SeG. This tract becomes lost among the arborizations of the MM, cells (Fig. 10a; open arrow), and, therefore, cannot not be traced to the somata from which they originate. However, a similar


dorsal diagonal tract leading caudally into each cervical connective can be observed in the posterior region of the SeG (Fig. 10a; arrowhead), and this tract comes from PBAN-ir cells in the thoracic ganglia. These thoracic cells may be serial homologs of the InMx cells and contribute to the origin of the bans PBAN-ir arborizations in the median protocerebrum (see discussion of PBAN-ir thoracic neurons below).

In Vibratome sections of the adult brain, we also were able trace fine immunoreactive processes from the tritocerebrum, around the esophageal foramen, and toward the median bundle of the brain. These fibers seem to terminate in an area lateral to the esophagus. A second laterally ascending fiber bundle gives rise to ramifications in lateral areas of the antenna1 mechanosensory and motor center (Fig. l l c , arrows). From there, the fibers continue in a loose bundle into the protocerebrum and form wide arborizations in the ventro-median and inferior median protocerebrum and around the pedunculi of the mushroom bodies (Fig. l l b ; CB, Pd, VMP). Fine side branches ascend medially around the pedunculi of the mushroom bodies (Fig. l l b ) to terminal arborizations in the anterior optic tu- bercles.

PBAN-ir Neurons in Thoracic and Abdominal Ganglia. In larvae there are two pairs of mid-ventral, PBAN-ir ascending interneurons (INTG) in the pro- and mesothoracic ganglia, and one pair in the metathoracic ganglion (Fig. 10d; INTG). These neurons project dorsally and then anteriorly in tracts on the inner side of each cervical connective (Fig. 10d; arrowhead). Ante- riorly they project in a diagonal tract in the dorsal neuropil of the SeG and become lost among the arborizations of the MM, neuroendocrine cells (Fig. 10a; arrowhead). The possible projection of these cells to aborizations in the protocerebrum has been discussed above, and their location and projections suggest that they are serial homologues of the INMx cells.

In abdominal ganglia 2-6 of larvae, an additional set of neuroendocrine cells were found to be PBAN-ir, and, as can be seen in Figure 10e, these cells project anteriorly to form neurohemal-like varicosities in the transverse nerve (Fig. 10e; M,, arrowhead, TN). These cells are identifiable as the M, cells because the M, cells are the only abdominal neuroendocrine cells that project anteriorly through the median procurrent nerve to the transverse nerve (Davis et al., 1993; Tublitz and Tru- man, 1985). The transverse nerve is known as the perivisceral organ and serves for neurohemal release of products of the neuroendocrine cells of the abdominal ganglia (Taghert and Truman, 1982; Truman, 1973). The PBAN-ir M, cells were not found in the first and terminal abdominal ganglia.

Proctolin-Like Immunostaining In larvae and adults the MMd and MM, cells (Fig.

12a,b; short arrow, long arrow) were immunostained for proctolin. We were able to trace the projections of both groups of immunoreactive cells into their contralateral maxillary nerve and from there into NCC-V to the CC. Furthermore, by immunostaining of adjacent sections of the SeG of adults, we found that proc-

tolin and FMRFamide immunoreactivities are colocalized in the MMd and MMx cells (see Fig. 15a,a’,c,c’).

The ML, cells of the SeG of larvae and adults did not show proctolin immunoreactivity (Fig. 12a,b), and in adults’ adjacent sections immunostained for proctolin and FMRFamide, only FMRFamide immunoreactivity was seen in the MLb cells (Fig. 15c,c’).

SPB-Like Immunostaining In larvae the SCP monoclonal antibody recognized

the somata of only two kinds of cells, the IN,, neurons and a distinctive ventral, median unpaired neuron, the WMM, cell, located just posterior to the INM, cells (Fig. 12c, INM,, VUMM,). The antibody was ineffective in staining processes of these neurons.

Starting early in pupation and continuing into the adult, the SCP antibody immunostained the MLb cells (Fig. 12d; arrowhead). Immunostaining of SCP-ir cells in the SeG of larvae and adults, combined with immunostaining of each of the other peptides used in this study, demonstrated that SCP immunoreactivity is colocalized with that of PBAN in the INMx cells (Fig. 12e,f; double-headed arrow) but not in the median neuroendocrine cells (Fig. 12e; short arrow, long arrow, arrowheads). The SCP immunoreactivity is colocalized with immunoreactivities of PSK and FMRFamide in the INM, cells and with PBAN, PSK, and FMRFamide immunoreactivities in the MLb cells of the adult (see below). In contrast, SCP and proctolin immunoreactivities are not colocalized in any of the cells of the SeG.

FMRFamide-Like Immunostaining The anti-FMRFamide antiserum stained several

cells in the larval SeG, including weak staining of the MMd, MMx, and MLb neuroendocrine cells, and was especially effective in staining the IN,, interneurons (Fig. 13a; short arrows, long arrows, arrowhead, INM,). Because many processes were FMRFamide-ir, it was not possible to distinguish the processes of the neuroendocrine cells in the larval SeG, but in the maxillary nerve and NCC-V, we could see immunostained processes corresponding to those of the MMd and MMMx cells.

During the wandering stage of the fifth-instar larva, FMRFamide-immunostaining of most somata in the SeG disappeared, and there was little or no staining of the median neuroendocrine cells. Immunostaining of the somata of the INMx neurons, however, remained strong (Fig. 13b; INM,) but declined by the end of development of the pharate adult (Fig. 13b,c,d; INMx). Also in the pharate adult, the VUMMx cell, an unpaired neuron locatedjust posterior to the INMx cells was FMRF- amide-ir (Fig. 13b,c,d; VUM). Moderate FMRFamide- immunostaining of the MMd and MMx cells returned late in the development of the pharate adult (Fig. 13c,d; short and long arrows), and staining of the MLb cells became strong (Fig. 13c; arrowhead).

Immunostaining of adjacent sections of the adult SeG demonstrated that FMRFamide and PBAN immunoreactivities are colocalized in the MMd, MMx, and MLb cells (Figs. 15e,e’; 16a,a’). While the intensity of PBAN-immunostaining was similar in the Mvd, MMx, and MLb cells, FMRFamide immunoreactivity was most intense in the ML, neurons, and faintest, though

Fig. 12. Fluorescent-immunostaining of wholemounts of the SeG for proctolin (a,b), PBAN (e), and SCP (c,d,f) immunoreactivities (ventral aspect, anterior is up). a,b SeGs of a third-instar larva and a stage-18 pharate adult, showing proctolin-immunostaining of the M,, and the M,, cells (short arrows, long arrows); note that the MLb neurons are not stained. c,d SCP-immunostaining of the IN,, and WM,. neurons in the SeG of a third-instar larva and of a stage-12 pharate adult. Note that in the adult the M,, neurons have become

strongly SCP-ir (arrowhead). e,fi SeG of a third-instar larva with double-staining for PBAN (e) and SCP (f) immunoreactivities. e: The three groups of median neuroendocrine cell (short arrow, long arrow, arrowheads) and the (out of focus) IN, interneurons (double-headed arrow) show Fluorescein labeling of PBAN immunoreactivity. f: In the same ganglion, only the IN,, neurons show Rhodamine labeling of SCP immunoreactivity. Scale bars: 50 pm.


still substantial, in the MM, cells. We also found strik- ing similarities between PBAN- and FMRFamide-immunostaining of processes in some regions of the adult brain (Fig. llc,d).

CCK-Like Immunostaining The antiserum against CCK did not stain any cells in

the SeG of larvae. However, in the adult this antiserum stained the MMd and MMx cells, but not the MLb cells (Fig. 13e; short and long arrows). Staining of adjacent sections of the adult SeG demonstrated that CCK and proctolin immunoreactivities are colocalized in the MMd and M M x cells (Fig. 15b,b’).

PSK-Like Immunostaining In larvae and in the development of pharate adult

the INMx and VUMM, interneurons were immunostained by the anti-PSK antiserum (Fig. 14a,b; short and long arrows), and before eclosion of the adult, the MLb cells also became PSK-ir (Fig. 14b; arrowhead).

PDH-Like Immunostaining The anti-PDH antiserum did not stain any cells in

the SeG of larvae. In adults there was moderate staining of the MMd and M, cells and very weak staining of the MLb cells (Fig. 13f; short and long arrows, arrowhead). There was no staining of the projections of these cells. The identity of the PDH-ir cells was confirmed by showing that in adjacent sections PDH and PBAN immunoreactivities in the MMd, MMx, and MLb cells were colocalized, but, as in wholemounts, the PDH immunoreactivity in the MLb cells was very faint (Figs. 145,d’, e,e’; 16b,b’).

DISCUSSION Neuroanatomy

The anatomy of the SeG and its nerves has been included in studies of M . sextu by Eaton (19741, Eaton and Dickens (1974), Copenhaver and Truman (1986), and Griss (1990); we have provided additional details and depictions of this anatomy and have described un- usual features of the larval NCC-V. In addition, the two peripheral cells located on NCC-V of adults (Copenhaver and Truman, 1986; Eaton and Dickens, 1974) have been shown to be present on this nerve in larvae. These peripheral cells were not recognized by any of the primary antisera used in this study. They appear to be neuroendocrine cells, but the nature of their neuroeffector has not been determined.

By means of cobalt-backfilling we have established that the MMd and MMx neuroendocrine cells project through the maxillary nerve and then via NCC-V to reach the CC. This pathway has been confirmed by immunostaining of MMd and M, axons extending to the CC through these nerves. Immunostaining also demonstrates that these axons and their collateral branches form neurohemal endings on the NCC-V and adjacent nerves.

In M . sexta the projection pathway of the MMd and M M x cells to the CC is similar to that previously reported by Davis et al. (1989) in the gypsy moth, Ly- mantria dispar, and by Ichikawa et al. (1995) in the silkworm, Bombyx mori. Our cobalt-backfilling and im-

munostaining also confirms the observations of Copen- haver and Truman (1986) that the LMx and MLb neuroendocrine cells of M . sextu project to the CC via the contralateral circumesophageal connectives, tritocerebrum, and NCC-3.

The patterns of dendritic arborization of the median neuroendocrine cells of the SeG have been demonstrated by cobalt backfills. While there can be no certainty that the filling of the finer branches was complete, the principal patterns of the arborizations are clear. The arborizations of the M M d and M,, cells are in the median dorsal neuropil, and this location is characteristic of neuroendocrinal neuropil (Taghert, 1981). Moreover, cobalt fills of these cells indicate that they do not project to the brain or posteriorly in the ventral nerve cord. The arborizations of the ML, cells demonstrated here, confirm the pattern previously described by Copenhaver and Truman (1986). This pattern is un- usual for neuroendocrine cells in that the arborizations are located mostly in the neuromere anterior to that of their somata, and rather than being in the dorso-median neuroendocrinal neuropil (Taghert, 19811, they extend laterally as transverse arcs. As in the case of the MMd and MMx cells, there is no indication that these cells project into the brain or into the ventral nerve cord.

PBAN-Like Immunoreactivity The PBANs that have been isolated and sequenced

(H. zea, Raina et al., 1989; B . mori, Kitamura et al., 1989, 1990; Lymuntria dispar, Masler et al., 1994) are members of a family of neuropeptides characterized by a common C-terminal sequence of Phe-Xaa-Pro-Arg- Leu-NH,, in which Xaa may be Gly, Thr, Ser, or Val. In addition to the PBANs, this family contains various myotropic peptides (myotropins and pyrokinins), the diapause hormone ofB. mori, and the melanization and reddish coloration hormone of the armyworm, Pseuah- Zetia separata (reviewed by Nachman et al., 1993; Teal et al., 1996). Various studies have shown that the common FXPRLamide C-terminal of these peptides is responsible for their physiological functions and that a FXPRLamide with a known function in one species can be very effective in stimulating unrelated physiological responses in another species (Nachman et al., 1993). Peptides of this family, including PBAN, have not yet been isolated and identified from M . sexta, but such peptides, if present, may be expected to have the FXPRLamide C-terminus characteristic of this family. Therefore, anti-PBAN antisera which recognize the FXPRLamide C-terminus should recognize PBAN-like peptides of M . sexta.

The K592 anti-PBAN antiserum, although previously used in demonstrating PBAN-ir cells in H . zea (Kingan et al., 19921, was ineffective in staining comparable cells in M . sextu. This antiserum has been shown to be most effective when recognizing the complete peptide of H. zea PBAN, and was ineffective in recognizing any of several PBAN fragments, including C-terminal fragments (Kingan et al., 1992). Possibly the K592 antiserum was not suitable for demonstrating PBAN immunoreactivity in M . sexta due to weak recognition of the conserved C-terminus. These results


Fig. 13. Legend on facing page.

NEUROENDOCRINE CELLS OF THE SEG O F M. SEXTA 221

Fig. 14. Confocal images of the SeG of a third-instar larva (a) and stage-18 pharate adult (b), showing PSK-immunostaining of the IN, and VUM, cells (long and short arrows). In the pharate adult (b) the M, cells also have become PSK-ir (arrowhead). Scale bars: 100 km.

also suggest that the structure of M. sexta PBAN may be substantially different from that of H . zea.

Teal et al. (1997) demonstrated that in ELISA the pT2 antiserum recognizes the C-terminal decapeptide of H . zea PBAN, and Gazit et al. (1992) showed that in ELISA the YG17 antiserum also recognizes the C-ter-

Fig. 13. SeG wholemounts with fluorescent-staining of FMRF- amide-like, CCK-like, and PDH-like immunoreactivities (dorsal views, anterior is up). a 4 show changes in FMRFamide-immunostaining intensity at successive stages of development. a: FMRFa- mide-immunostaining of the SeG of a third-instar larva, showing weak staining of the M,,, M,, and M,, cells (short arrow, long arrow, arrowhead), and distinct staining of the IN,, cells. b: FMR- Famide-immunostaining of the SeG of a day-2 wandering, fifth-instar larva, showing intense staining of the IN,, and VUM,, cells, very weak staining of the M,, cells (arrowhead), and no staining of the M,, and M,, cells. c: FMRFamide-immunostaining of the SeG of a stage-12 pharate adult, showing a decrease in staining intensity of the IN,, and VUM, cells, strong staining of the M,, cells, and weak staining of the M,, and M, cells (short arrow, long arrow). d En- larged view of the M, (arrows), IN,,, and VUM,, somata, showing their relative staining intensity in the SeG of a stage-14 pharate adult. e: CCK-immunostaining of the M,, (short arrow), M, (long arrow), and M,, (arrowhead) neuroendocrine cells of the SeG of a stage-18 pharate adult. E SeG of a stage-18 pharate adult, showing PDH-immunostaining of the somata of M,, and M,, neurons (short and long arrows), and very weak staining of the M,, neurons (arrowhead). Scale bars: 100 pm.

minal end of H . zea PBAN. Therefore, the recognition of a M. sexta PBAN by these antisera is to be expected. The preadsorption of these antisera with the C-terminal fragment of leucopyrokinin resulted in elimination of immunostaining in M. sextu. Because the leucopyrokinin fragment is very similar to the C-terminus of PBAN, these results also indicate that the PT2, YG16, and YG17 antisera recognize the C-terminus of a M. sexta PBAN.

Our results show that in the SeG of larval, pupal, and adult stages of M. sexta, the MMd, MMX, and MLb neuroendocrine cells and the INMx neurons are recognized by three antisera (PT2, YG16, YG17) raised against H . zea PBAN. Immunoreactivity to PBAN has been observed in comparable cells in the SeG of H . zea (Blackburn et al., 1992; Kingan et al., 1992; Ma et al., 19951, in B . mori (Ichikawa et al., 19951, and in the gypsy moth, Lymntria dispar (Golubeva et al., 1997). Because PBAN has been isolated and identified from heads of H . zea, B . mori, and L. dispar, the PBAN immunoreactivity observed in the MMd, MMx, and ML, neuroendocrine cells and the INMx neurons of M. sexta probably indicates that these cells also produce PBAN- like neuropeptides.

The significance of the PBAN-immunostaining of other neurons in M. sexta is more equivocal. The IN,,

Fig. 15. Colocalization of peptide immunoreactivities in M,, and M, neuroendocrine cells of the SeG. Adjacent sections (a,a’; b,b’; c,c’; d,d’; e,e‘) were treated with two different antisera. a,a’: Colocalized proctolin (a) and FMRFamide immunoreactivities (a’) in the two pairs of M,, cells. Arrowhead in a‘ points to FMRFamide-ir neuron that does not exhibit proctolin irnmunoreactivity. b,b’: Colocalized proctolin (b) and CCK immunoreactivities (b‘) in the MMq cells. c,c’: Colo- calized proctolin (c) and FMRFamide immunoreactwities (c’) in the three pairs of M, cells. d,d’: Colocalized PBAN (d) and PDH imrnu- noreactivities (d‘) in the M,, cells (arrowheads) and M, cells. Only

the two M,, cells of the right hemisphere are shown in this section. e,e’: Colocalization of PBAN (e) and FMRFamide immunoreactivities (e‘) in the M,, cells (arrowheads) and M,, cells. Two M,, and five M,, cells are shown in this section. Arrows point to an IN,, neuron that is intensely FMRFarnide-ir (e’) and exhibits weak PBAN immunoreactivity (e). Double-headed arrows point to neurons that are FMRFamide-ir (e‘) but not PBAN-ir (el. Note in e’ that FMRFamide immunoreactivity in the M,, neurons is more intense than in M,, neurons, while in e no difference is seen in PBAN immunoreactivity. Scale bar: 50 prn.


Fig. 16. Colocalization of peptide immunoreactivities in the M,, neuroendocrine cells. Adjacent sections (a,a‘; b,b’; c,c’) were treated with two different antisera. a,a’: Colocalized PBAN (a) and FMRF- amide (a’) immunoreactivities in the two MLb cells. Arrows point to an adjacent FMRFamide-ir neuron (a‘) which is not PBAN-ir (a). b,b’:

neurons of the thoracic ganglia of M. sexta were recognized by the PT2 and YG17 antisera, but hardly at all by the YG16 antiserum. Comparable PBAN-ir neurons were demonstrated in the thoracic ganglia of H . zea (Kingan et al., 1992; Ma et al., 1996) and in L. dispar (Golubeva et al., 1997).

The PT2, YG16, and YG17 antisera all recognized the M, neuroendocrine cells of the larval abdominal ganglia of M. sexta, and Ma et al. (1996) demonstrated comparable PBAN-ir cells in the abdominal ganglia of H . zeu. The neurons in the brain of M. sextu were demonstrated only by the PT2 antiserum. These various results suggest that, in addition to PBAN, there are other peptides of the FXPRLamide family in various parts of the CNS of M. sexta.

It is known that in vertebrates, groups of related neuropeptides are synthesized as components of larger preprohormone molecules, and it has been demonstrated that the release of a variety of mammalian neuropeptides is determined by differential post-translational processing of the precursor molecule (see Altstein and Gainer, 1988, and references therein). There also is evidence that some insect neuropeptides, including the FXPRLamides, may be produced by this mechanism (see Ma et al., 1994, and references therein). Kawano et al. (1992) and Sato et al. (1993) have characterized the cDNA encoding the preprohormone of the PBAN and of diapause hormone of B . mori and found that the corresponding mRNA also encodes PBAN and three other FXPRLamide peptides. In situ hybridization demon-

The M,, cells immunostained with the anti-PBAN antiserum (b) show only faint PDH immunoreactivity (b‘). c,c‘: The MLb cells and a smaller adjacent neuron immunostained with the anti-FMRFamide antiserum (c’) do not exhibit proctolin immunoreactivity (c, arrowheads). Scale bar: 50 pm.

strated that expression of this gene in B . mori is limited to three sets of cells in the SeG of larvae and adults (Sato et al., 1994). These cells, comparable to the MMd, MMx, and MLb neurons of M. sexta, have been shown to be immunoreactive to antisera to PBAN and the diapause hormone of B . mori (Ichikawa et al., 1995).

Similarly, Ma et al. (1994) have isolated and characterized the cDNA of H . zea PBAN; the corresponding mRNA appears to encode a preprohormone that might be processed to produce PBAN and four other peptides having C-terminal characteristic of members of the FXPRLamide family. This mRNA was found to be present in the SeG of H . zea adults but was not found in other ganglia. The results of these studies of B . mori and H . zeu suggest that in adults of M. sexta, PBAN also is produced only in the SeG, and the source of PBAN immunoreactivity in other parts of the CNS remains to be determined. As Ma et al. (1994) note, it is possible that peptides related to PBAN are encoded by other genes.

The origin of some of the processes leading to PBAN-ir arborizations in the brain of M. sextu could not be determined with complete certainty. Kingan et al. (1992) have suggested that comparable arborizations in the adult brain of H . zea originate from PBAN-ir cells in the maxillary cluster. We have shown that in M. sexta one set of cells in the maxillary cluster, the INMv cells, do project to the PBAN-ir arborizations in the tritocerebrum and possibly to arborizations in the protocerebrum as well. In addition, some of these


arborizations in the brain might originate from ascending projections of the INT, cells of the thoracic ganglia. This possibility is suggested by lines of evidence as follows: (1) PBAN-ir tracts extend into the SeG from the IN,, cells, and similar tracts extend from the SeG to the arborizations in the median protocerebrum; these tracts may be the pathway from the INTG cells to the brain; (2) the YG16 antiserum did not recognize the INTG cells, and, correspondingly, recognized very few brain arborizations; those that were stained appeared to originate from the IN,, cells; (3) in each circumesophageal connective, there are five or more processes leading to the PBAN-ir arborizations of the brain- more processes than the two INM, cells would be expected to contribute; and (4) cobalt backfills of the median neuroendocrine cells of the SeG of larva and adults never resulted in the staining of projections into the brain, and, therefore, these neuroendocrine cells apparently are not the source of the PBAN-ir arborizations of the brain.

The IN,, neurons, in addition to projecting to the brain, send prominent processes down the ventral nerve cord and into the terminal abdominal ganglion. In each ventral ganglion, these descending processes give rise to varicose collaterals extending into the dor- solateral neuropil, and the descending processes terminate in fine, extensive arborizations in posterior neuropil of the terminal ganglion. Similar projections of PBAN-ir descending neurons have been described in H . zea (Kingan et al., 1992; Ma et al., 1995) and in L. dispar (Golubeva et al., 1997). The anatomy of these neurons suggests that they may exert a broad modulatory function in the CNS. The distribution and structure of the collaterals of the descending processes of the INM, neurons in the ventral ganglia have the appearance of neurosecretory endings, and, thus, they may be involved in paracrine release of PBAN-like peptides in the CNS. In contrast, the appearance of the arborizations of these processes in the caudal neuromere of the terminal abdominal ganglion is more suggestive of parasynaptic release of the peptide. Comparative Aspects of PBAN-Immunostaining

The results of our PBAN-immunostaining of neurons in the SeG of M. sexta are generally similar to those described from the SeG of H . zea (Blackburn et al., 1992; Kingan et al., 1992; Ma et al., 1995) but differ in several significant respects. In larvae and adults of H . zea there are two median pairs of PBAN-ir cells in the labial neuromere, and their axonal projections are to the CC via NCC-3 (Blackburn et al., 1992; Ma et al., 1996). Therefore, these cells are comparable to the Mqb cells of M. sexta, but in M . sexta there is only one pair of M, cells, except, possibly in very early instars (see Fig. 8f; arrow). One of the two pairs of ML, cells of H . zea is FMRFamide-ir (Blackburn et al., 19921, as is the single pair of M, cells of M. sexta.

In H . zea, as in M . sexta, there are two pairs of PBAN-ir cells in the mandibular neuromere and a cluster of cells in the maxillary neuromere; these two groups of cells project into the maxillary nerve (Black- burn et al., 1992; Kingan et al., 1992; Ma et al., 1995). Therefore, these cells of H . zea are comparable to the

MMd and MM, cells of M . sexta. In larvae of H . zea the maxillary cluster contains eight PBAN-ir somata, and this number increases to as many as 14 in the adult (Blackburn et al., 1992; Kingan et al., 1992), while we have found that there are only 10 somata (six MMx and four INM,) in the maxillary cluster of larvae and adults of M. sexta. In H . zea it was noted that some of the cells in the maxillary cluster project posteriorly to the terminal abdominal ganglion (Blackburn et al., 1992; Kingan et al., 1992; Ma et al., 19951, but, perhaps due to the small size of this insect, these authors could not distinguish between the somata of the maxillary neuroendocrine cells and those of the descending interneurons.

The PBAN-immunostaining of neuroendocrine cells in M. sexta is essentially the same as that reported by Ichikawa et al. (1995) in B. nori, but these authors did not report PBAN-immunostaining of other cells in the CNS. In adult females of L. dispar, Golubeva et al. (1997) found four pairs of PBAN-ir cells in the mandibular neuromere, seven pairs in the maxillary neuromere, and one pair in the labial neuromere. The maxillary cluster included interneurons that project to the terminal abdominal ganglion. Thus, the PBAN-ir cells of the SeG of L. dispar appear to be comparable to those of M. sexta. In addition, Golubeva et al. (1997) found several sets of PBAN-ir neuroendocrine cells in the abdominal ganglia of L. dispar, all cells not found in M. sexta.

Proctolin-Immunostaining The proctolin immunoreactivity demonstrated here

probably represents authentic proctolin, because (1) proctolin has been shown to be present in lepidopterans (Brown, 1977; Davis et al., 19891, (2) no peptides related to proctolin have been isolated, and (3) our antiserum has been shown to be specific to proctolin (Davis et al., 1989). Proctolin-ir neuroendocrine cells have also been demonstrated in brains of M. sexta larvae and adults (Homberg et al., 1991; 8itiian et al., 1995). The pattern of proctolin-immunostaining in the SeG is similar to that of PBAN in that proctolin immunoreactivity is found in the M M d and MM, cells, but differs in that this immunoreactivity is not in the MLb cells. This staining pattern is also characteristic of the CCK-like immunoreactivity observed in the SeG of adults. Staining of adjacent sections confirmed that immunoreactivities to proctolin and PBAN are colocalized only in the MMd and M,, cells.

Immunoreactivities of RFamide-Related Peptides

The FMRFamide-related peptides (RFaRPs) com- prise a somewhat diverse group of neuropeptides that in insects includes the extended FMRFamides, extended FLRFamides (a.k.a., myosupressins), LXYRFamides (a.k.a., head peptides), and sulfakinins (DYGHRF- amides) (see reviews by Walker, 1992, and Nassel, 1993, and the study of Veenstra et al., 1995). These types of peptides structurally related in that they have an amidated RFamide C-terminus, and, consequently, they have the potential of being recognized by the same antisera. However, RFaRPs have diverse functions and in-


clude several families of peptides. The LXYRFamides have been identified in mosquitoes (Matsumoto et al., 1989) and cockroaches (Veenstra and Lambrou, 1995), sulfakinins have been identified in cockroaches (Nach- man et al., 1986; Veenstra, 1989) and D. melanogaster (Nichols et al., 19881, but these two types of peptides have not yet been isolated from M . sexta. The FMRFa- mides have been found only in D. melanogaster (Nambu et al., 1988; Schneider and Taghert, 1988), and these peptides are not found in M . sexta (Kingan et al., 1990, 1996). The FLRFamides were first identified from cockroaches (Holman et al., 1986) and locusts (Robb et al., 1989). Three FLRFamides (MasFLRFamide I, 11, and 111) have been isolated and identified from M. sexta, and there are indications of at least three additional FLR- Famide-like fractions in this insect (Kingan et al., 1990, 1996).

The anti-FMRFamide antiserum used in this study was raised to the molluscan cardioacceleratory tetrapeptide, FMRFamide (O’Donahue et al., 1984). Be- cause this and other anti-FLRFamide antisera recognize the amidated RFamide C-terminus of peptides, they cannot be relied upon to distinguish between the various types of RFaRPs. Consequently, specific immunostaining of the RFaRPs of M. sexta must await development of antisera directed to the individual N-termini of these various peptides. Even so, some pre- liminary interpretation of the significance of our FMRFamide-like immunostaining may be possible. Be- cause FMRFamides are not found in M. sexta (Kingan et al., 1990, 19961, FMRFamides are probably not responsible for the FMRFamide-like immunostaining in this insect. Kingan et al. (1996) demonstrated that Mas- FLRFamide I is, by far, the principal FLRFamide in the corpora cardiaca of fifth-instar larvae and of adults of M . sextu, but they found that this organ also contains small amounts of MasFLRFamide 11. Consequently, they suggested that MasFLRFamide I and I1 are neurohormones of cephalic origin. Therefore, our FMRFamide-like immunostaining of the median neuroendocrine cells of the SeG may indicate the presence of these FLRFamides.

Kingan et al. (1996) found that in bioassays, Mas- FLRFamide I1 is very effective in stimulating the rate of myogenic contractions of the hindgut of adults, but that MasFLRFamide I has no effect on this organ. However, MasFLRFamide I has been shown to inhibit contractions of the midgut of another sphingid moth, Agrius conuoluuli (Fujisawa et al., 1993). Because the functions of these two peptides appear to be different, it seems likely that MasFLRFamide I and I1 are not produced by the same neuroendocrine cells. In addition to the neuroendocrine cells of the SeG of M . sexta, immu- nocytological studies have shown that in the brains of larvae and adults, two groups of lateral neuroendocrine cells, Ia, and 111, are strongly FMRFamide-ir (Hom- berg et al., 1991; %than et al., 1994). Thus, it is possible that the secretions of MasFLRFamides I and I1 are partitioned amongst these various FMRFamide-ir neuroendocrine cells of the brain and SeG.

Kingan et al. (1996) also demonstrated that the levels of MasFLRFamides I and I1 in the corpora cardiaca and braidSeG increase dramatically from larva to adult. Similarly, we found that the FMRFamide-like

immunostaining of the three sets of median neuroendocrine cells of the SeG is weak in larvae and that in adults the staining of these cells, especially the M,, cells, becomes much stronger. Therefore, the changes that we observed in intensity of FMRFamide-like immunostaining of the median neuroendocrine cells may reflect the changes that Kingan et al. (1996) reported in levels of MasFLRFamide I and 11. This observation, however, does not preclude the possibility that some of the immunostaining could be due to other RFaRPs.

In addition to being recognized by anti-FMRFamide antisera, RFaRPs may be recognized by antisera against certain vertebrate peptides (pancreatic polypeptide, gastrin, and CCK) (Veenstra and Schooneveld, 1984; Veenstra et al., 19851, and this recognition is believed to be due to the structural similarity of the C-termini of these vertebrate peptides to those of the RFaRPs (reviewed by Nassel, 1993). Therefore, the question may arise as to whether or not our CCK-immunostaining is due to such cross-reactivity. However, the anti-CCK antiserum used in our study was raised to a CCK fragment which lacks the last six C-terminal peptides of CCK (J. Polak, personal communication), and, consequently, is not likely to recognize the C-terminus of RFaRPs. However, insect sulfakinins, as well as the CCK fragment, have an -Asp-Tyr-group, and so it possible that the anti-CCK antiserum can recognize insect sulfakinins. In M . sexta the CCK-like immunostaining was limited to the MMd and M M x neuroendocrine cells of adults, and no neuroendocrine cells of the larval SeG were recognized. Thus, the CCK-like immunostaining is distinctive and appears to show a certain specificity.

These results suggest that the MMd and MM, neuroendocrine cells of adults contain a sulfakinin, but this conclusion may be inconsistent with the results we ob- tained by the use of the anti-PSK antiserum. This antiserum has been tested rigorously in competitive ELISA and immunocytochemistry and has been shown to recognize sulfakinins but not other RFaRPs (Veen- stra et al., 1995). In M. sexta the anti-PSK antiserum stained only the INMx interneurons of larvae and these and the MLb neuroendocrine cells of adults. This pattern is very similar to that of SCP-immunostaining (see below), and is in contrast to CCK-like immunostaining of only the M M d and MMMx neuroendocrine cells of adults. We were unable to resolve this apparent con- flict in the identification of sulfakinin-ir cells of the SeG of M . sexta.

SCP-Like Immunostaining The SCP monoclonal antibody recognizes the INMx

and VUMMx interneurons of larvae and adults and the ML, neuroendocrine cells of adults. As noted above, this pattern of staining is very similar to that of the anti-PSK antiserum. The SCP-immunostaining also is found in neurons of the SeG that are strongly FMRF- amide-ir. This capacity of the SCP antibody to recognize neurons that are also FMRFamide-ir has been observed in studies on many other arthropods (see Arbiser and Beltz, 1991, and references therein), and, therefore, the question arises as to whether the colo-


calized FMRFamide-like and SCP-like immunoreactivities seen in the SeG of M. sexta represent recognition of the same or different peptides. Arbiser and Beltz (1991) have shown in lobsters that SCP-like immunostaining is not abolished by preadsorption with the FMRFamide tetrapeptide, but that this staining is eliminated by preadsorption with an extended FLRF- amide. Because extended FLRFamides, but not FMRF- amide, occur in M. sexta (Kingan et al., 1990,1996), the SCP antibody may recognize cells that contain Mas- FLRFamides. However, this immunostaining does not eliminate the possibility that the SCP antibody also may label a SCP-like peptide in these cells.

Masinovsky et al. (1988) have shown that the SCP monoclonal antibody recognizes all or part of the six amino acids (LAFPRM) of the C-terminus of SCP. The SCP-like peptide identified from M. sextu (CAP,,; Huesmann et al., 19951, has a C-terminus identical to that of SCP, except that in M. sexta, valine replaces methionine as the terminal amino acid. Our tests dem- onstrate that immunostaining by the SCP antibody can be blocked by preadsorption with synthetic CAP,,. These results indicate that the SCP antibody can recognize a SCP-like peptide in M. sextu, and, therefore, it is possible, but not proven, that the INMx and MLb cells contain both SCP-like and FLRFamide-like peptides.

Changes in Immunoreactivities During Postembryonic Development

By the completion of metamorphosis, the median neuroendocrine and INMx cells of the SeG express those peptide immunoreactivities observed in the larval SeG. In addition, the MLb cells become relatively larger and more strongly FMRFamide-ir, the MMd and M M x neuroendocrine cells acquire CCK-like immunoreactivity, the MLb cells acquire PSK- and SCP-like immunoreactivities, and all of the median neuroendocrine cells become PDH-ir. These changes in expression of peptide immunoreactivity may indicate that the median neuroendocrine cells acquire the capacity to produce additional hormones in the adult. Because the behavioral and physiological requirements of the adults are so different from that of the larvae, it is to be expected that such changes will occur, and several other examples of this plasticity of neuropeptide expression in the development of M. sexta have been described (see Witten and Truman, 1996, and examples cited therein). The mech- anisms underlying these changes in immunoreactivities of the median neuroendocrine cells of the SeG remain to be determined. Similar changes in peptide synthesis have been shown to be regulated by steroid hormones, and such regulation suggests an alteration of phenotypic expression (reviewed by Tublitz, 1993; Witten and Truman, 1996).

During the metamorphosis of M. sexta the INMx cells temporarily lose their PBAN immunoreactivity but remain strongly FMRFamide-ir. In contrast the median neuroendocrine cells of the SEG remain PBAN-ir but temporarily lose their FMRFamide-like immunoreactivity during this period. Late in the development of pharate adults the FMRFamide immunoreactivity re- turns and becomes more intense than it was in larvae. Blackburn et al. (1992) observed a similar decline in

FMRFamide-like immunostaining of neuroendocrine cells of the SeG of H . zea and, using a competitive ELISA, confirmed that there was a quantitative decline in FMRFamide-ir material in the SeG at the time of pupation. The functional significance of this cycle of change in levels of FMRFamide immunoreactivity is not known, and these changes may simply reflect down- and up-regulation of peptide synthesis associated with the changing requirements in metamorphosis. However, Kingan et al. (1996) have shown that metamorphosis brings about dramatic changes in the content and profiles of kinds of MasFLRFamides in the brain/SeG of M. sexta, and these changes are believed to be associated with neurosecretory cells. Therefore, it is intriguing to speculate that the loss and return of FMRFamide-ir that occurs during metamorphosis may reflect a switch from synthesis of one type of FLRF- amide to another.

It has been shown by Xu et al. (1995) that in B. mori the level of the mRNA of the preprohormone of PBAN reaches a large peak at about the midpoint of development of the pharate adult and that by adult eclosion, this mRNA is no longer present; consequently, there is no biosynthesis of PBAN in adults of this species. How- ever, Ando et al. (1988) have shown that there is a diurnal fluctuation of sex pheromone titers in virgin females of B. mori, and Arima et al. (1991) and Ichikawa et al. (1996) have provided evidence of hormonal regulation of pheromone biosynthesis by PBAN. Because PBAN is not synthesized in adults of B. mori (Xu et al., 1995), any hormonal release of PBAN in virgin females is assumed to be from stores produced in the pharate adult. We have observed that during the development of the pharate adult of M. sextu, there is an accumulation of PBAN-ir material in the neurohe- ma1 system (NCC-V) of the MMd and MM, cells. Thus, it is possible that in M. sextu, the sex-pheromone gland is controlled by release of a PBAN-like peptide produced and stored by the MM, and M,, cells of the pharate adult. The M,, cells do not appear to have much capacity for such storage.

Areas for Further Study Our study indicates that the median neurosecretory

cells of the SeG are a very significant part of the neuroendocrine system of M. sexta, and points to certain areas wherein further studies are needed, especially studies of various functions of the presumptive neurohormones of this system. Immunostaining suggests that the median neurosecretory cells of the SeG of M. sextu produce several neuropeptides and that some these peptides may be coreleased. In addition to PBAN- like peptides, these peptides may include proctolin and peptides related to FLRFamides, sulfakinins, and SCP. These peptides are known to be myoactive but probably have other functions as well. The demonstration of PSK and PBAN immunoreactivities in the median neurosecretory cells of the SeG points to the special need to isolate and identify these peptides in M. sexta.

Immunostaining indicates that in larvae and in adult males the median neuroendocrine cells also produce a PBAN-like hormone (or hormones), and while there have been extensive studies of the pheromono-


tropic function of PBAN in adult females, very little is known of the function of PBAN-like peptides in larvae and males.

Functional Differentiation of the Median Neuroendocrine Cells

In B. mori, Ichikawa et al. (1996) have demonstrated that the three groups of median neurosecretory cells of the SeG are recognized both by an antiserum to PBAN and by an antiserum to B. mori diapause hormone, a neuropeptide closely related to PBAN. These authors also have evidence indicating that only the mandibular and maxillary neurosecretory cells are responsible for pheromonotropic activity and that the labial neurosecretory cells are responsible only for the induction of diapause. Thus, there is a functional differentiation between these two groups of neuroendocrine cells in B. mori. Because the type of diapause found in M . sextu is very different from that ofB. mori, there is no reason to suppose that the labial neurosecretory cells of M . sextu are involved in diapause. However, the immunostaining characteristics of the MLb cells of M. sextu are distinctly different from those of the MM, and M M x cells, especially in adults, and so it is entirely possible that the M,, cells have a function distinctly different from that of the MMd and Mhlx cells.

PBAN and the Regulation of the Sex-Pheromone Gland

Our demonstration of PBAN-ir in the neurohemal system of adult females of M . sextu and the report by Fang et al. (1995) that the injection of PBAN into de- capitated females results in release of sex pheromone, indicates that PBAN can function as a pheromonotropic hormone in this insect. Conclusive proof that PBAN does have this function in M . sextu requires that PBAN be shown to be in the hemolymph in sufficient quanti- ties and at appropriate times for regulation of the sex- pheromone gland. Because PBAN may be coreleased with other neuropeptides, especially proctolin and FLRFamides, the possibility of a synergistic or modulatory effect of these peptides on PBAN activity should now be examined.

Hormonal regulation of the sex-pheromone gland of M. sextu does not preclude the possibility that the gland is also regulated, in part, by a descending neural pathway. The evidence for neural regulation of the sex- pheromone gland of moths has been reviewed by Chris- tensen and Hildebrand (1995), and these authors report that bath application of PBAN to the terminal abdominal ganglion of M. sextu results in activation of certain efferent cells of this ganglion. Moreover, Thy- agaraja and Raina (1994) have shown that in L. dispar, severance of the ventral nerve cord blocks pheromone production and application of PBAN to the severed terminal abdominal ganglion results in significant pheromone production. Therefore, it is perhaps significant that in M. sextu, as well as in H . zeu (Kingan et al., 1992) and L. dispur (Golubeva et al., 19971, PBAN-ir interneurons do project from the SeG to arborizations in the terminal abdominal ganglion. Christensen et al. (1994) have shown that orthodromic stimulation of the ventral nerve cord of M. sextu results in the immediate

release of sex pheromone; this release requires synap- tic transmission, is blocked by severance of the terminal nerves, and does not require bioactive factors in the hemolymph. Therefore, there is evidence of both hormonal and neural regulation of the sex-pheromone gland of M. sextu. One possible explanation for this apparent duplication of control is as follows: at the start of calling behavior, the neural activation of the gland enables a very rapid onset of pheromone biosynthesis and release, and then, during the long period of calling behavior, hormonal regulation enables the sus- tenance of these processes.

ACKNOWLEDGMENTS We are grateful to J . Polak, W.H. Watson 111, J.A.

Veenstra, H. Dircksen, T.G. Kingan, and A.K. Raina for their generous donations of antisera used in this study. Also, we gratefully extend our thanks to Dr. A.A. Os- man for rearing the M . sextu; to Charles Hedgcock, R.B.P., and S. Buchhauser for their excellent photo- graphic assistance; to Patricia Jansma for expert assistance in the use of the confocal microscope; to Eva Lodde for expert technical assistance in the laboratory; and to Drs. Tom Christensen and Tim Kingan for their very useful discussions and suggestions regarding the manu- script. Unfortunately, this paper was in press for almost two years, and, if, as a consequence of this long delay, we have failed to cite any relevant publications, we express our sincere regrets. This research was sup- ported in part by a Whitehall Foundation grant (N.T.D.), by DFG grant Ho 950/6-1 (U.H.), by the Charles H. Revson Foundation Endowment Fund for Basic Research in Life Sciences (M.A.), by Bundesmin- isterium fur Forschung und Technologie project number 0316919A (H.J.A), and by NIH grant AI-23253 (J.G.H.).

REFERENCES Altstein, M., and Gainer, H. (1988) Differential biosynthesis and pos-

translational processing of vasopressin and oxytocin in rat brain during embryonic and postnatal development. J. Neurosci., 8:3967- 3977.

Ando, T., Hase, T., Funayoshi, A., Arima, R., and Uchiyama, M. (1988) Sex pheromone biosynthesis from 14C-hexadecanoic acid in the silkworm moth. Agric. Biol. Chem., 52:141-147.

Arbiser, Z.K., and Beltz, B.S. (1991) SCP,- and FMRFamide-like immunoreactivities in lobster neurom: Colocalization of distinct peptides or colabeling of the same peptideb). J. Comp. Neurol., 306: 417-424.

Arima, R, Takahara, K., Kadoshima, T., Numazaki, F., Ando, T., Uchi- yama, M., Nagasawa, H., Kitamura, A., and Suzuki, A. (1991) Hor- monal regulation of pheromone biosynthesis in the silkworm moth, Bombyx mori (Lepidoptera: Bombycidae). Appl. Entomol. Zool., 26: 137-147.

Bell, R.A., and Joachim, F.A. (1976) Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Ann. Ent. Soc. Am., 69:365-373.

Blackburn, M.B., Kingan, T.G., Bodnar, W., Shabanowitz, J., Hunt, D.F., Kempe, T., Wagner, R.M., Raina, A.K., Schnee, M.E., and Ma, M.C. (1991) Isolation and identification of a new diuretic peptide from the tobacco hornworm, Manduca serta. Biochem. Biophys. Res. Commun., 181:927-932.

Blackburn, M.B., Kingan, T.G., Raina, A.K., and Ma, M.C. (1992) Colocalization and differential expression of PBAN- and FMRF- amide-like immunoreactivity in the subesophageal ganglion of He- licouerpa zea (Lepidoptera: Noctuidae) during development. Arch. Insect Biochem. Physiol., 21:225-238.

Boer, H.H., Schot, L.P.C., Roubos, E.W., Maat, A. ter, Lodder, J.C., and Reichelt, D. (1979) ACTH-like immunoreadivity in two elec-


tronically coupled giant neurons in the pond snail Lymnaeu stag- nalis. Cell Tissue Res., 202231-240.

Brown, B.E. (1977) Occurrence of proctolin in six orders of insects. J . Insect Physiol., 23:861-864.

Christensen, T.A., and Hildebrand, J.G. (1995) Neural regulation of sex-pheromone glands in Lepidoptera. Invert Neurosci., 1:97-103.

Christensen, T.A., Lashbrook, J.M., and Hildebrand, J.G. (1994) Neu- ral activation of the sex-pheromone gland in the moth Munducu sexta: Real-time measurement of pheromone release. Physiol. En- tomol., 19265 -270.

Copenhaver, P., and Truman, J.W. (1986) Metamorphosis of the cerebral neuroendocrine system in the moth Manduca sextu. J . Comp. Neurol., 249: 186 -204.

Davis, N.T. (1982) Improved methods for cobalt filling and silver in- tensification of insect motor neurons. Stain Technol., 57239-244.

Davis, N.T. (1985) Serotonin-immunoreactive nerves and neurohemal system in the cockroach Peripluneta umericuna (L.). Cell Tissue

Davis, N.T., Velleman, S.G., Kingan, T.G., and Keshishian, H. (1989) Identification and distribution of a proctolin-like neuropeptide in the nervous system of the gypsy moth, Lymuntrin dispur and in other Lepidoptera. J. Comp. Neurol., 283:71-85.

Davis, N.T., Homberg, U., Dircksen, H., Levine, R.B., and Hildebrand, J.G. (1993) Crustacean cardioactive peptide-immunoreactive neurons in the hawkmoth Manducu sexta and changes in their immunoreactivity during postembryonic development. J. Comp. Neurol.,

Dircksen, H., Zahnow, C.A., Gaus, G., Keller, R., Rao, K.R., and Riehm, J.P. (1987) The ultrastructure of nerve endings containing pigment-dispersing hormone (PDH) in crustacean sinus glands: Identification by an antiserum against synthetic PDH. Cell Tissue Res. 250:377-387.

Eaton, J.L. (1974) Nervous system of the head and thorax of the adult tobacco hornworm, Munduca sexta (Lepidoptera: Sphingidae). Int. J . Insect Morphol. Embryol., 3:47-66.

Eaton, J.L., and Dickens, J.C. (1974) Retrocerebral endocrine glands and the brain of the adult tobacco hornworm, Munduca sexta (L.). Int. J . Insect Morphol. Embryol., 3:237-278.

Fang, N., Teal, P.E.A., and Tumlinson, J.H. (1995) PBAN regulation of pheromone biosynthesis in female tobacco hornworm moths, Manduca sextu (L.). Arch. Insect Biochem. Physiol., 29:35-44.

Fujisawa, Y., Shimoda, M., Kiguchi, K., Ichikawa, T., and Fijita, N. (1993) The inhibitory effect of a neuropeptide, Manduca FLRF- amide, on the midgut activity of the sphingid moth, Agrilus conuoluuli. Zool. Sci., 10:773-777.

Fukuda, S., and Takeuchi, S. (1967) Studies on the diapause factor- producing cells in the suboesophageal ganglion of the silkworm, Bombyx mori L. Embryologica, 9:333-353.

Gazit, Y., Dunkelblum, 0.B.-A., and Altstein, M. (1992) Immuno- chemical and biolopical analvsis of Dheromone biosvnthesis activat-

Res., 240:593-600.

338~612-627.

ing neuropeptide inHeliothis peltz&ru. Arch. Insect Biochem. Phys- iol. 19947-260.

Golubeva, E., Kingan, T.G., Blackburn, M.B., Masler, E.P., and Raina, A.K. (1997) The distribution of PBAN (pheromone biosynthesis activating neuropeptidel-like immunoreactivity in the nervous system of the gypsy moth, Lymantria dispar. Arch. Insect Bio- chem. Physiol. (in press).

Griss, C. (1989) Serotonin-immunoreactive neurons in the suboesophageal ganglion of the caterpillar of the hawk moth Munduca sextu. Cell Tissue Res., 258:lOl-109.

Griss, C. (1990) Mandibular motor neurons of the caterpillar of the hawk moth Manducu sexta. J . Comp. Neurol., 296:393-402.

Holman, G.M., Cook, B.J., and Nachman, R.J. (1986) Isolation, primary structure and synthesis of leucomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut. Comp. Biochem. Physiol., 85C:329-333.

Homberg, U. (1994) Distribution of Neurotransmitters in the Insect Brain. Gustav Fischer, Stuttgart.

Homberg, U., and Hildebrand, J.G. (1989) Serotonin-immunoreactive neurons in the median protocerebrum and subesophageal ganglion of the sphinx moth Munduca sextu. Cell Tissue Res., 2581-24.

Homberg, U., Kingan, T.G., and Hildebrand, J.G. (1987) Immunocy- tochemistry of GABA in the brain and suboesophageal ganglion of Munducu sexta. Cell Tissue Res., 248:l-24.

Homberg, U., Kingan, T.G., and Hildebrand, J.G. (1990) Distribution of FMRFamide-like immunoreactivitv in the brain and suboesouh-

with SCP,-, BPP-, and GABA-like immunoreactivity. Cell Tissue Res., 259:401-419.

Homberg, U., Davis, N.T., and Hildebrand, J.G. (1991) Peptide-immunocytochemistry of neurosecretory cells in the brain and retrocerebral complex of the sphinx moth Munduca sextu. J . Comp. Neu- rol., 303:335-352.

Huesmann, G.R., Cheung, C.C., Loi, P.K., Lee, T.D., Swiderek, K.M., and Tublitz, N.J. (1995) Amino acid sequence of CAP,,, an insect cardioacceleratory peptide from the tobacco hawkmoth Manduca sexta. FEBS Lett., 371:311-314.

Ichikawa, T., Hasegawa, K., Shimizu, I., Katsuno, K., Kataoka, H., and Suzuki, A. (1995) Structure of neurosecretory cells with immunoreactive diapause hormone and pheromone biosynthesis activating neuropeptide in the Silkworm, Bombyx mori. Zool. Sci., 12:703- 712.

Ichikawa, T., Shiota, T., Shimizu, I., and Kataoka, H. (1996) Func- tional differentiation of neurosecretory cells with immunoreactive diapause hormone and pheromone biosynthesis activating neuropeptide of the moth, Bombyx mori. Zool. Sci., 13:21-25.

Kataoka, H., Troetschler, R.G., Kramer, S.J., Cesarin, B.J., and Schooley, D.A. (1987) Isolation and primary structure of the eclosion hormone of the tobacco hornworm, Manduca sexta. Biochem. Biophys. Res. Commun., 146:746-750.

Kataoka, H., Troetschler, R.G., Li, J.P., Kramer, S.J., Carney, R.L., and Schoolev. D.A. (1989a) Isolation and identification of a diuretic hormone from the tobacco hornworm, Manducu sextu. Proc. Natl. Acad. Sci. U.S.A., 86:2976-2980.

Kataoka, H., Toschi, A., Li. J.P.. Carnev. R.I.. Schoolev. D.A.. and Kramer, S.J. (1989b) 'Identification of"an allatotropin 'from adult Munducu sextu. Science, 243:1481-1483.

Kawano, T., Kataoka, H., Nagasawa, H., Isaogai, A,, and Suzuki, A. (1992) cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun., 189:221-226.

Kelly, T.J., Masler, E.P., and Menn, J.J . (1994) Insect neuropeptides: Current status and avenues for pest control. In: Natural and Engi- neered Pest Management Agents. P.A. Hedin, J .J . Menn, and R.M. Hollingworth, eds. American Chemical Society Symposium Series, 551:292-318.

Kent, K. (1985) Metamorphosis of the antenna1 center and the influ- ence of sensory innervation on the formation of glomeruli in the hawkmoth Munducu sexta. PhD Thesis, Harvard University, Cam- bridge, MA.

Kingan, T.G., Teplow, D.B., Phillips, J.M., Riehm, J.P., Rao, K.R., Hildebrand, J.G., Homberg, U., Kammer, A.E., Jardine, I., Griffin, P.R., and Hunt, D.F. (1990) A new peptide of the FMRFamide family isolated from the CNS of the hawkmoth, Manduca sexta. Pep- tides, 11:849-856.

Kingan, T.G., Blackburn, M.B., and Raina, A.K. (1992) The distribution of pheromone-biosynthesis-activating neuropeptide (PBAN) immunoreactivity in the central nervous system of the corn earworm moth, Helicouerpa zeu. Cell Tissue Res., 270:229-240.

Kingan, T.G., Shabanowitz, J., Hunt, D.F., and Witten, J.L. (1996) Characterization of two myotropic neuropeptides in the FMRF- amide family from segmental ganglia of the moth Munduca sextut Candidate neurohormones and neuromodulators. J . Exp. Biol., 199: 1095-1104.

Kitamura, A., Nagasawa, H., Kataoka, H., Inoue, T., Matsumoto, S., Ando, T., and Suzuki, A. (1989) Amino acid sequence of pheromone- biosynthesis-activating neuropeptide (PBAN) of the silkmoth Bom- byx rnori. Biochem. Biophys. Res. Commun., 163:520-526.

Kitamura, A., Nagasawa, H., Kataoka, H., Ando, T., and Suzuki, A. (1990) Amino acid sequence of pheromone biosynthesis activating neuropeptide-I1 (PBAN-11) of the silkmoth, Bombyx mori. Agric. Biol. Chem., 54:2495-2497.

Klukas, K.A., Brelje, T.C., and Mesce, K.A. (1996) Novel mouse IgG- like immunoreactivity expressed by neurons in the moth Munducu sexta: Developmental regulation and colocalization with crustacean cardioactive peptide. Microsc. Res. Tech., 352422264,

Kramer, S.J., Toschi, A., Miller, C.A., Kataoka, H., Quistad, G.B., Li, J.P., Carney, R.L., and Schooley, D.A. (1991) Identification of an allatostatin from the tobacco hornworm Manduca sexta. Proc. Natl. Acad. Sci. U.S.A., 88:9458-9462.

Lazar, G. (1978) Application of cobalt-filling technique to show retinal projections in the frog. Neuroscience, 3:725-737.

Ma, P.W.K., Knipple, D.C., and Roelofs, W.L. (1994) Structural orga- ageal ganglion of the sphinx moth Mukuca sexta and colocalizacon nization of the Helicouerpa zea gene encoding the precursor protein

NEUROENDOCRINE CELLS O F THE SEG O F M . SEXTA 229

for pheromone biosynthesis-activating neuropeptide and other neuropeptides. Proc. Natl. Acad. Sci. U.S.A., 91:6506-6510.

Ma, P.W.K., and Roelofs, W.L. (1995) Sites of synthesis and release of PBAN-like factor in the female European corn borer, Ostrinu nu- bilulis J . Insect Physiol. 41:339-350.

Ma, P.W.K., Roelofs, W.L., and Jurenka, R.A. (1996) Characterization of PBAN and PBAN-encoding gene neuropeptides in the central nervous system of the corn earworm moth, Helicouerpu zeu. J. Insect Physiol., 42:257-266.

Marti, T., Takio, K., Walsh, K.A.. Terzi. G.. and Truman, J.W. (1987) Microanalysis of the amino acid sequence of the eclosion hormone from the tobacco hornworm, Munducu sextu. F.E.B.S. Lett., 219: 415-418.

Masinovsky, B., Kempf, S.C., Calloway, J.C., and Willows, A.O.D. (1988) Monoclonal antibodies to the molluscan small cardioactive peptide SCP,: Immunolabeling in diverse invertebrates. J . Comp. Neurol . , 273: 500 -5 12.

Masler, E.P., Raina, A.K., Wagner, R.M., and Kochansky, J.P. (1994) Isolation and identification of a pheromonotropic neuropeptide from brain-subesophageal ganglion complex of Lymantria dispur. A new member of the PBAN family. Insect Biochem. Mol. Biol., 24:829- 836.

Matsumoto, S., Brown, M.R., Crim, J.W., Vigna, S.R.,and Lea, A.O. (1989) Isolation and primary structure of neuropeptides from the mosquito, Aedes uegypti immunoreactive to FMRFamide antiserum. Insect Biochem. 19:277-283.

Morita, M., Hatakoshi, M., and Tojo, S. (1988) Hormonal control of cuticular melanization in the common cutworm, Spodopteru litum. J . Insect Physiol., 34:751-758.

Nachman, R.J., Holman, G.M., Haddon, W.F., and Ling, N. (1986) Leucosulfakinin, a sulfated neuropeptide with homology to gastrin and cholecystokinin. Sci. 234:71-73.

Nachman, R.J., Holman, G.M., Schoofs, L., and Yamashita, 0. (1993) Silkworm diapause induction activity of myotropic pyrokinin (FXPRLamide) insect neuropeptides. Peptides, 14:1043-1048.

Nambu, J.R., Murphy-Erdosh, C., Andrews, P.C., Gottfried, J., Feist- ner, G.J., and Scheller, R.H. (1988) Isolation and characterization of a Drosophilu neuropeptide gene. Neuron 155-61.

Nassel, D.R. (1993) Neuropeptides in the insect brain: A review. Cell Tissue Res., 273:l-29.

Nichols, R. Schneuwly, S.A., and Dixon, J.E. (1988) Identification and characterization o f a Drosophilu homologue to the vertebrate neuropeptide cholecystokinin. J . Biol. Chem. 263:12176-12170.

OBrien, M.A., Katahira, E.J., Flanagan, T.R., Arnold, L.W., Haugh- ton, G., and Bollenbacher, W.E. (1988) A monoclonal antibody to the insect prothoracicotropic hormone. J. Neurosci., 8:3247-3257.

ODonahue, T., Bishop, J.F., Chronwall, B.M., Groome, J.R., and Wat- son 111, W.H. (1984) Characterization and distribution of FMRF- amide immunoreactivity in the rat central nervous system. Pep- tides, 5563-568.

Ogura, N. (1975) Hormonal control of larval colouration in the armyworm, Leucunia sepurutu. J. Insect Physiol., 21559-576.

Raabe, M. (1989) Recent Developments in Insect Neurohormones. Ple- num Press, New York.

Raina, A.K., Jaffe, H., Kempe, T.J., Keim, P., Blacher, R.W., Fales, H.M.. Rilev. T.C.. Klun. J.A.. Ridaewav. and Haves. D.K. (1989) Identification of aneuropeptide hormone that regdates pheromone production in female moths. Science, 224796-798.

Robb, S., Packman, L.C., and Evans, P.D. (1989) Isolation, primary structure and bioactivity of SchistoFLRFamide, a FMRFamide-like neuropeptide from the locust, Schistocercu greguriu. Biochem. Bio- phys. Res. Commun., 160:850-856.

Sato, Y. Oguchi, M., Menjo, N., Imai, K., Saito, H., Ikeda, M., Isobe, M., and Yamashita, 0. (1993). Precursor polyprotein for multiple neuropeptides secreted from the subesophageal ganglion of the silkworm Bombyx mori: Characterization of the cDNA encoding the diapause hormone precursor and identification of additional peptides. Proc. Natl. Acad. Sci. U.S.A., 90:3251-3255.

Sato, Y., Ikeda, M., and Yamashita, 0. (1994) Neurosecretory cells expressing the gene for common precursor for diapause hormone and pheromone biosynthesis-activating neuropeptide in the suboesophageal ganglion of the silkworm, Bombyx mori. Gen. Comp. En- docrinol., 9627-36.

Schneider, L.E., and Taghert, P.H. (1988) Isolation and characteriza-

tion of a Drosophila gene that encodes multiple neuropeptides related to Phe-Met-Arg-Phe-NH, (FMRFamide). Proc. Natl. Acad. Sci. U.S.A., 851993-1997.

Sternberger, L.A. (1986) Immunocytochemistry. 3rd ed. John Wiley & Sons, New York.

Taghert, P.H. (1981) Identification and analysis of neurosecretory neurons in the insect. In: Neurosecretion: Molecules, Cells, and Sys- tems. D.S. Farner and K. Lederis, eds. Plenum Press, New York, pp.

Taghert, P.H., and Truman, J.W. (1982) Identification of the bursicon- containing neurones in abdominal ganglia of the tobacco hornworm, Munducu sextu. J . Exp. Biol., 98:385-401.

Teal, P.E.A., R.L. Abernathy, R.J. Nachman, N. Fang, J.A. Meredith, and J.H. Tumlinson (1996). Pheromone biosynthesis activating neuropeptides: functions and chemistry. Peptides, 17:337-344.

Teal, P.E.A., Meridith, J.A., Davis, J.T., and Tumlinson, J.A. (1997) Isolation, partial purification and characterization of pheromonotropic substances in the terminal abdominal ganglion of Heliothis uirescens (F) (Lepidoptera: Noctuidae). J. Insect Physiol., (submit- ted)

Thyagaraja, B.S., and Raina, A.K. (1974) Regulation of pheromone production in the gypsy moth, Lymantriu dispur, and development of an in uiuo bioassay. J . Insect Physiol. 40:969-974.

Truman, J.W. (1973) Physiology of insect ecdysis. 111. Relationship between the hormone control of eclosion and tanning in the tobacco hornworm, Munducu sexta. J . Exp. Biol., 57:365-379.

Truman, J.W., and Copenhaver, P.F. (1989) The larval eclosion hormone neurones in Munducu sertu: Identification of the brain-proc- todeal neurosecretory system. J. Exp. Biol., 147:457-470.

Tublitz, N.J. (1993) Steroid-induced transmitter plasticity in insect peptidergic neurons. Comp. Biochem. Physiol., 105C:147-154.

Tublitz, N.J., and Truman, J.W. (1985) Identification of neurons containing cardioacceleratory peptides (CAPS) in the ventral nerve cord of the tobacco hawkmoth Munducu sata. J. Exp. Biol., 116 395-410.

Veenstra, J.A. (1989) Isolation and structure of two gastridCCK-like neuropeptides from the American cockroach homologous to the leu- cosulfakinins. Neuropeptides, 14145-149.

Veenstra, J.A. (1991) Presence of corazonin in three insect species, and isolation and identification of [Hi~~lcorazonin from Schzstocercu umericunu. Peptides, 12:1285-1289.

Veenstra, J.A., and Hagedorn, H.H. (1991) Identification of neuroendocrine cells producing a diuretic hormone in the tobacco hornworm moth, Munducu sertu. Cell Tissue Res., 266:359-364.

Veenstra, J.A., and Lambrou, G. (1995) Isolation of a novel RF amide peptide from the midgut of the American cockroach, Periplunetu umericuna. Biochem. Biophys. Res. Commun., 213519-524.

Veenstra, J.A., and Schooneveld, H. (1984) Immunocytochemical localization of neurons in the nervous system of the Colorado potato beetle with antisera against FMRFamide and bovine pancreatic polypeptide. Cell Tissue Res., 325303-308.

Veenstra, J.A., Romberg-Privee, H.M., Schooneveld, H., and Polak, J.M. (1985) Immunocytochemical localization of peptidergic neurons and neurosecretory cells in the neuro-endocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides. Histochemistry, 82:9-18.

Veenstra, J.A., Lau, G.W., Agricola, H.J., and Petzel, D.H. (1995) Immunohistological localization of regulatory peptides in the midgut of the female mosquito Aedes uegypti. Histochem. Cell Biol., 104:337-347.

Walker, R.J. (1992) Neuroactive peptides with an RFamide or Famide carboxyterminal. Comp. Biochem. Physiol. C, 102:213-222.

Witten, J.L., and Truman, J.W. (1996) Developmental plasticity of neuropeptide expression in motoneurons of the moth, Munducu sertu: Steroid hormone regulation. J. Neurobiol., 29:99-114.

Xu, W.-H., Sato, Y., Ikeda, M., and Yamashita, 0. (1995) Stage-de- pendent and temperature-controlled expression of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis activating neuropeptide in the silkworm, Bombyx mori. J. Biol. Chem., 270:3804-3808.

Zitiian, D., Kingan, T.G., Kramer, S.J., and Beckage, N.E. (1995) Accumulation of neuropeptides in the cerebral neurosecretory system of Munducu ser tu larvae parasitized by the braconid wasp Cote- sia congregutu. J . Comp. Neurol., 356:83-100.

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