Retrocerebral Complex Research Articles

Distribution of myomodulin-like immunoreactivity in the brain and retrocerebral complex of the locust, Schistocerca gregaria.

The distribution of myomodulin-like immunoreactivity is described for the brain and retrocerebral complex of an insect, the locust, Schistocerca gregaria. The locust brain contains 70-100 neuronal cell bodies and numerous neuropilar processes exhibiting myomodulin-like immunoreactivity. The most marked feature of the staining is a group of lateral tritocerebral neurones that form a highly immunoreactive tract that gives rise to a complex neuropile of stained processes in the dorsal tritocerebrum. This tract continues dorsally and bifurcates into a major branch that exists the brain via nervi corpora cardiaca 1 (NCC1) to innervate the corpora cardiaca and the corpora allata. A minor branch, consisting of several individual axons, combines with immunoreactive processes from the ventral nerve cord and generates a complex immunoreactive neuropile in the anterior and posterior regions of the protocerebrum. Immunoreactive processes are also found in the structured neuropile of the central body complex. Immunoreactive cell bodies are also found in the antennal lobes, in the lateral margins of the protocerebrum, in the optic lobes, and in a few cells in the pars intercerebralis. The results suggest that myomodulin-like neuropeptides may play roles as central neurotransmitters or neuromodulators in insects as well as being released into the circulation as neurohormones or acting as releasing agents for neurohormones in neurohaemal areas. They also further strengthen the idea that myomodulins, which were first identified in molluscs, may represent another interphyletic family of neuropeptides.

Postembryonic development of corazonin‐containing neurons and neurosecretory cells in the blowfly, Phormia terraenovae

An antiserum against the cockroach cardioactive peptide corazonin was used to investigate the distribution of immunoreactive neurons and neurosecretory cells in the nervous system of the blowfly, Phormia terraenovae, during postembryonic development. A small number of corazonin-immunoreactive neurons was found at larval, pupal, and adult stages. At all postembryonic stages two cell groups were found in the protocerebrum of the brain: 1) two lateral cell clusters and 2) two median cells. In the larva eight bilateral cell pairs were found in thoracic and abdominal neuromeres of the fused ventral ganglion. The lateral brain neurons are located in the lateral neurosecretory cell group and extend axons with branches in several components of the retrocerebral neuroendocrine complex, in the stomatogastric nervous system of larvae and adults, and additionally in muscles of the alimentary canal in the adult. The most prominent element of these peripheral processes is a large plexus of varicose fibers located in the wall of the aorta, the main site for the release of neurohormones produced in the brain of blowflies. The presence of corazonin-immunoreactive material in the aortic plexus suggests that this peptide functions as a neurohormone. During metamorphosis, the immunoreactive neurons found in the thoracic-abdominal ganglion of the larva disappear, and in the brain new immunoreactive neurons are added to those that persist from larval stages. The bulk of the corazonin-immunoreactive material extracted from adult brains and corpora cardiaca-aorta complexes was found to co-elute with synthetic corazonin in reversed-phase high-performance liquid chromatography as monitored with enzyme-linked immunosorbent assay.

Stimulatory effect of octopamine on juvenile hormone biosynthesis in honey bees ( Apis mellifera): Physiological and immunocytochemical evidence

The effect of octopamine on the activity of corpora allata of adult worker honey bees has been examined in vitro and correlated to the local distribution of this biogenic amine in brain and retrocerebral complex as studied immunocytochemically by means of an highly specific antiserum. Octopamine causes a dose-dependent increase in juvenile hormone release from corpora allata. Maximum increase is obtained with concentrations of 10 −6 M in nurse and foraging bees by 45.3 or 32.3%, respectively. Octopamine-like immunoreactivity occurs in about 45 somata of the median neurosecretory cells in the pars intercerebralis of the bee brain. They project via immunopositive nervus corporis cardiaci I into the corpora cardiaca, where interspersed varicose structures and 8–10 cell bodies in the ventral part of this gland are stained. A network of immunoreactive fine varicose nerve fibres surrounds each gland cell of the corpora allata. Immunoreactivity in these neuronal structures is detectable if bees were starved over night, a condition in which corpora allata elicit the highest juvenile hormone production ever observed in bees. Both, the stimulatory effect of octopamine and the presence of immunoreactive nerve fibers in the corpora allata, strongly indicate a physiological role of this biogenic amine in the regulation of juvenile hormone biosynthesis in adult honey bees.

Nervous system of Triatoma infestans

The anatomy of the adult nervous system of the haematophagous bug Triatoma infestans has been studied by means of dissections and histology. The central nervous system comprises three nervous masses: the brain+suboesophageal ganglion, the prothoracic ganglion, and the posterior fused ganglion (meso+metathoracic+abdominal ganglia). The form of the brain is determined by the tubular head and the highly developed muscles of the pharyngeal pump. The prothoracic ganglion is located near the posternum, the posterior ganglionic mass near the mesosternum. A significative variation of the branching pattern of abdominal nerves is reported. The innervations of mouth parts, salivary glands, muscles, retrocerebral complex, spiracles, rectum, reproductive organs, alary muscles, and peripheral nerves are described.

HAEMOLYMPH AND TISSUE TITRES OF ACHETAKININS IN THE HOUSE CRICKET ACHETA DOMESTICUS: EFFECT OF STARVATION AND DEHYDRATION

Achetakinin-like immunoreactive material in tissues and haemolymph of adult male crickets was quantified by radioimmunoassay. Achetakinin-like material was found in the brain, suboesophageal ganglia and the thoracic and abdominal ganglia, but the largest amount was within the retrocerebral complex. A Ca2+-dependent release of achetakinin-like immunoreactive material was demonstrated from retrocerebral complexes incubated in vitro in saline containing a high concentration of K+. The concentration of achetakinin-like material in haemolymph from fed crickets was estimated to be 2.8 nmol l-1 and increased more than 10-fold in insects starved for 48 h without access to water. The presence of achetakinin-like material in haemolymph suggests that these peptides are released in vivo and function as circulating neurohormones.

Control of diapause and supercooling by the retrocerebral complex in Pyrrhocoris apterus

AbstractEffects of external (photoperiod, temperature) and internal (retrocerebral complex, diapause condition) factors on supercooling were studied in Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae) adults. An increase in supercooling capacity is associated with the induction of diapause by short‐day photoperiod or cardiacallatectomy of long‐day non‐diapause insects in spite of a high temperature of 26 °C. The induction of diapause is a prerequisite for a further increase in supercooling capacity by cold acclimation.Post‐diapause adults show low values of the supercooling point also in late January or early February, although their developmental potential is fully restored. Evidently the ability to supercool associated with diapause induction is maintained by a low ambient temperature in spite of diapause termination. The supercooling point increases rapidly when these adults are transferred to a high temperature of 26 °C and cold re‐acclimation is then no longer possible. The inhibition of morphogenesis (maturation of gonads) by cardiacallatectomy does not prevent the supercooling point from increasing in post‐diapause adults at the high temperature.

Allatotropin is a cardioacceleratory peptide in Manduca sexta

ABSTRACT Allatotropin is a neuropeptide that was originally isolated from the sphinx moth Manduca sexta, in which it stimulates the synthesis of juvenile hormone in adults (Kataoka et al. 1989). An antiserum raised against this peptide was characterized, shown to be specific for allatotropin and used in a competitive enzyme-linked immunosorbent assay (ELISA) to demonstrate that allatotropin is present not only in the brain and retrocerebral complex but also in the ventral nerve cord of Manduca sexta (Veenstra and Hagedorn, 1993). In the present study, allatotropin-immunoreactive neurons were located using the allatotropin antiserum in a whole-mount immunofluorescence method adapted from Davis et al. (1989). Several cell types were found to be immunoreactive, including three pairs of intensely staining median neurons in abdominal ganglia 3–6 of the pharate adult. The location of these median pairs of cells in each ganglion is anterior, mid-dorsal and posterio-ventral (Fig. 1A). The cells project bilaterally via the ventral nerves of each ganglion to the next transverse nerve (Fig. 2), where their axons terminate in numerous superficial varicosities (Fig. 1D). It is well established that the transverse nerve is a neurohemal organ in Lepidoptera (Provansal, 1972; Taghert and Truman, 1982; Tublitz and Truman, 1985a–d). The location and projection pattern of these three pairs of median neurons indicate that they belong to a group of abdominal median neurosecretory cells identified previously (Taghert and Truman, 1982; Tublitz and Truman, 1985a). Using the nomenclature of Davis et al. (1993), these are the M1, M2 and M5 neurosecretory cells. The M1 and M2 cells mature during adult development, but the M5 cells are already differentiated in the larval stages (Taghert and Truman, 1982; Tublitz and Truman, 1985a). The M5 cells of larvae were not stained by the allatotropin antiserum and only became immunoreactive, along with the M1 and M2 cells, during adult metamorphosis. Two pairs of the abdominal median neurosecretory cells were not allatotropin-immunoreactive: (1) the M3 cells, which are ventral and mature during adult metamorphosis (Taghert and Truman, 1982); and (2) the M4 cells, which are already differentiated in the larval stages (Tublitz and Truman, 1985a).

Isolation, sequence, and bioactivity of FMRFamide-related peptides from the locust ventral nerve cord

The ventral nerve cord of the locust, Locusta migratoria, was examined for the presence of FMRFamide-related peptides (FaRPs). RP-HPLC coupled to an RIA specific for extended-RFamides revealed the presence of several FaRPs eluting at different percentages of acetonitrile. The sequences of five of these peptides were determined. Two sequences are identical to the two peptides previously sequenced from brain and retrocerebral complex of Locusta. These two peptides (PDVDHVFLRFamide and ADVGHVFLRFamide) were inhibitory when tested on locust oviduct contractions. The other peptides are novel with sequences of GQERNFLRFamide, AXXRNFIRFamide, and AFIRFamide. The synthesized peptides were stimulatory when tested on locust oviduct contractions, increasing the frequency and amplitude of spontaneous contractions and resulting in a basal contraction.

Immunocytochemical localization of a diuretic peptide Manduca diuresin (MasDPII) in the brain and suboesophageal ganglion of the tobacco hawkmoth Manduca sexta (Lepidoptera: Sphingidae)

AbstractA purified polyclonal antibody against synthetic Manduca diuresin (formerly named MasDPII) was used to localize immunoreactive cell clusters in the protocerebrum and the suboesophageal ganglion of adult, larval, and pupal stages of Manduca sexta. In adults and pharate adults, strong immunostaining was found in type IIb median neurosecretory cells and IIa1–3 cells. Moderate staining was observed in the maxillary, mandibular, and labial cell clusters in the suboesophageal ganglion. In larvae, immunostaining was found in four IIa cells and in the maxillary, mandibular, and labial cell clusters of the suboesophageal ganglion. In the early pupal stage, six to eight type IIa cells were immunopositive and two clusters of developing type IIb cells showed positive staining. Furthermore, maxillary, mandibular, and labial cell clusters in the pupal suboesophageal ganglion also contained immunoreactive material. In all stages, nervi corporis cardiaci–1+2 and the retrocerebral complex showed immunoreactivity. While preadsorption of purified antibody with another diuretic peptide from M. sexta brain, MasDH, slightly reduced the immunostaining by the Manduca diuresin antibody, preadsorption with Manduca diuresin completely abolished immunoreactivity in all of the cells previously mentioned. The findings suggest the presence of Manduca diuresin or Manduca diuresin‐like peptides in larval and pupal type IIa cells and in the suboesophageal ganglion of M. sexta in all three developmental Stages. © 1994 Wiley‐Liss, Inc.

Isolation, sequence, and bioactivity of PDVDHVFLRFamide and ADVGHVFLRFamide peptides from the locust central nervous system

The brain and the retrocerebral complex of the locust, Locusta migratoria, were examined for the presence of FMRFamide-related peptides (FaRPs) using an RIA specific for -RFamide. RP-HPLC of these extracts using both C18 and phenyl columns revealed the presence of several FaRPs eluting at different percentages of acetonitrile. The sequences of two peptides were determined. One sequence is identical to the previously described SchistoFLRFamide (PDVDHVFLRFamide), whereas a second peptide is novel and differs from SchistoFLRFamide in positions 1 and 4 (ADVGHVFLRFamide). The bioactivity of these native and synthetic FaRPs on locust oviduct contractions has been examined. Both peptides showed inhibitory activity on the locust oviduct. Truncated versions of PDVDHVFLRFamide revealed that the essential features for inhibition lay in the sequence HVFLRFamide.

Characterization of an antiserum against an achetakinin I-analog and its use for the localization of Culekinin Depolarizing Peptide II in the mosquito, Culex salinarius

ELISA experiments revealed that an antiserum raised against an achetakinin-analog could specifically detect the recently isolated Culekinin Depolarizing Peptide (CDP)-II from the mosquito, Culex salinarius. The characterization indicated that two different epitopes in the C-terminal region of achetakinin I and CDP-II are recognized. One epitope is the -F-Y-region, the other is the -P-W-region. Among the peptides isolated from C. salinarius, the antiserum reacts only with CDP-II. Pre-absorption tests of the antiserum with CDP-II in immunohistological stainings abolished the reaction, while tests with pre-immune sera did not cause any immunopositive reactions. In the mosquito head ganglia, immunoreactive neurons were detected in the pars lateralis, the optic lobe and the suboesophageal ganglion. Although some immunopositive axons extended into the nervi corporis cardiacii II, no immunoreactivity was observed in the retrocerebral complex. In the thoracic ganglia, immunoreactive neurons were found in the pro-, meso- and metathoracic neuromeres. No immunoreactivity was found elsewhere. With this study we demonstrate that CDP-II, isolated from a whole body extract, is truly a neuropeptide, and the data suggest that its function is neuromodulating or neurotransmitting rather than neurohormonal.

FMRFamide‐like activity in the female locust during vitellogenesis

The control of oviposition in the locust involves the expulsion of eggs from the lateral oviducts, a process believed to be under neurohormonal control. In this paper we have attempted to identify this putative hormone. Immunohistochemical staining of the brain retrocerebral complex and suboesophageal ganglion of Locusta migratoria with antiserum against FMRFamide revealed a number of FMRFamide-immunopositive cells. FMRFamide-like immunoreactivity was present in median neurosecretory cells and lateral neurosecretory cells of the protocerebrum. Other FMRFamide-immunoreactive cells were detected in the deutocerebrum and tritocerebrum. Immunoreactive cell processes were observed in the mushroom bodies, the central body, the optic lobes, and in the axon tracts leaving the pars intercerebralis and tritocerebrum. FMRFamide-like material was also seen in the circumoesophageal commissures. Further FMRFamide-like material was present in cell bodies of the suboesophageal ganglion. FMRFamide-like staining activity changed dramatically during the oviposition cycle in mature adult females. The median neurosecretory cells stained lightly immediately after oviposition and remained pale until the third day, when staining of perikarya and axon tracts increased. The staining intensity decreased on days 4 and 5. The titre of FMRFamide-like material in the hemolymph increased during the vitellogenic cycle but plummeted after oviposition. A single band of FMRFamide-like material was evident on immunoblot following sodium dodecyl sulphate-polyacrylamide gel electrophoresis of adult female hemolymph. The approximate molecular weight of this molecule was 8,000. Gel permeation chromatography of hemolymph revealed a FMRFamide-immunoreactive fraction with a molecular weight of 8,000. This fraction possessed myotropic activity when applied to the locust oviduct.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of corazonin in the nervous system of the cockroach Periplaneta americana

Antisera raised to the cardioactive peptide corazonin were used to localize immunoreactive cells in the nervous system of the American cockroach. Sera obtained after the seventh booster injection were sufficiently specific to be used for immunocytology. They recognized a subset of 10 lateral neurosecretory cells in the protocerebrum that project to, and arborize and terminate in the ipsilateral corpus cardiacum. They also reacted with bilateral neurons in each of the thoracic and abdominal neuromeres, a single dorsal unpaired median neuron in the suboesophageal ganglion, an interneuron in each optic lobe, and other neurons at the base of the optic lobe, in the tritocerebrum and deutocerebrum. The presence of corazonin in the abdominal neurons and the lateral neurosecretory cells was confirmed by HPLC fractionation of extracts of the abdominal ganglia, brains and retrocerebral complexes, followed by determination of corazonin by ELISA, which revealed in each tissue a single immunoreactive peak co-eluting with corazonin in two different HPLC systems. Antisera obtained after the first three booster injections recognized a large number of neuroendocrine cells and neurons in the brain and the abdominal nerve cord. However, the sera from the two rabbits reacted largely with different cells, indicating that the majority of this immunoreactivity was due to cross-reactivity. These results indicate that the production of highly specific antisera to some neuropeptides may require a considerable number of booster injections.

The pulsatile appearance of FMRFamide-related peptides in the haemolymph and loss of FMRFamide-like immunoreactivity from neurohaemal areas of Rhodnius prolixus following a blood meal

The release of FMRFamide-related peptides (FaRPs) from nervous tissue and corpus cardiacum/dorsal vessel and their appearance in the haemolymph of Rhodnius prolixus after a blood meal was investigated using immunohistochemistry, RIA and HPLC. Release of FaRPs was indicated by a decrease in the intensity of FMRFamide-like immunoreactive staining of neurohaemal areas associated with the retrocerebral complex and abdominal nerves, and confirmed, using RIA, by the tissue-specific decreases and appearance of FaRPs in the haemolymph. Titres of FaRPs in the haemolymph rose and fell in a pulse-like manner at fairly fixed times during the first 12 h after feeding (period of observation). Titres in the haemolymph rose from 0.88 nM to post-feeding pulse titres which ranged from 2.4 to 19.6 nM. The highest titres found in the haemolymph occurred at about 3, 6 and 10 h after feeding. Results from HPLC and RIA indicate that FaRPs may be synthesized in the nervous system and then released into the haemolymph, possibly as a mixture, which might subsequently affect various physiological processes involved in digestion and development.

Quantitative Determination of Myotropic Neuropeptide in the Nervous System of the Cockroach Leucophaea Maderae: Distribution and Release of Leucokinins

ABSTRACT Being interested in the distribution and function of insect myotropic peptides, we developed a sensitive radioimmunoassay (RIA) using an antiserum directed against the cockroach neuropeptide leucokinin I. The levels of leucokinin immunoreactivity were measured in extracts of different portions of the nervous system, in the retrocerebral complex and in the hemolymph of the cockroach Leucophaea maderae. The brain contains about 1.9pmol of immunoreactive material while the ganglia of the ventral nerve cord each contain less than 10% of this amount. Large amounts of leucokinin immunoreactivity (6.6pmol per tissue) were found in the corpora cardiaca–corpora allata (CC–CA) complex and in the hemolymph the titer was in the nanomolar range. When levels of leucokinins were compared in male and female cockroaches, no differences could be detected in any of the sample tissues. A calcium-dependent release of leucokinin-immunoreactive material could be induced from isolated CC–CA complexes by high-potassium depolarization. The amount of released immunoreactivity, as measured in the bathing saline by RIA, was about 2% of the extractable leucokinin immunoreactivity in the CC–CA complex. In Leucophaea maderae, the leucokinins apparently act both as neuroactive substances in the central nervous system and as neurohormones released into the circulation.

Monoclonal antibody immunohistochemistry in the American cockroach identifies a novel neuropeptidergic system similar to, but distinct from this insect's (FM)RFamide-like system

A monoclonal antibody to peptidergic neurons in the neuroendocrine system of the Colorado potato beetle immunohistochemically labels neuropeptide-like substances throughout the cephalic neurosecretory system of the American cockroach, Periplaneta americana. The dictyopteran antigen shows a histological distribution similar to that of the neuropeptide-like material which we have described earlier using (FM)RFamide specific antibodies. It was conclusively demonstrated, particularly by means of a convenient double labelling procedure, that the (FM)RFamide- and the coleopteran neuropeptide-like antigens are not localized in the same neuronal structures, although both neurochemicals are in close vicinity to one another. The relative abundance of the immunoreactive products in neurons throughout the cockroach nervous system and retrocerebral complex, in addition to its apparently homologous distribution in species of different insect orders, suggests an important role of this material in insect neuro(endocrine)-physiology.

Sensitive enzyme immunoassay for Manduca allatotropin and the existence of an allatotropin‐immunoreactive peptide in Periplaneta americana

AbstractAntisera to Manduca sexta allatoropin were raised in rabbits and were used to develop a competitive enzyme immunoassay for this neuropeptide. The detection limit of the assay is less than 2 fmol/well. A useful quantification can be obtained from 2 to 30 fmol/well. No cross‐reactivity was observed with several insect peptides, but the enzyme‐linked immunosorbent assay does recognize [Ala6, Leu7, Ser8]‐allatotropin, a myotropin recently isolated from Locusta migratoria. The assay was used to study the distribution of allatotropin within the nervous system of Manduca sexta. The peptide is present in the retrocerebral complex, the brain, and the ventral nerve cord of this species, in quantities of respectively 0.01, 1.2, and 1.7 pmol per insect. An allatotropin‐immunoreactive peptide was found in the nervous system of Periplaneta americana. It is present in the ventral nerve cord (3.3 pmol/insect), brain (1.9 pmol/insect), and retrocerebral complex (0.09 pmol/insect). These data suggest that peptides of this family are generally present in insects. © 1993 Wiley‐Liss, Inc.

Development of an in vitro assay for prothoracicotropic hormone of the gypsy moth, Lymantria dispar (L.) following studies on identification, titers and synthesis of ecdysteroids in last-instar females

Hemolymph ecdysteroid titers and in vitro prothoracic gland ecdysteroid synthesis have been examined in last-instar larval (5th instar) females of Lymantria dispar. Ecdysteroids were quantified by radioimmunoassay and characterized by co-elution with known standards of ecdysteroids on reverse-phase high-performance liquid chromatography. Analysis of hemolymph yielded ecdysone and 20-OH-ecdysone in ratios of 1:1 (day 6, shortly after attainment of maximum weight) and 1:28 (day 10, molting peak). Analysis of in vitro culture media from glands challenged with extracts of brains or retrocerebral complexes, or left unchallenged, revealed only immunoreactive material co-eluting with a known standard of ecdysone. Time-course studies of in vitro prothoracic gland ecdysone secretion demonstrated a major peak on day 10, 1–2 days prior to pupal ecdysis, and a small elevation on days 5–6. On days 5 and 6, 2.29±0.41 and 2.65±0.72 ng ecdysone per gland, respectively, were secreted in 6-h cultures. On day 10, 25.69±4.36 ng was secreted in 6-h culture. The ability of prothoracic glands of various ages to respond to brain extracts containing prothoracicotropic hormone activity was tested by determining an activation ratio for each day of the instar. The activation ratio was determined over a 90-min period by dividing the amount of ecdysone secreted by one member of a pair of prothoracic glands in the presence of brain extract by that of its contralateral control gland in Grace's medium. Prior to the addition of brain extract, the activity of the glands was allowed to subside to basal level for 180 min in Grace's medium. The activition ratio was highest on days 3–7 and fell throughout the remainder of the instar as the inherent ability of the prothoracic gland to maintain high levels of ecdysteroid synthesis in vitro in the absence of prothoracicotropic hormone increased. A two-phase in vitro assay for prothoracicotropic hormone was established using activition ratios. This assay showed saturable doseresponse kinetics for prothoracic gland ecdysone secretion and specificity to extracts prepared from brain or retrocerebral complexes. A comparable assay for prothoracicotropic hormone purification, based on net synthesis and requiring half the number of prothoracic glands was also established.

Kopec revisited—Neuroendocrine regulation of metamorphosis in the gypsy moth ( Lymantria dispar): Development of a larval in vivo assay for prothoracicotropic hormone

The role of the brain as a neuroendocrine regulator of metamorphosis in the gypsy moth, Lymantria dispar L. was originally demonstrated by Kopec more than 70 years ago. We have reexamined this role by determining haemolymph ecdysteroid titres, activity and responsiveness of the prothoracic glands, and growth of female last (i.e. fifth)-instar larvae in relation to secretion of the brain prothoracicotropic hormone (PTTH), the factor known to trigger moulting and metamorphosis in insects. The head critical period for secretion of PTTH in Lymantria reared at 25°C in a 16 h light-8 h dark cycle was shown by neck ligation to occur on day 7–8 of the fifth instar. The critical period occurred shortly after a small peak of haemolymph ecdysteroid that reached 600 pg 20-hydroxyecdysone equivalents/μl, as determined by radioimmunoassay. Feeding was completed by day 7, thus neck ligation did not affect nutrient intake. Animals starved from day 5 onward completed pupation at the same time as non-starved controls and showed normal increases in haemolymph ecdysteroid titres. Animals neck-ligated on day 7 and injected 5 days later with extracts of brain, retrocerebral complex, or brain tissues containing lateral or median neurosecretory cells were stimulated to pupate within 5 days, and brain-extract-injected animals showed an increase in haemolymph ecdysteroid titre of over 4000 pg/μl. Animals neck-ligated earlier had lower haemolymph ecdysteroid titres than day 7, neck-ligated larvae and their prothoracic glands showed lower activity in vitro. Using the in vivo assay, PTTH activity was detected in pre-hatch eggs and in brains taken from day-5 last-instar larvae, day-1 pupae and day-1 adults. The assay appears specific to tissues from the brain and retrocerebral complex, since no activity was found with extracts of the subesophageal ganglion, fat body or muscle.

The distribution of pheromone-biosynthesis-activating neuropeptide (PBAN) immunoreactivity in the central nervous system of the corn earworm moth, Helicoverpa zea

Production of sex pheromone in several species of moths has been shown to be under the control of a neuropeptide termed pheromone-biosynthesis-activating neuropeptide (PBAN). We have produced an antiserum to PBAN from Helicoverpa zea (Lepidoptera: Noctuidae) and used it to investigate the distribution of immunoreactive peptide in the brain-suboesophageal ganglion complex and its associated neurohemal structures, and the segmental ganglia of the ventral nerve cord. Immunocytochemical methods reveal three clusters of cells along the ventral midline in the suboesophageal ganglion (SOG), one cluster each in the presumptive mandibular (4 cells), maxillary (12–14 cells), and labial neuromeres (4 cells). The proximal neurites of these cells are similar in their dorsal and lateral patterns of projection, indicating a serial homology among the three clusters. Members of the mandibular and maxillary clusters have axons projecting into the maxillary nerve, while two additional pairs of axons from the maxillary cluster project into the ventral nerve cord. Members of the labial cluster project to the retrocerebral complex (corpora cardiaca and cephalic aorta) via the nervus corpus cardiaci III (NCC III). The axons projecting into the ventral nerve cord appear to arborize principally in the dorsolateral region of each segmental ganglion; the terminal abdominal ganglion is distinct in containing an additional ventromedial arborization in the posterior third of the ganglion. Quantification of the extractable immunoreactive peptide in the retrocerebral complex by ELISA indicates that PBAN is gradually depleted during the scotophase, then restored to maximal levels in the photophase. Taken together, our findings provide anatomical evidence for both neurohormonal release of PBAN as well as axonal transport via the ventral nerve cord to release sites within the segmental ganglia.