Pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster

The pyrokinin/pheromone biosynthesis activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including stimulation of pheromone biosynthesis in female moths, stimulation of muscle contraction, induction of embryonic diapause in Bombyx mori, and stimulation of melanization in some larval moths. Recently, this family of peptides has been implicated in accelerating the formation of the puparium in a dipteran. Using bioassay and immunocytochemical techniques, we demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster. Pheromonotropic activity was shown in the moths Helicoverpa zeaand Helicoverpa armigera by using dissected larval nervous systems and adult heads and bodies of D. melanogaster. Polyclonal antisera against the C-terminal ending of PBAN revealed the location of cell bodies and axons in the central nervous systems of larval and adult flies. Immunoreactive material was detected in at least three groups of neurons in the subesophageal ganglion of 3rd instar larvae, pupae, and adults. The ring gland of both larvae and adults contained immunoreactivity. Adult brain-subesophageal ganglion complex possessed additional neurons. The fused ventral ganglia of both larvae and adults contained three pairs of neurons that sent their axons to a neurohemal organ connected to the abdominal nervous system. These results indicate that the D. melanogasternervous system contains pyrokinin/PBAN-like peptides and that these peptides could be released into the hemolymph.     

PBAN/pyrokinin peptides in the central nervous system of the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

The pyrokinin/pheromone-biosynthesis-activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including the stimulation of pheromone biosynthesis in female moths, muscle contraction, induction of embryonic diapause, melanization, acceleration of puparium formation, and termination of pupal diapause. We have used immunocytochemical techniques to demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of the fire ant, Solenopsis invicta. Polyclonal antisera against the C-terminal end of PBAN have revealed the location of the peptide-producing cell bodies and axons in the central nervous system. Immunoreactive material is detectable in at least three groups of neurons in the subesophageal ganglion and corpora cardiaca of all adult sexual forms. The ventral nerve cord of adults consists of two segmented thoracic ganglia and four segmented abdominal ganglia. Two immunoreactive pairs of neurons are present in the thoracic ganglia, and three neuron pairs in each of the first three abdominal ganglia. The terminal abdominal ganglion has no immunoreactive neurons. PBAN immunoreactive material found in abdominal neurons appears to be projected to perisympathetic organs connected to the abdominal ganglia. These results indicate that the fire ant nervous system contains pyrokinin/PBAN-like peptides, and that these peptides are released into the hemolymph. In support of our immunocytochemical results, significant pheromonotropic activity is found in fire ant brain-subesophageal ganglion extracts from all adult fire ant forms (queens, female and male alates, and workers) when extracts are injected into decapitated females of Helicoverpa zea. This is the first demonstration of the presence of pyrokinin/PBAN-like peptides and pheromonotropic activity in an ant species. This research was supported in part by a US-Israel Binational Science Foundation Grant (no. 2003367).     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.     

CNS microstructure in the wandering wolf spider Arctosa kwangreungensis (Araneae: Lycosidae)

The supraesophageal ganglion of the wolf spider Arctosa kwangreungensis is made up of a protocerebral and tritocerebral ganglion, whereas the subesophageal ganglionic mass is composed of a single pair of pedipalpal ganglia, four pairs of appendage ganglia, and a fused mass of abdominal neuromeres. In the supraesophageal ganglion, complex neuropile masses are located in the protocerebrum which include optic ganglia, the mushroom bodies, and the central body. Characteristically, the only nerves arising from the protocerebrum are the optic nerves, and the neuropiles of the principal eyes are the most thick and abundant in this wandering spider. The central body which is recognized as an important association center is isolated at the posterior of the protocerebrum and appears as a complex of highly condensed neurons. These cells give off fine parallel bundles of axons arranged in the mushroom bodies. The subesophageal nerve mass can be divided into two main tracts on the basis of direction of the neuropiles. The dorsal tracts are contributed to from the motor or interneurons of each ganglion, whereas the ventral tracts are from incoming sensory axons.     

Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I

Summary An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electronmicroscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hindgut.     

Biochemical and cytoimmunological evidence for the control of Aedes aegypti larval trypsin with Aea-TMOF

Trypsin and chymotrypsin-like enzymes were detected in the gut of Aedes aegypti in the four larval instar and pupal developmental stages. Although overall the amount of trypsin synthesized in the larval gut was 2-fold higher than chymotrypsin, both enzymes are important in food digestion. Feeding Aea-Trypsin Modulating Oostatic Factor (TMOF) to Ae. aegypti and Culex quinquefasciatus larvae inhibited trypsin biosynthesis in the larval gut, stunted larval growth and development, and caused mortality. Aea-TMOF induced mortality in Ae. aegypti, Cx. quinquefasciatus, Culex nigripalpus, Anopheles quadrimaculatus, and Aedes taeniorhynchus larvae, indicating that many mosquito species have a TMOF-like hormone. The differences in potency of TMOF on different mosquito species suggest that analogues in other species are similar but may differ in amino acid sequence or are transported differently through the gut. Feeding of 29 different Aea-TMOF analogues to mosquito larvae indicated that full biological activity of the hormone is achieved with the tetrapeptide YDPA. Using cytoimmunochemical analysis, intrinsic TMOF was localized to ganglia of the central nervous system in larvae and male and female Ae. aegypti adults. The subesophageal, thoracic, and abdominal ganglia of both larval and adult mosquitoes contained immunoreactive cells. Immunoreactive cells were absent in the corpus cardiacum of newly molted 4th instar larvae but were found in late 4th instar larvae. In both males and females, the intrinsic neurosecretory cells of the corpus cardiacum were filled with densely stained immunoreactive material. These results indicate that TMOF-immunoreactive material is synthesized in sugar-fed male and female adults and larvae by the central nervous system cells.     

Leucokinin and diuretic hormone immunoreactivity of neurons in the tobacco hornworm,Manduca sexta,and co-localization of this immunoreactivity in lateral neurosecretory cells of abdominal ganglia

Because leucokinins stimulate diuresis in some insects, we wished to identify the neurosecretory cells in Manduca sexta that might be a source of leucokinin-like neurohormones. Immunostaining was done at various stages of development, using an antiserum to leucokinin IV. Bilateral pairs of neurosecretory cells in abdominal ganglia 3–7 of larvae and adults are immunoreactive; these cells project via the ipsilateral ventral nerves to the neurohemal transverse nerves. The immunoreactivity and size of these lateral cells greatly increases in the pharate adult, and this change appears to be related to a period of intensive diuresis occurring a few days before adult eclosion. Relationships of these neurons to cells that are immunoreactive to a M. sexta diuretic hormone were also investigated. Diuretic hormone and leucokinin immunoreactivity are co-localized in the lateral neurosecretory cells and their neurohemal projections. A median pair of leucokinin-immunoreactive, and a lateral pair of diuretic hormone-immunoreactive neurons in the larval terminal abdominal ganglion project to neurohemal release sites within the cryptonephridium. The immunoreactivity of these cells is lost as the cryptonephridium is eliminated during metamorphosis. This loss appears to be related to the change from the larval to adult pattern of diuresis.     

FMRFamide-like immunoreactivity in the isolated,in vivo cultured ventral ganglion of the fly,Sarcophaga bullata (Parker) (Diptera: Sarcophagidae)

The influence of peripheral connectivity on the survival and differentiation of Phe-Met-Arg-Phe-amide-like immunoreactive (FLI) neurons in the ventral ganglion (VG) of the fly Sarcophaga bullata (Diptera: Sarcophagidae) was examined. Isolated larval VG were cultured in vivo for 13 days. The ganglia had undergone metamorphosis and resembled in situ metamorphosed VG in morphology and in the number and location of FLI neurons. The 3 pairs of large thoracic FLI neurons survived and became translocated to the midventral position extending immunoreactive axons into the dorsal neuropil. The 5 pairs of small FLI neurons also appeared de novo in the abdominal ganglion. However, the dorsal neural sheath of the cultured VG was devoid of FMRFamide-like immunoreactivity that was so characteristic of adult VG, which suggests the importance of peripheral connectivity for the metamorphic modification of FLI neurons.     

DPKQDFMRFamide expression is similar in two distantly related Drosophila species

Drosophila melanogaster DPKQDFMRFamide was isolated and its expression reported. Distribution of DPKQDFMRFamide immunoreactivity is now described in Drosophila virilis. DPKQDFMRFamide antibody stained a cell in the subesophageal ganglion in embryo. DPKQDFMRFamide antibody stained cells in the superior protocerebrum, subesophageal ganglion, thoracic ganglia, and an abdominal ganglion in larva, pupa, and adult. DPKQDFMRFamide antibody stained an additional pair of cells in the optic lobe and a cell in the lateral protocerebrum in adult. Structure identity and similar distribution of DPKQDFMRFamide in D. virilis and D. melanogaster, two distantly related Drosophila species, suggests an important and conserved activity for the peptide.     

Immunocytochemical localization of an allatotropin in developmental stages of Heliothis virescens and Apis mellifera

Juvenile hormone biosynthesis by the corpora allata is regulated by stimulatory neuropeptides called allatotropins and inhibitory neuropeptides called allatostatins. This study localized Manduca sexta allatotropin-like material in developmental stages of the noctuid moth Heliothis virescens and the honeybee Apis mellifera. Immunocytochemical methods using both fluorescence-tagged antibodies and enzyme-coupled antibodies were used to stain the central nervous tissue of both species. H. virescens contains M. sexta allatotropin (Manse-AT)-like material consistently throughout larval development. The distribution patterns of Manse-AT immunoreactive cell bodies in the CNS persisted from one larval instar to the next. It will be discussed how larval Manse-AT distribution patterns differed from those in adults. The total number of AT-containing cells in brain and subesophageal ganglion gradually increased during larval development, whereas in the thoracic and abdominal ganglia, the number of AT-containing neurons remained constant. In the honeybee A. mellifera, Manse-AT immunoreactive cells were only found in a few brains from late last instar larvae (prepupae). Manse-AT-like material was present in a group of 6-8 cells in the pars intercerebralis. However, we did not find any Manse-AT-like material in brains of early last instar larvae, whose corpora allata (CA) are more sensitive to in vitro stimulation by Manse-AT than prepupal CA.     

Distribution and characterization of Corazonin in the central nervous system of Triatoma infestans (Insecta: Heteroptera)

The distribution of corazonin in the central nervous system of the heteropteran insect Triatoma infestans was studied by immunohistochemistry. The presence of corazonin isoforms was investigated using MALDI-TOF mass spectrometry in samples containing the brain, the subesophageal ganglion, the corpora cardiaca-corpus allatum complex and the anterior part of the aorta. Several groups of immunopositive perikarya were detected in the brain, the subesophageal ganglion and the thoracic ganglia. Regarding the brain, three clusters were observed in the protocerebrum. One of these clusters was formed by somata located near the entrance of the ocellar nerves whose fibers supplied the aorta and the corpora cardiaca. The remaining groups of the protocerebrum were located in the lateral soma cortex and at the boundary of the protocerebrum with the optic lobe. The optic lobe housed immunoreactive somata in the medial soma layer of the lobula and at the level of the first optic chiasma. The neuropils of the deutocerebrum and the tritocerebrum were immunostained, but no immunoreactive perikarya were detected. In the subesophageal ganglion, immunostained somata were found in the soma layers of the mandibular and labial neuromeres, whereas in the mesothoracic ganglionic mass, they were observed in the mesothoracic, metathoracic and abdominal neuromeres. Immunostained neurites were also found in the esophageal wall. The distribution pattern of corazonin like immunoreactivity in the central nervous system of this species suggests that corazonin may act as a neurohormone. Mass spectrometric analysis revealed that [Arg⁷]-corazonin was the only isoform of the neuropeptide present in T. infestans tissue samples.     

Localization of allatostatin-immunoreactive material in the central nervous system,stomatogastric nervous system,and gut of the cockroach Blattella germanica

Immunoreactivity against peptides of the allatostatin family having a typical YXFGL-NH₂ C-terminus has been localized in different areas of the central nervous system, stomatogastric nervous system and gut of the cockroach Blattella germanica. In the protocerebrum, the most characteristic immunoreactive perikarya are situated in the lateral and median neurosecretory cell groups. Immunoreactive median neurosecretory cells send their axons around the circumesophageal connectives to form arborizations in the anterior neuropil of the tritocerebrum. A group of cells in the lateral aspect of the tritocerebrum project to the antennal lobes in the deutocerebrum, where immunoreactive arborizations can be seen in the periphery of individual glomeruli. Nerve terminals were shown in the corpora allata. These terminals come from perikarya situated in the lateral neurosecretory cells in the pars lateralis and in the subesophageal ganglion. Immunoreactive axons from median neurosecretory cells and from cells positioned in the anteriormost part of the tritocerebrum enter together in the stomatogastric nervous system and innervate foregut and midgut, especially the crop and the valve between the crop and the midgut. The hindgut is innervated by neurons whose perikarya are located in the last abdominal ganglion. Besides immunoreactivity in neurons, allatostatin-immunoreactive material is present in endocrine cells distributed within the whole midgut epithelium. Possible functions for these peptides according to their localization are discussed. Arch. Insect Biochem. Physiol. 37:269–282, 1998. © 1998 Wiley-Liss, Inc.     

Crustacean cardioactive peptide-immunoreactive neurons innervating brain neuropils,retrocerebral complex and stomatogastric nervous system of the locust,Locusta migratoria

The distribution and morphology of crustacean cardioactive peptide-immunoreactive neurons in the brain of the locust Locusta migratoria has been determined. Of more than 500 immunoreactive neurons in total, about 380 are interneurons in the optic lobes. These neurons invade several layers of the medulla and distal parts of the lobula. In addition, a small group of neurons projects into the accessory medulla, the lamina, and to several areas in the median protocerebrum. In the midbrain, 12 groups or individual neurons have been reconstructed. Four groups innervate areas of the superior lateral and ventral lateral protocerebrum and the lateral horn. Two cell groups have bilateral arborizations anterior and posterior to the central body or in the superior median protocerebrum. Ramifications in subunits of the central body and in the lateral and the median accessory lobes arise from four additional cell groups. Two local interneurons innervate the antennal lobe. A tritocerebral cell projects contralaterally into the frontal ganglion and appears to give rise to fibers in the recurrent nerve, and in the hypocerebral and ingluvial ganglia. Varicose fibers in the nervi corporis cardiaci III and the corpora cardiaca, and terminals on pharyngeal dilator muscles arise from two subesophageal neurons. Some of the locust neurons closely resemble immunopositive neurons in a beetle and a moth. Our results suggest that the peptide may be (1) a modulatory substance produced by many brain interneurons, and (2) a neurohormone released from subesophageal neurosecretory cells.     

Localization of an insulin-like peptide in brains of two flies

A nucleotide sequence (GenBank AI134194) was identified in the database of the Berkeley Drosophila Genome Project based on the similarity of its translated sequence to insulin-like peptides known for other insects. The putative A chain of the Drosophila peptide was synthesized and used to produce an antiserum for immunocytochemistry. Medial neurosecretory cells and their axons were immunostained in whole brains of Drosophila larvae and adults. In larvae, immunostained axons from these cells extended to the corpus cardiacum in the ring gland and the hypocerebral ganglion and along the aorta in the head. In adults, axons from these cells extended along the aorta to the corpus cardiacum-hypocerebral ganglion complex near the cardiac valve and branched along the midgut and crop. In contrast, two clusters of lateral neurosecretory cells and their axons were immunostained weakly in brains of female mosquitoes, Aedes aegypti. No other cells were stained in the nervous systems or midguts of Drosophila larvae and adults or Ae. aegypti females. These specific localizations substantiate the existence of an endogenous insulin-like peptide in Drosophila and suggest that a similar peptide is present in the distantly related mosquitoes.     

Immunohistochemical localization of gastrin-cholecystokinin-like material in the central nervous system of the migratory locust

Brain, corpora cardiaca (CC)-corpora allata (CA) complex, suboesophageal ganglion, thoracic and abdominal ganglia of adults, larvae and embryos of Locusta migratoria have been immunohistochemically screened for gastrin cholecystokinin (CCK-8(s]-like material. In adult, numerous immunoreactive neurons and nerve fibres are located, with a marked symmetry, in various parts of the brain and throughout the ventral nerve cord. In the median part of the brain, cell bodies belonging neither to cellular type A1 nor A2 (following Victoria blue-paraldehyde fuchsin staining) are immunopositive; their processes terminate in the upper protocerebral neuropile. In lateral parts of the brain, external cell bodies send axons into CC and some up to CA, other internal have processes which terminate in the neuropile of the brain. Two of these latter cells react also with methionine-enkephalin antiserum. In the ventral nerve cord, in addition to numerous perikarya, immunoreactive arborizations terminate in the neuropile or in close association with the sheath, at the dorsal part of all ganglia. This CCK-8(s) distribution pattern is observed only at the two last larval instars, but is precociously detected in the abdominal nerve cord of embryos, one day before hatching.     

FMRfamide-like immunoreactivity in the ventral nerve cord of the larval eastern spruce budworm,Choristoneura fumiferana (clemens) (Lepidoptera : Tortricidae)

Distribution of FMRFamide-like immunoreactivity was examined in the larval ventral nerve cord of the eastern spruce budworm, Choristoneura fumiferana (Lepidoptera : Tortricidae). Indirect immunofluorescent methods revealed the existence of 3 groups of FLI neurons in each ganglion. The neurons are distributed in a bilaterally symmetrical fashion at the anterodorsal, midlateral and posteroventral regions of the ganglia. There are 4 FMRFamide-like immunoreactive fiber tracts on the dorsal surface of the ganglia to which the anterodorsal FLI neurons project ipsilaterally, while the midlateral pair projects both ipsi-, and contralaterally. The last abdominal ganglion (AG8) has 4 additional pairs of FLI neurons; and axons from some of these extend into the median abdominal nerve, which suggests some role for this neuropeptide in the control of posterior structures of the larva.     

The identification and distribution of progesterone receptors in the brain and thoracic ganglion in the mud crab Scylla paramamosain (Crustacea: Decapoda: Brachyura)

The existence of progesterone receptors (PR) in the Scylla paramamosain (mud crab) was studied using immunological techniques. By Western blotting, PR with an apparent molecular weight of 70 kDa is identified in both the brain and the thoracic ganglion. By immunohistochemistry, PR immunoreactive neurons are detected mainly in the protocerebrum, the subesophageal ganglion and the leg ganglion. PR immunoreactivity is localized mainly in the nuclei of these neurons, while only a few neurons show such activities in their cytoplasm. Our results provide evidence that progesterone modulates the neuroendocrine system mainly via nucleus receptors.     

Serotonin immunoreactivity of neurons in the gastropod Aplysia californica

Serotonergic neurons and axons were mapped in the central ganglia of Aplysia californica using antiserotonin antibody on intact ganglia and on serial sections. Immunoreactive axons and processes were present in all ganglia and nerves, and distinct somata were detected in all ganglia except the buccal and pleural ganglia. The cells stained included known serotonergic neurons: the giant cerebral neurons and the RB cells of the abdominal ganglion. The area of the abdominal ganglion where interneurons are located which produce facilitation during the gill withdrawal reflex was carefully examined for antiserotonin immunoreactive neurons. None were found, but two bilaterally symmetric pairs of immunoreactive axons were identified which descend from the contralateral cerebral or pedal ganglion to abdominal ganglion. Because of the continuous proximity of this pair of axons, they could be recognized and traced into the abdominal ganglion neuropil in each preparation. If serotonin is a facilitating transmitter in the abdominal ganglion, these and other antiserotonin immunoreactive axons in the pleuroabdominal connectives may be implicated in this facilitation.