Metamorphic changes in the neurosecretory cells of Diacrisia obliqua Walker (Lepidoptera: Arctiidae)

4 PF-positive NSC appear for the first time in each brain hemisphere on 4th day of exclosion. B-, C- and D-cells appear in the pars intercerebralis region on the second day in the 2nd instar. 3 lateral NSC appear for the first time in this instar. 2 A4-cells appear in the tritocerebral region of the 4th instar. An increase in the number and size of the NSC is noted in the successive instars. A3-cells appear for the first time in 5th instar. The secretory phases in the pupal period are completed in three "stages" which last for 3.3 and 4 days respectively. Adults lack PF-positive cells in the lateral and tritocerebral regions. Females show accumulation of NSM before mating which depletes gradually as the mating time reaches.     

Neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae

Anatomical study of neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae, was done by NiCl2 filling and immunocytochemistry. Retrograde filling through the cardiac-recurrent nerve labeled three groups of neurons in the brain/subesophageal ganglion: (1) paramedial clusters of the pars intercerebralis, (2) neurons in each pars lateralis, and (3) neurons in the subesophageal ganglion. The pars intercerebralis neurons send prominent axons into the median bundle and exit from the brain via the contralateral nervus corporis cardiaci. Based on the projection pattern, two types of the pars lateralis neurons can be distinguished: the most lateral pairs of neurons contralaterally extend through the posterior lateral tract and the remainder ipsilaterally extend through the posterior lateral tract. The neurons in the subesophageal ganglion run through the contralateral nervus corporis cardiaci. The dendritic arborization of the pars intercerebralis and pars lateralis neurons is restricted to the superior protocerebral neuropil and to the anterior neuropil of the subesophageal ganglion where the neurons in the subesophageal ganglion also project. Retrograde filling from the corpus allatum indicated that the pars lateralis neurons and a few pars intercerebralis neurons project to the corpus allatum, but that the neurons in the subesophageal ganglion do not. Orthograde filling from the pars intercerebralis and staining by paraldehyde-thionin/paraldehyde-fuchsin indicated that the pars intercerebralis neurons project primarily to the corpus cardiacum/hypocerebral ganglion complex. Immunostaining with a polyclonal antiserum against diapause hormone, a member of the FXPRLamide family, suggests that some of the subesophageal ganglion neurons contain FXPRLamide-like peptides.     

Embryonic development of the pars intercerebralis/central complex of the grasshopper

 We have studied the embryonic development of the pars intercerebralis/central complex in the brain of the grasshopper using immunocytochemical and histochemical techniques. Expression of the cell-surface antigen lachesin reveals that the neuroblasts of the pars intercerebralis first differentiate from the neuroectoderm at around 26% of embryogenesis. Differentiation of medial and lateral neuroblasts occurs first. By the 28% stage a more or less uniform sheet of 20 neuroblasts has formed. As a result of both cell proliferation and cell translocation, the pars intercerebralis proliferative cluster in each hemisphere expands so that at 30% the most medial neuroblasts lie apposed at the midline. We followed the further development of the pars intercerebralis of each brain hemisphere using bromo-deoxy-uridine incorporation and osmium-ethyl-gallate staining. Within the pars intercerebralis itself, the neuroblasts redistribute into discrete subsets. The neuroblasts of each subset generate clusters of progeny which extend in a stereotypic, subset-specific direction in the brain. We have used this feature to identify one subset of four neuroblasts as being the likely progenitor cells for four clusters of embryonic neurons (W, X, Y, Z) which develop at around 55% of embryogenesis. We show that these progeny project axons via four discrete fascicles (w, x, y, z) into the embryonic central complex. At the single cell level, Golgi impregnation reveals that the axons from these neighbouring cell clusters remain discrete, and those from the same cluster tightly fasciculated, as they project into the central complex, consistent with a modular organization for this brain region. Received: 16 June 1997 / Accepted: 25 June 1997     

Rôle of eyes,optic lobes,and pars intercerebralis in locomotory and stridulatory circadian rhythms of Teleogryllus commodus

Locomotory and stridulatory activity rhythms of male crickets (Teleogryllus commodus) were assayed simultaneously following various experimental procedures. These included (a) severing the pathways between the ocelli and the brain, between the ommatidia and the optic lobes, or between the optic lobes and the brain, and (b) RF cauterization of the pars intercerebralis. The results indicate that (1) light-dark cycles which entrain both rhythms are perceived by the compound eyes and not by the ocelli; (2) loss of connexion between the brain and the optic lobes leads to arrhythmicity in both behaviours, but a single optic lobe can maintain rhythmicity; (3) absence of neurosecretory cells in the pars intercerebralis is correlated with loss of stridulatory activity and arrhythmicity in locomotory behaviour. It is suggested that the pars intercerebralis serves as a site of coupling between a circadian pacemaker and various overt behaviours. However, intermediate control by the pars intercerebralis is assumed to be exerted via channels that can be either hormonal or purely neuronal in nature.     

Simplified staining in situ of neurosecretory cells of the supraoesophageal ganglion of the female Culex pipiens molestus (Forskal) (Diptera: Culicidae)

The author describes the morphology and distribution of the neurosecretory cells in the supraoesophageal ganglion of the adult female Culex pipiens molestus, using paraldehyde fuchsin and paraldehyde thionine-paraldehyde fuchsin as vital staining techniques. The brain of Culex pipiens molestus has three regions (the proto-, deuto- and tritocerebrum) in which principally two types of neurosecretory cells (A and B) can be detected. Both A (dark) and B (light) cells are to be found in the protocerebrum, where they are termed medial neurosecretory cells, as they are distributed in the pars intercerebralis and only a few occur more laterally. A small group of type A and B neurosecretory cells is to be found in the posterior part of the brain (the tritocerebrum). These cells display characteristics differences in their tinctorial affinity.     

Comparison between pars intercerebralis neurosecretory cells of Glossina and those of other higher Diptera

Three types of the A-neurosecretory cells (NSCs) were found in the pars intercerebralis (PI) of the adult brain of four species of tsetse flies (Glossina palpalis, G. pallidipes, G. austeni and G. morsitans). The typical formula of their composition is: 8 A1-, 14 A2- and 4 A3-cells. After permanganate oxidation, the neurosecretory materials (NSMs) of Al- and A3-cells are rich in strong acid groups. The A2-NSM contains both strong and weak acid groups after this treatment; besides, it retains a weak affinity to acid dyes. In addition to the A-cells, the PI of Glossina species seems to include a number of NSCs of the type B.
The data obtained indicate a great similarity of the NSC composition in the PI of Glossina and other related higher dipterans.
In addition to the NSCs, giant neurons and particular cells with large cytoplasmic inclusions were also found in the PI of Glossina. The cells with large cytoplasmic inclusions correspond apparently to vacuolated cells previously observed in the brain of some flies. It is suggested that the considerable complication in reproductive biology which took place in tsetse flies had no effect on the composition of the NSCs in their PI when compared to that in related species with more ordinary cycles of reproduction.     

Effects of ablation of the pars intercerebralis on ecdysteroid quantities and yolk protein expression in the blowfly Protophormia terraenovae

Quantities of ecdysteroid are compared in the haemolymph and ovaries of the blowfly Protophormia terraenovae Robineau‐Desvoidy (Diptera: Calliphoridae) under reproductive (LD 18 : 6 h at 25 °C) and diapause (LD 12 : 12 h at 20 °C) conditions. The effects of ablation of the pars intercerebralis or ovaries on ecdysteroid quantities and of ablation of the pars intercerebralis on yolk protein expression are examined. Under reproductive conditions, the levels of ecdysteroid in vitellogenic females are high, although the levels in previtellogenic females and females with mature ovaries are low. Under diapause conditions, there are low quantities of ecdysteroid in both the haemolymph and ovaries. Ecdysteroid titres in the haemolymph are not significantly affected by the removal of the ovaries, suggesting that tissues other than the ovaries are also involved in the production of ecdysteroids. Reproductive females in which the pars intercerebralis of the brain is experimentally ablated have ecdysteroid levels that are not significantly different from sham‐operated or intact females. However, yolk protein expression in the fat body is suppressed after removal of the pars intercerebralis. These results suggest that the suppression of ecdysteroid levels in the haemolymph and ovaries is associated with reproductive diapause, and that the pars intercerebralis could play a role in yolk protein synthesis without mediating ecdysteroid production.     

Corpus allatum regulation during the metamorphosis of Periplaneta americana: Axon pathways

The anatomy of the retrocerebral complex was studied after supravital staining with methylene blue, and axonal tracts within the corpora allata (CA) were traced after applying the CoCl₂ technique together with Timm's sulfide-silver enhancement. Cobalt chloride fills of the nerves to and from the CA revealed two major sources of innervation: the brain and the subesophageal ganglion. Three cell clusters in the brain contribute axons that reach each nervus corporis allati I (NCA I) and, apparently, pass to or beyond the CA. These are: a cluster of 8 to 12 cells in the contralateral pars lateralis, a cluster of 16 to 20 cells in the ipsilateral pars lateralis, and a cluster of 50 to 60 cells in the contralateral pars intercerebralis. PAF-stained sections of other brains revealed a corresponding number of PAF-positive cells in these same regions. The medial and lateral neurons arborize in the neuropile adjacent to the pars intercerebralis, and may associate there. The lateral group also arborizes extensively in the neuropile surrounding the pedunculus of the mushroom body. At least four cell bodies located antero-ventrad in the subesophageal ganglion send axons to the CA via each nervus corporis allati II (NCA II).To determine possible inhibitory pathways to the CA, the NCA I, NCA II, and postallatal nerves of last instar larvae were severed; either singly, or in combination. Additional experiments were performed on last instar larvae to substantiate that superlarvae were a direct result of an enhanced or sustained juvenile hormone titre. These experiments included: implanting two or more CA, extirpating one CA, or applying 100 μg of Altosid topically onto allatectomized larvae. The experiments indicated that only NCA I is an inhibitory pathway and that superlarvae were a direct consequence of CA activation. NCA II does not seem to provide the CA with an essential excitatory innervation; when it and NCA I are severed a supernumerary apolysis will still result. Some of the cells in the brain stainable by the CoCl₂ method are most probably identical to those that are PAF-positive. These cells may inhibit the CA in last instar larvae via neurosecretomotor junctions.     

Rupture de la diapause ovarienne d'Anacridium aegyptium par stimulation électrique des cellules neurosécrétrices médianes de la pars intercerebralis

Oögenesis and the physiological activity of the corpora allata were studied in adult females of the Egyptian locust (Anacridium aegyptium), in ovarian diapause, after electrical stimulation in vivo of the pars intercerebralis. This stimulation provokes (1) a decrease in the quantity of fuchsinophilic material present in the median neurosecretory cell bodies and in the internal cardiac tract, (2) an increase in the physiological activity of the corpora allata (measured by its chromatropic effect on larvae of Locusta), and (3) rupture of the ovarian diapause (advance of maturation of the oöcytes and oviposition by 5 months, and initiation of the ovarian cycle).In the control animals, the same electrical stimulations of various regions of the central nervous system (tritocerebrum, first ganglion of the abdominal cord) have no effect on these phenomena.In allatectomized females, electrical stimulations of the pars intercerebralis are followed by a slight growth of oöcytes, without a deposit of yellow vitellus. The diapause is not broken. Section of the allatocardiac nerves or rupture of the allatocardiac and allato-suboesophageal nervous connexions do not change the physiological state of the corpora allata. In the case of females in which the corpora allata have been disconnected, electrical stimulations of the pars intercerebralis succeed in activating the corpora allata and breaking the ovarian diapause. The aggregate of these results confirms that in locusts the control of the brain over the physiological activity of the corpora allata is above all neuroendocrine.     

A comparative study of leucokinin-immunoreactive neurons in insects

Antisera were raised against leucokinin IV, a member of the leucokinin peptide family. Immunohistochemical localization of leucokinin immunoreactivity in the brain of the cockroach Nauphoeta cinerea revealed neurosecretory cells in the pars intercerebralis and pars lateralis, several bilateral pairs of interneurons in the protocerebrum, and a group of interneurons in the optic lobe. Several immunoreactive interneurons were found in the thoracic ganglia, while the abdominal ganglia contained prominent immunoreactive neurosecretory cells, which projected to the lateral cardiac nerve. The presence of leucokinins in the abdominal nerve cord was confirmed by HPLC combined with ELISA. Leucokinin-immunoreactive neurosecretory cells were also found in the pars intercerebralis of the cricket Acheta domesticus and the mosquito Aedes aegypti, but not in the locust Schistocerca americana or the honey bee Apis mellifera. However, all these species have leucokinin-immunoreactive neurosecretory cells in the abdominal ganglia. The neurohemal organs innervated by abdominal leucokinin-immunoreactive cells were different in each species.     

Studies on the neuroendocrine system of the mangohopper, Idiocerus atkinsoni Leth. (Homoptera : Jassidae)

The neuroendocrine system of the homopteran, Idiocerus atkinsoni has been described, employing a neurosecretory stain. Two groups of medial neurosecretory cells (NSC) of one tinctorial type are present in the pars intercerebralis of the brain. Processes believed to be dendrites of the neurosecretory neurons lie superficially underneath the neurilemma and enclose neurosecretory material (NSM). Both the nervi corporis cardiaci, NCCI and NCCII, are branched. The branches of the former join to form an oesophageal nerve that runs on the oesophageal surface and terminates on the midgut, and those of the latter, innervate the oesophageal dilator muscles. Besides being present in the dendrite-like processes and NSC, the NSM is also seen in the NCCI, anterior part of the aorta and oesophageal nerve but not in the NCCII, corpora cardiaca (CC) and the corpus allatum (CA). It is suggested that the release of NSM into the circulation in this insect occurs through two main routes: the dendrites and the aorta. The evolution of the aorta as an exclusive neurohaemal organ in Hemiptera is discussed.     

Distribution of circadian clock-related proteins in the cephalic nervous system of the silkworm, Bombyx mori

In the circadian timing systems, input pathways transmit information on the diurnal environmental changes to a core oscillator that generates signals relayed to the body periphery by output pathways. Cryptochrome (CRY) protein participates in the light perception; period (PER), Cycle (CYC), and Doubletime (DBT) proteins drive the core oscillator; and arylalkylamines are crucial for the clock output in vertebrates. Using antibodies to CRY, PER, CYC, DBT, and arylalkylamine N-acetyltransferase (aaNAT), the authors examined neuronal architecture of the circadian system in the cephalic ganglia of adult silkworms. The antibodies reacted in the cytoplasm, never in the nuclei, of specific neurons. A cluster of 4 large Ia(1) neurons in each dorsolateral protocerebrum, a pair of cells in the frontal ganglion, and nerve fibers in the corpora cardiaca and corpora allata were stained with all antibodies. The intensity of PER staining in the Ia(1) cells and in 2 to 4 adjacent small cells oscillated, being maximal late in subjective day and minimal in early night. No other oscillations were detected in any cell and with any antibody. Six small cells in close vicinity to the Ia(1) neurons coexpressed CYC-like and DBT-like, and 4 to 5 of them also coexpressed aaNATlike immunoreactivity; the PER- and CRY-like antigens were each present in separate groups of 4 cells. The CYC- and aaNAT-like antigens were further colocalized in small groups of neurons in the pars intercerebralis, at the venter of the optic tract, and in the subesophageal ganglion. Remaining antibodies reacted with similarly positioned cells in the pars intercerebralis, and the DBT antibody also reacted with the cells in the subesophageal ganglion, but antigen colocalizations were not proven. The results imply that key components of the silkworm circadian system reside in the Ia(1) neurons and that additional, hierarchically arranged oscillators contribute to overt pacemaking. The retrocerebral neurohemal organs seem to serve as outlets transmitting central neural oscillations to the hemolymph. The frontal ganglion may play an autonomous function in circadian regulations. The colocalization of aaNAT- and CYC-like antigens suggests that the enzyme is functionally linked to CYC as in vertebrates and that arylalkylamines are involved in the insect output pathway.     

Characterization of a new neurosecretory cell type containing gastrin-cholecystokinin-like peptide in the locust pars intercerebralis: ultrastructural and immunocytochemical studies,in situ and in vitro

A new neurosecretory cell type of the locust pars intercerebralis, immunolabelled with an antiserum against a vertebrate peptide related to gastrin-cholecystokinin (CCK-8(s)), was characterized both in situ and in primary cell cultures. Semithin sections of pars intercerebralis were first immunostained in order to identify neurosecretory cells containing CCK-like material and then examined by electron microscopy. The neurosecretory cells containing CCK-like material were paraldehyde fuchsin negative and were unequivocally identified in ultrathin sections adjacent to immunostained semithin sections. They exhibited neurosecretory vesicles of variable electron density, ranging in diameter from 150 to 250 nm. Immunogold labelled ultrathin sections adjacent to unlabelled ultrathin sections allowed for the unambiguous localization of CCK-like immunoreactive material over the neurosecretory vesicles of the cells containing CCK-like material. Immunoreactivity towards CCK-8(s)-like peptide could also be detected in pars intercerebralis neurosecretory neurons grown in vitro. The CCK-like positive neurons showed a multipolar morphology with fine processes radiating from the cell body. The positive cells had the same ultrastructural characteristics as the in situ CCK-like neurons. The pattern of neurite outgrowth on reactive CCK-like neurosecretory cells in vitro and the neuroanatomical pathway of the CCK-like immunoreactive neurosecretory cells in situ could be correlated. On the basis of their number, size and localization in the locust pars intercerebralis, it is possible that the CCK-like neurosecretory cells correspond to neurosecretory cell type C, which has not, to date, been identified at the ultrastructural level.     

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.     

Circadian activity rhythms in cockroaches. 3. The role of endocrine and neural factors

The control of circadian activity rhythms (diurnal rhythms) in insects has been suggested to result by periodic neuroendocrine secretions. More specifically, Harker ('56) claimed that the locomotor rhythm in the cockroach, Periplaneta americana, is timed by a secretory “clock” located in the subesophageal ganglion. Later experiments by Harker indicated that this “clock” function failed unless the retrocerebral organs were left intact; allatectomy was said (no evidence given) to abolish a rhythm. The procedure for demonstrating a “clock” function in the ganglion involved transplanting it from a rhythmic donor into the hemocoel of an arrhythmic host and observing that the host subsequently became rhythmic. This result (without explicit information about the phase of the rhythm) does not warrant the conclusion that the ganglion acts as a clock. Therefore, I have attempted to confirm and extend these important results. Employing techniques essentially identical to Harker's, and using the same species of roach, I have been unable to find any evidence to support the original claim: (1) in 20 test animals, implantation of ganglia from rhythmic donors failed to re-instate a rhythm, and (2) allatectomy (22 cases) or removal of the entire retrocerebral complex (20 cases) did not interfere with the rhythm. The results of another series of experiments show that the cockroach brain is involved in the control of the activity rhythm. When the brain is surgically bisected (mid-sagittal) through the pars intercerebralis, arrhythmic activity patterns are immediately evoked. These continue for many weeks, but in a few cases rhythms ultimately “regenerate”.     

Characterization of the dCaMKII-GAL4 driver line whose expression is controlled by the <Emphasis Type="Italic">Drosophila</Emphasis> Ca<Superscript>2+</Superscript>/calmodulin-dependent protein kinase II promoter

Transgenic flies that can drive GAL4 expression under the control of the 7 kb 5'-region of the Drosophila Ca(2+)/calmodulin-dependent protein kinase II (dCaMKII) gene (dCaMKII-GAL4) were established. Characteristic features of this dCaMKII-GAL4 driven reporter expression were compatible with the endogenous dCaMKII expression pattern: The dCaMKII-GAL4 driven reporter gene was expressed preferentially in the central nervous system of the embryo and larvae. Reporter expression was also observed in the brain, thoracic ganglion, and gut of the adult. The whole-brain distribution and projections of dCaMKII-GAL4-expressing cells in the adults were visualized three-dimensionally by using UAS-linked reporter genes. Prominent signals of nuclear-localized beta-Gal reporter gene expression were found in extensive brain regions, especially in the Kenyon cells of the mushroom body (MB), cells in the pars intercerebralis, and subesophageal ganglion (SOG). tau reporter gene expression highlighting neurite projections was detected in the MB lobes, median bundle, antennal lobe glomeruli, and fibers of clusters in the SOG, ventrolateral protocerebrum and superior lateral protocerebrum. These observations agree with those of a previous study mapping the dCaMKII-dependent memory circuits in courtship conditioning. Interestingly, green fluorescent protein reporter gene expression in adult MB lobes was predominantly observed in the alpha and beta lobes with a core-deficient pattern, but not in the alpha' and beta' lobes, similar to Fasciclin II immunoreactivity.     

Proctolin in the brain and ganglia of Triatoma infestans (Hemiptera: Reduviidae)

The distribution of proctolin in the central nervous system of the hemipteran bug, Triatoma infestans, was studied by immunohistochemistry using the sensitive avidin‐biotin technique combined with nickel salt intensification of the reaction product. Proctolin was present in cells and fibers of the brain and ganglia. In the brain, protocerebral proctolin‐immunoreactive cell bodies were found in the pars intercerebralis, the optic lobes, and the lateral soma rind. The deutocerebrum showed positive somata in relation to the antennal motor center and the tritocerebrum had intense immunoreactive fibers but few positive cells. Proctolin‐immunoreactive cell bodies of different sizes were observed in the subesophageal ganglion. Large cell bodies were found mainly rostrally and beaded positive processes were present around the ventral border of the esophageal foramen and in the rostrolateral neuropil of this ganglion. Small‐ to medium‐sized positive somata were found in the posterior part of the prothoracic ganglion; some of these cells were sending immunoreactive processes to the central neuropil. The meso‐metathoracic‐abdominal ganglionic mass showed positive cells in all the neuromeres, where some of them were large and had thick immunoreactive granules. The results show that the labeling pattern of proctolin‐like immunoreactivity in Triatoma i. appears to be widespread and unique for its central nervous system. It is suggested that proctolin may serve neuroendocrine, integrative, and motor functions in the brain of T. infestans. J. Morphol. 240:39–47, 1999. © 1999 Wiley‐Liss, Inc.     

Morphology of identified neurosecretory and non-neurosecretory cells in the cockroach pars intercerebralis

Electrophysiologically identified cells of the cockroach pars intercerebralis (Periplaneta americana) were injected with the dye Lucifer Yellow for morphological examination and with horseradish peroxidase for ultrastructural marking. In addition to this, uninjected cells were also studied to elaborate the findings from the injected material. The two electrophysiologically distinct classes of cells (type I and type II) correspond to two distinct morphological and ultrastructural classes. Type I cells are the medial neurosecretory cells of the pars intercerebralis, which project their axons to the retrocerebral neuro-hemal complex. Their cell bodies have a mean diameter of 17 microns, and they contain neurosecretory granules 200 nm in diameter. Arborizations emanate from the axon in the anterior part of the protocerebral neuropil. The type II cell bodies are larger (38 microns in diameter). Their axons project into the contralateral circumesophageal connective. These cells were usually multipolar, having somatic arborizations in the anterior portocerebral neuropil. The cell bodies contain vesicles 40 nm in diameter, numerous trophospongia, and a multi-layered glial envelope.     

Immunoreactivities to three circadian clock proteins in two ground crickets suggest interspecific diversity of the circadian clock structure

The closely related crickets Dianemobius nigrofasciatus and Allonemobius allardi exhibit similar circadian rhythms and photoperiodic responses, suggesting that they possess similar circadian and seasonal clocks. To verify this assumption, antisera to Period (PER), Doubletime (DBT), and Cryptochrome (CRY) were used to visualize circadian clock neurons in the cephalic ganglia. Immunoreactivities referred to as PER-ir, DBT-ir, and CRY-ir were distributed mainly in the optic lobes (OL), pars intercerebralis (PI), dorsolateral protocerebrum, and the subesophageal ganglion (SOG). A system of immunoreactive cells in the OL dominates in D. nigrofasciatus, while immunoreactivities in the PI and SOG prevail in A. allardi. Each OL of D. nigrofasciatus contains 3 groups of cells that coexpress PER-ir and DBT-ir and send processes over the frontal medulla face to the inner lamina surface, suggesting functional linkage to the compound eye. Only 2 pairs of PER-ir cells (no DBT-ir) were found in the OL of A. allardi. Several groups of PER-ir cells occur in the brain of both species. The PI also contains DBT-ir and CRY-ir cells, but in A. allardi, most of the DBT-ir is confined to the SOG. Most immunoreactive cells in the PI and in the dorsolateral brain send their fibers to the contralateral corpora cardiaca and corpora allata. The proximity and, in some cases, proven identity of the PER-ir, DBT-ir, and CRY-ir perikarya are consistent with presumed interactions between the examined clock components. The antigens were always found in the cytoplasm, and no diurnal oscillations in their amounts were detected. The photoperiod, which controls embryonic diapause, the rate of larval development, and the wing length of crickets, had no discernible effect on either distribution or the intensity of the immunostaining.     

Evidence for a crustacean hyperglycemic hormone-like molecule in the nervous system of the stick insect,Carausius morosus

Summary Histological sections of the brain, suboesophageal ganglion, and the corpora cardiaca/corpora allata complex were examined for the presence of crustacean hyperglycemic hormone-like immunoreactive material. With the use of an antiserum directed against the hyperglycemic hormone of Carcinus maenas, immunofluorescence was found in the median portion of the pars intercerebralis, and the corpora cardiaca. Extracts of corpora cardiaca were examined by radioimmunoassay for competitive binding to the antiserum; one pair of corpora cardiaca contains at least 7 pg crustacean hyperglycemic hormone-like material.