Corpora Cardiaca Research Articles

Differential Receptor Activation by Cockroach Adipokinetic Hormones Produces Differential Effects on Ion Currents, Neuronal Activity, and Locomotion

Adipokinetic hormone (AKH) peptides in insects serve the endocrine control of energy supply. They also produce, however, neuronal, vegetative, and motor effects, suggesting that AKHs orchestrate adaptive behavior by multiple actions. We have cloned, for Periplaneta americana, the AKH receptor to determine its localization and, based on current measurements in neurons and heterologous expression systems, the mechanisms of AKH actions. Apart from fat body, various neurons express the AKH receptor, among them abdominal dorsal unpaired median (DUM) neurons, which release the biogenic amine octopamine. They are part of the arousal system and are involved in the control of circulation and respiration. Both the two Periplaneta AKHs activate the Gs pathway, and AKH I also potently activates Gq. AKH I and--with much less efficacy--AKH II accelerate spiking of DUM neurons through an increase of the pacemaking Ca2+ current. Because the AKHs are released from the corpora cardiaca into the hemolymph, they must penetrate the blood-brain barrier for acting on neurons. That this happens was shown electrophysiologically by applying AKH I to an intact ganglion. Systemically injected AKH I stimulates locomotion potently in striking contrast to AKH II. This behavioral difference can be traced back conclusively to the different effectiveness of the AKHs on the level of G proteins. Our findings also show that AKHs act through the same basic mechanisms on neuronal and nonneuronal cells, and they support an integration of metabolic and neuronal effects in homoeostatic mechanisms.

Tissue localization and partial characterization of pheromone biosynthesis activating neuropeptide inAchaea janata

Female sex pheromone production in certain moth species have been shown to be regulated by a cephalic endocrine peptidic factor: pheromone biosynthesis activating neuropeptide (PBAN), having 33 amino acid residues. Antisera against synthetic Heliothis zea-PBAN were developed. Using these polyclonals, immunoreactivity was mapped in the nervous system of Achaea janata. Three distinct groups of immunopositive secretory neurons were identified in the suboesophageal ganglion; and immunoreactivity was observed in the corpora cardiaca, thoracic and in the abdominal ganglia. From about 6000 brain sub-oesophageal ganglion complexes, the neuropeptide was isolated; and purified sequentially by Sep-pak and reversed phase high performance liquid chromatographic methods. Identity of purified PBAN fraction was confirmed with polyclonal antibody by immunoblotting. Molecular mass of the isolated peptide was determined by matrix-assisted laser desorption/ionization mass spectrometry, and was found to be 3900 Da, same as that of known H. zea-PBAN. Radiochemical bioassay confirmed the pheromonotropic effect of the isolated neuropeptide in this insect.

Activation of triacylglycerol lipase in the fat body of a beetle by adipokinetic hormone

The activation of triacylglycerol lipase and the stimulation of proline synthesis in the fat body of the fruit beetle Pachnoda sinuata by the endogenous octapeptide hormone Melme-CC (pQLNYSPDWa), which belongs to the family of insect adipokinetic hormones, were studied, and the correlation of both events investigated. At rest, the activity of triacylglycerol lipase in the fat body of the beetle was higher than in the fat body of the American cockroach, Periplaneta americana, but lower than in the migratory locust, Locusta migratoria. Triacylglycerol lipase of the beetle is activated by: (a) injection of synthetic Melme-CC and (b) the stimulus of flight. Activation of lipase by Melme-CC is time-dependent. Injection of cpt-cAMP activates triacylglycerol lipase in the fat body and causes an increase in the concentration of proline in the haemolymph at the expense of alanine. In contrast, injection of F-inositol-1,4,5-phosphate does not affect the activation state of lipase, nor the levels of amino acids in the haemolymph. High doses of octopamine do not activate lipase. Furthermore, activity of fat body lipase and proline concentration in the haemolymph both follow a circadian rhythm: both parameters are high in the morning, whereas they are low in the evening. When transfer of Melme-CC, released from the corpora cardiaca, to the thorax/abdomen is prevented by neck-ligation, the activity of lipase, as well as the circulating proline levels are low. Regression analysis revealed that activity of triacylglycerol lipase is positively correlated to proline concentration in the haemolymph, whereas there is a negative correlation of the enzyme activity and alanine level in the haemolymph. From these results we conclude that the activation of fat body triacylglycerol lipase by Melme-CC in P. sinuata stimulates proline synthesis. Proline is one of the major substrates to power flight activity in the beetle.

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.

New insights into the evolution of the GRF superfamily based on sequence similarity between the locust APRPs and human GRF

In a manual sequence alignment experiment we accidentally observed that the locust adipokinetic hormone (AKH) 1 Abbreviations used: AKH, adipokinetic hormone; APRP, adipokinetic hormone precursor-related peptide; GIP, glucose-dependent insulinotropic polypeptide; GLP, glucagon-like peptide; GLUC, glucagon; GRF, growth hormone-releasing factor; Lom, Locusta migratoria; MYA, million years ago; PACAP, pituitary adenylate cyclase-activating polypeptide; PHM, peptide histidine-methionine; Scg, Schistocerca gregaria; Scn, Schistocerca nitens; VIP, vasoactive intestinal polypeptide. 1 precursor-related peptides (APRPs), which are peptides contained in the AKH-preprohormones, display sequence similarity with human growth hormone-releasing factor (GRF). So far, a counterpart for GRF, either structurally or functionally, has not yet been reported in any non-chordate. This could give new insights into the origin of the GRF superfamily, as it would mean that in some invertebrates (locusts), the structural homologues of glucagons (AKHs) and GRFs (APRPs) are located on the same precursor. No function could as yet be attributed to APRPs, although these neuropeptides occur abundantly in the corpora cardiaca, a neurohaemal organ from which many neuropeptides are released. Therefore, similar to GRF, a role as releasing factor may be envisaged for APRPs.

AKH-producing neuroendocrine cell ablation decreases trehalose and induces behavioral changes in Drosophila.

Adipokinetic hormone (AKH) is a metabolic neuropeptide principally known for its mobilization of energy substrates, notably lipid and trehalose during energy-requiring activities, such as flight and locomotion. Drosophila melanogaster AKH cell localization in corpora cardiaca, as in other insect species, was confirmed by immunoreactivity and by a genetic approach using the UAS/GAL4 system. To assess AKH general physiological rules, we ablated AKH endocrine cells by specifically driving the expression of apoptosis transgenes in AKH cells. Trehalose levels were decreased in larvae and starved adults, when the stimulation by AKH of the production of trehalose from fat body glycogen is no longer possible. Moreover, we show that these adults without AKH cells become progressively hypoactive. Finally, under starvation conditions, those hypoactive AKH-knockout cell flies survived approximately 50% longer than control wild-type flies, suggesting that the slower rate at which AKH-ablated flies mobilize their energy resources extends their survival.

Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells

Antagonistic activities of glucagon and insulin control metabolism in mammals, and disruption of this balance underlies diabetes pathogenesis. Insulin-producing cells (IPCs) in the brain of insects such as Drosophila also regulate serum glucose, but it remains unclear whether insulin is the sole hormonal regulator of glucose homeostasis and whether mechanisms of glucose-sensing and response in IPCs resemble those in pancreatic islets. Here we show, by targeted cell ablation, that Drosophila corpora cardiaca (CC) cells of the ring gland are also essential for larval glucose homeostasis. Unlike IPCs, CC cells express Drosophila cognates of sulphonylurea receptor (Sur) and potassium channel (Ir), proteins that comprise ATP-sensitive potassium channels regulating hormone secretion by islets and other mammalian glucose-sensing cells. They also produce adipokinetic hormone, a polypeptide with glucagon-like functions. Glucose regulation by CC cells is impaired by exposure to sulphonylureas, drugs that target the Sur subunit. Furthermore, ubiquitous expression of an akh transgene reverses the effect of CC ablation on serum glucose. Thus, Drosophila CC cells are crucial regulators of glucose homeostasis and they use glucose-sensing and response mechanisms similar to islet cells.

Hemolymph Sugar Homeostasis and Starvation-Induced Hyperactivity Affected by Genetic Manipulations of the Adipokinetic Hormone-Encoding Gene in Drosophila melanogaster

Adipokinetic hormones (AKHs) are metabolic neuropeptides, mediating mobilization of energy substrates from the fat body in many insects. In delving into the roles of the Drosophila Akh (dAkh) gene, its developmental expression patterns were examined and the physiological functions of the AKH-producing neurons were investigated using animals devoid of AKH neurons and ones with ectopically expressing dAkh. The dAkh gene is expressed exclusively in the corpora cardiaca from late embryos to adult stages. Projections emanating from the AKH neurons indicated that AKH has multiple target tissues as follows: the prothoracic gland and aorta in the larva and the crop and brain in the adult. Studies using transgenic manipulations of the dAkh gene demonstrated that AKH induced both hypertrehalosemia and hyperlipemia. Starved wild-type flies displayed prolonged hyperactivity prior to death; this novel behavioral pattern could be associated with food-searching activities in response to starvation. In contrast, flies devoid of AKH neurons not only lacked this type of hyperactivity, but also displayed strong resistance to starvation-induced death. From these findings, we propose another role for AKH in the regulation of starvation-induced foraging behavior.

The Tribolium homologue of glass and the evolution of insect larval eyes

While non-arthropod orthologues have been found for many Drosophila eye developmental genes, this has not been the case for the glass ( gl) gene, which encodes a zinc finger transcription factor required for photoreceptor cell specification, differentiation, and survival. This study reports sequence and expression analysis of the gl orthologue of the flour beetle Tribolium castaneum. A strongly conserved C-terminal zinc finger binding region and a moderately conserved N-terminal transcriptional activation domain characterize the putative Tribolium gl protein. Tribolium gl transcripts were detected in the developing photoreceptors of the larval and adult visual system, the corpora cardiaca, and subsets of cells in the developing brain. This suggests that the gl function of specifying predominantly neuronal cells is strongly conserved. Using gl as a marker for the onset of larval photoreceptor differentiation, we studied the embryonic development of the Tribolium visual system. We find that the Tribolium larval eyes originate at the posterior margin of the embryonic eye lobes as defined by eye-field-specific wingless expression domains. This is consistent with the hypothesis that the larval visual organs (stemmata) of holometabolous insects were derived from and are therefore homologous to the posterior-most ommatidia of the adult retina in primitive nonholometabolous insects.

Substrate usage and its regulation during flight and swimming in the backswimmer, Notonecta glauca

Abstract. The metabolites that are generally used by insects during exercise are present in quite different concentrations in the haemolymph of the backswimmer Notonecta glauca L. Lipids are most abundant (between 10 and 20 mg/mL), whereas carbohydrates (2–3 mg/mL) and proline (approximately 1 mg/mL) are at very low concentrations. Injection of an extract of conspecific corpora cardiaca causes pronounced hyperlipaemia in the backswimmer. A neuropeptide with the same effect was isolated from the corpora cardiaca in a single high‐performance liquid chromatography (HPLC) step; the primary sequence was deduced from mass spectrometric measurements (matrix‐assisted laser desorption/ionization‐time of flight and electrospray quadrupol time‐of‐flight mass spectrometry) of whole corpora cardiaca, and the mass was confirmed in the HPLC fraction that had adipokinetic activity. The biologically active octapeptide has the sequence pGlu‐Val‐Asn‐Phe‐Ser‐Pro‐Ser‐Trp amide, which was characterized previously from the corpora cardiaca of the Emperor dragonfly, Anax imperator, and denoted Anaim‐adipokinetic hormone (AKH). The synthetic Anaim‐AKH peptide causes lipid mobilization when injected at a dose of 1 pmol into N. glauca. When other synthetic AKH members that occur in Hemiptera are injected into N. glauca at the same dose, the hyperlipaemic responses are significantly lower than after injection of Anaim‐AKH. Because only lipids increase upon activity, such as continuous swimming for 1 h or during a 1‐h rest period after a 3‐min flight episode in the laboratory, it is assumed that Anaim‐AKH serves as a true adipokinetic hormone in the backswimmer during bouts of natural swimming and flight.

Helicostatins: Brain-gut peptides of the moth,Helicoverpa armigera (Lepidoptera: Noctuidae)

Gene expression and immunolocalisation studies have determined that the helicostatins are brain-gut peptides in larvae of the lepidopteran, Helicoverpa armigera. Mapping of the distribution of these peptides in the nervous system and alimentary canal has provided evidence for multifunctional regulatory roles. In situ hybridisation studies have shown that the helicostatin precursor gene is expressed in neurones of the central and stomatogastric nervous systems, and endocrine cells of the midgut demonstrating that the helicostatins are true brain-gut peptides. Antisera raised against Leu-callatostatin 3 (ANRYGFGL-NH(2)), a peptide isolated from the blowfly, Calliphora vomitoria was used to map the distribution of allatostatin-like immunoreactive (Ast-ir) material in H. armigera to elucidate possible functions of the helicostatins. In situ hybridisation studies verified that the helicostatin precursor gene is expressed in neurones shown to contain Ast-ir, providing strong evidence that the Ast-ir material is helicostatins. Extensive immunoreactive axonal projections into complex regions of neuropile indicate that the helicostatins may have a neuromodulatory role in the brain and segmental ganglia of the ventral nerve cord. The presence of large amounts of immunoreactive material in axons within the corpora cardiaca (CC) and transverse nerves of the perisympathetic nervous system, two known neurohaemal organs, provides evidence for a neurohormonal role. The corpora allata (CA) were innervated only sparsely by Ast-ir axons suggesting that the CA are not a neurohaemal release site or a target. Thus, it is unlikely that the helicostatins regulate juvenile hormone (JH) biosynthesis or release. Ast-ir axons extended from the frontal ganglion through the recurrent nerve and many branches were closely associated with muscles of the foregut, stomodeal valve, and anterior midgut, implicating helicostatins in regulation of foregut motility. Ast-ir material was also present in nerves associated with muscles of the pyloric valve and rectum, and in endocrine cells of the midgut.

A comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry

The occurrence of neuropeptides in the retrocerebral complexes of adult male and females of the tobacco hawkmoth, Manduca sexta, was investigated using matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry (MS), post source decay (PSD) and collision-induced dissociation (CID) MS/MS. From fractions of methanol extracts of corpora cardiaca (CC)/corpora allata (CA), separated by reversed-phase high performance liquid chromatography (RP-HPLC), a total of 11 mass ions were assigned to known peptides from M. sexta. These peptides were adipokinetic hormone (AKH), FLRFamides I, II and III, crustacean cardioactive peptide (CCAP), cardioactive peptide 2b (CAP 2b), three myoinhibitory peptides, corazonin, and M. sexta allatostatin (Manse-AS). A further six masses were in agreement with Y/FXFGLamide allatostatins identified from other Lepidoptera. The sequence identities of FLRFamide I and AKH were confirmed using post source decay analysis. Fragmentation by collision-induced dissociation MS/MS identified an extended AKH peptide. The apparent differences in the peptides present in male and female retrocerebral complexes are most likely quantitative rather than sex specific.

Presence of [His<sup>7</sup>]- corazonin in the central nervous system of a newly isolated albino strain of Schistocerca gregaria (Orthoptera: Acrididae) - mass spectrometric and immunocytochemical evidence

An inbred strain of a newly isolated spontaneous albino mutant of Schistocerca gregaria (Forsk.) was examined for the presence of the neuropeptide [His7]-corazonin by immunocytochemical and mass spectrometric methods. It was concluded that this peptide is definitely present in a limited number of neurosecretory cells in the pars lateralis as well as in the corpora cardiaca (CC). Injection of either synthetic [His7]-corazonin or of extracts of CC of the normal coloured phenotype of S. gregaria failed to induce darkening of the cuticle, while albino Locusta migratoria, used as a positive control, turned dark. The conclusion is that the cause of albinism in the new S. gregaria albino is probably due to a defect in the receptor system for [His7]-corazonin or in the biosynthetic pathway of melanin.

Osmotic lysis of corpora cardiaca using distilled water reveals the presence of partially processed peptides and peptide fragments

Abstract. A simple, single‐step aqueous extraction method has been developed to study the neuropeptide content of small neuroendocrine organs. Perifusion of these tissues with deionized water causes osmotic bursting of the cells and release of their content into the surrounding fluid. The neuropeptides are immediately retained from the perifusion fluid using disposable C18 cartridges. After one separation step and mass spectrometry, it was possible to identify a large number of known neuropeptides from the corpora cardiaca of Locusta migratoria (L). Also present in the extract were a number of neuropeptide fragments and two incompletely processed peptides. Using this method, a 959Da peptide present in the corpora cardiaca was sequenced de novo. The full sequence, deduced using Collision Induced Dissociation Tandem Mass Spectrometry (CID MS/MS), is Ser‐Pro‐Leu‐Asp‐Ala‐His‐His‐Leu‐Ala. This nonapeptide is predicted from the gene encoding the ion transport peptide precursor and from the gene encoding the ion transport‐like peptide precursor. In both cases, this nonapeptide, which was named ion transport peptide‐copeptide, is flanked by the signal sequence at the N‐terminus and a dibasic cleavage site (Lys‐Arg) at the C‐terminus. This structural feature is common to many physiologically important locust preproneuropeptides and indicates that this copeptide might have a physiological function, but this is not yet known.

The endocrine control of phase transition: some new aspects

Abstract. The present article summarizes some recent findings relating to the underlying mechanism of phase transition in locusts, from the nonswarming solitarious phase to the swarming gregarious phase. These phases differ in many traits, such as colouration, morphometrics and behaviour. The most comprehensive theory at present to explain the switch from the nonswarming to the swarming form is that the locusts are brought together by the heterogeneity of the environment. They gather at preferential structures and food plants and physical contact then stimulates individuals to gregarize. Phase change can also be transferred across generations by maternal pheromones. The endocrine regulation of phase polymorphism is still not fully understood. The role of ecdysteroids has been studied, so far with no final conclusion. It is remarkable that the prothoracic glands persist longer in isolated‐reared adults, which implies that these glands continue to play a role, although they no longer release important amounts of ecdysteroids. Juvenile Hormone, without any doubt, induces certain solitarious characteristics, such as green colouration, but is not the primary causal factor. A real breakthrough was the discovery of [His7]‐corazonin, made possible by using a novel assay system, the Okinawa albino mutant of Locusta migratoria, which was known to be deficient in this hormone. This peptide, which is produced in the brain and is most likely released via the corpora cardiaca, promotes the gregarious black pigmentation. It also plays a role in morphometrical phase change as well as in behavioural alterations. Corazonin is apparently quite an important peptide not only in locusts, but also in insects in general.

Identification and function of the hypotrehalosaemic hormone (Mas-AKH) in workers, drones and queens of Apis mellifero ligustica and A. m. carnica

SUMMARYMethanolic extracts of the corpora cardiaca (CC) of foraging workers of the yellow Italian race of honey bees (Apis mellifera ligustica) contained on average 3.0 pmol/pCC (paired corpora cardiaca) of the hypertrehalosaemic hormone, Mas-AKH. The CC of A. m. ligustica nurse bees (8–10 days old) contained only about 1/20th (0.14 pmol/pCC) of the Mas-AKH as that of foragers, and very young bees (2–4 days old) contained no detectable hormone. CC extracts of A. m. ligustica virgin drones and older drones (> 8 days) also contained Mas-AKH, but much less (0.23 pmol/pCC) than foraging worker bees. CC extracts from both virgin and mated (egg laying) ligustica/carnica queens, as well as from foraging workers or drones of the brown European race of honey bees (Apis mellifera carnica) all lacked detectable Mas-AKH or any other related peptides. There was no elevation of the total blood sugar titre in any caste of either ligustica or carnica when injected with either synthetic Mas-AKH or with extracts of their respective corpora cardiaca. The amount of fat body and the amount of stored glycogen is small in all castes of both races. The crop in all castes is large and contained up to 30% of the total body weight. In conclusion, none of the castes of either race seemed to use a hormone to mobilize sugar from the fat body or other glycogen stores (such as the muscle), but rather they depend for all activities entirely on sugars in the crop.

A quantitative study of adipokinetic hormone of the firebug, Pyrrhocoris apterus

The development of an enzyme-linked immunoassay (ELISA) for the adipokinetic neuropeptide hormone, Pya-AKH, from the firebug Pyrrhocoris apterus L. is described. The ELISA measures as little as 20 fmol of Pya-AKH. Tested against a range of synthetic peptides, the assay has a high sensitivity for peptides containing the C-terminal motif FTPNWamide. The amounts of Pya-AKH in the brain, corpora cardiaca, suboesophageal ganglia, and fused thoracic and abdominal ganglionic mass are very small, with only the corpora cardiaca containing appreciable levels of the hormone (ca. 4 pmol per bug). Preliminary estimates of the persistence of the hormone in the haemolymph are consistent with values determined for AKHs in other insects, and suggest that Pya-AKH has a rapid turnover with a half-life of ca. 18 min. Measurements of circulating titres of AKH in Pyrrhocoris are only possible in the ELISA described here by using pooled samples of haemolymph, and after preliminary clean-up of the haemolymph samples. The titre of Pya-AKH in resting reproductive female Pyrrhocoris is ca. 1 fmol/μl.

Effects of [His 7]-corazonin on the phase state of isolated-reared (solitarious) desert locusts, Schistocerca gregaria

[His 7]-corazonin is a neuropeptide produced in the pars lateralis of the brain. It is stored in the corpora cardiaca and probably released from there. The only well-documented effect in locusts is increased melanization of the cuticle. We investigated whether this hormone might also be causally related to changes in behavior and morphometrics that, like melanization, occur during crowding-induced gregarization. Solitary fourth-instar nymphs of Schistocerca gregaria (Forsk.) were injected thrice with 1 nmol [His 7]-corazonin. After molting to the 5th stadium their behavioral phase state was measured in an arena assay and analyzed using multiple logistic regression analysis. The hormone was found not to induce behavioral phase changes. Upon reaching adulthood, morphometrics shifts occurred towards values typical for crowd-reared and regregarized animals. Our results thus indicate that [His 7]-corazonin is not involved in behavioral gregarization but may participate in morphometrical phase change.

Search for candidate gonadotropins in extracts of the corpus cardiacum of the fleshfly, Neobellieria bullata

Summary No single fly gonadotropin has as yet been identified with certainty. Dababase analysis fails to yield positive results when the genomic database of Drosophila is scanned for the presence of family members of gonadotropins that have already been identified in other insects. However, a candidate gonadotropic peptide (cAMP generating peptide, Neb-cGP) was purified some time ago from the fleshfly Neobellieria bullata. A short form of this peptide was found to stimulate cAMP production in the ovaries of the fleshfly. In the present study, we gathered additional arguments that suggest a role for Neb-cGP as a gonadotropic hormone. Brains of male and female adult Neobellieria bullata were stained with antiserum directed to the cAMP generating peptide. In both sexes four lateral neurosecretory cells in the dorsal part of each pars lateralis were stained. In addition, mass spectrometric read-outs of extracts of fleshfly corpora cardiaca displayed the full length form (48 amino acid residues) of the cAMP-generating peptide. These findings suggest that Neb-cGP is synthesised in the brain and stored in and released from the corpora cardiaca as a neurohormone. The -terminal 15-mer isoform of the cAMP generating peptide could not be identified. Injection experiments in the fleshfly with this 15-mer peptide, that were set up to examine the in vivo effect, suggest a putative stimulatory effect on oocyte development. Attempts to identify a fly neuroparsin-like peptide through mass spectrometry, purification and microsequencing by Edman degradation or advanced Blast searches in the Drosophila genome all failed.

Neuroanatomical studies of period gene expression in the hawkmoth, Manduca sexta.

In the nervous system of the hawkmoth, Manduca sexta, cells expressing the period (per)gene were mapped by in situ hybridization and immunocytochemical methods. Digoxigenin-labeled riboprobes were transcribed from a 1-kb M. sexta per cDNA. Monoclonal anti-PER antibodies were raised to peptide antigens translated from both M. sexta and Drosophila melanogaster per cDNAs. These reagents revealed a widespread distribution of per gene products in M. sexta eyes, optic lobes, brains, and retrocerebral complexes. Labeling for per mRNA was prominent in photoreceptors and in glial cells throughout the brain, and in a cluster of 100-200 neurons adjacent to the accessory medulla of the optic lobes. Daily rhythms of per mRNA levels were detected only in glial cells. PER-like immunoreactivity was observed in nuclei of most neurons and glial cells and in many photoreceptor nuclei. Four neurosecretory cells in the pars lateralis of each brain hemisphere exhibited both nuclear and cytoplasmic staining with anti-PER antibodies. These cells were positively identified as Ia(1) neurosecretory cells that express corazonin immunoreactivity. Anti-corazonin labeled their projections in the brain and their neurohemal endings in the corpora cardiaca and corpora allata. Four pairs of PER-expressing neurosecretory cells previously described in the silkmoth, Anthereae pernyi, are likely to be homologous to these PER/corazonin-expressing Ia(1) cells of M. sexta. Other findings, such as widespread nuclear localization of M. sexta PER and rhythmic expression in glial cells, are reminiscent of the period gene of D. melanogaster, suggesting that some functions of per may be conserved in this lepidopteran species.