Presence of corazonin in three insect species, and isolation and identification of [His7]corazonin from Schistocerca americana.

An ELISA for corazonin, a cardioactive neuropeptide from the American cockroach, Periplaneta americana, was developed. It was used to isolate corazonin from the cockroach Nauphoeta cinerea, the locust Schistocerca americana, and the hawkmoth Manduca sexta. The peptides from Nauphoeta and Manduca had the same retention times as Periplaneta corazonin, and their amino acid compositions also suggested that these peptides are identical with corazonin. The corazonin-immunoreactive peptide from Schistocerca eluted slightly earlier on HPLC than corazonin, and its structure was determined to be [His⁷]corazonin, or pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-Asn-amide. These results indicate that corazonin is generally present in insects and that its structure has been well conserved.     

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.     

Corazonin reduces the spinning rate in the silkworm,Bombyx mori

The insect neuropeptide, [Arg⁷]-corazonin was injected into larvae of the silkworm, Bombyx mori to investigate its influence on development and behavior. A single injection of 50 pmol of corazonin into the fourth and fifth instar larvae induced prolongation of the spinning period in all experimental groups except for those injected on day 10 of the fifth instar. The injection also caused a prolongation of the pupal period in some experimental groups, while it had no effect on the timing of larval ecdysis and the length of feeding period of the fifth instar. The spinning period was significantly prolonged even at a low dose of 1 pmol. Both the spinning rate and the rate of increase in hemolymph ecdysteroid level during the spinning stage were reduced by injection of corazonin. However, corazonin injection during days 5-7 of the fifth instar reduced the spinning rate without influencing the ecdysteroid level until the end of day 8, thereafter the rate of increase in hemolymph ecdysteroid level was slower in the corazonin-injected larvae than in the control larvae. Therefore, the suppressed ecdysteroid level observed in the corazonin-injected larvae appears to be a result rather than a cause of the reduced spinning rate. This study is the first published report for the corazonin effect on the behavior in insects.     

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.     

Neuropeptides of the cotton fleahopper, Pseudatomoscelis seriatus (Reuter)

The cotton fleahopper, Pseudatomoscelis seriatus (Reuter), is an economically important pest of cotton, and increasing concerns over resistance, detrimental effects on beneficial insects and safety issues associated with traditional insecticide applications have led to an interest in research on novel, alternative strategies for control. One such approach requires a more basic understanding of the neurohormonal system that regulates important physiological properties of the fleahopper; e.g. the expression of specific messenger molecules such as neuropeptides. Therefore we performed a peptidomic study of neural tissues from the fleahopper which led to the first identification of the sequences of native peptide hormones. These peptide hormones include the following neuropeptides: corazonin, short neuropeptide F (sNPF), myosuppressin, CAPA-pyrokinin and CAPA-PVK peptides. The CAPA-pyrokinin, sNPF, and CAPA-PVK peptides represent novel sequences. A comparison of fleahopper neuropeptides with those of related heteropteran species indicates that they are quite different. The sNPF of P. seriatus shows, among others, a novel substitution of Leu with Phe within the C-terminal region; a modification that sets it apart from the known sNPFs of not only other Heteroptera but of other arthropod species as well. The identity of the neuropeptides native to the fleahopper can aid in the potential development of biostable, bioavailable mimetic agonists and antagonists capable of disrupting the physiological functions that these neuropeptides regulate.     

Functional significance of the copper transporter ATP7 in peptidergic neurons and endocrine cells in Drosophila melanogaster

The Drosophila ATP7 copper transporter has sequence homology to the human copper transporters ATP7A and ATP7B, which are defective in Menkes and Wilson disease, respectively. We show here that in Drosophila ATP7 is expressed by many peptidergic neurons. As C-terminal amidation of neuropeptides depends on the copper-containing enzyme PHM, it seemed likely that in the absence of ATP7 the activity of PHM might be compromised. Indeed, inhibition of ATP7 expression by RNAi led to a decrease in mature amidated neuropeptides and the appearance of C-terminally Gly-extended neuropeptides. The strength of this effect differed from one cell type to another; it was very pronounced for AKH and corazonin, but much less so for SIFamide and myosuppressin. Nevertheless, down-regulation of ATP7 specifically in the SIFamide-expressing neurons resulted in male-male courtship behavior.     

Characterization, expression, and evolutionary aspects of Corazonin neuropeptide and its receptor from the House Fly, Musca domestica (Diptera: Muscidae)

In this article, we characterized structure and expression of genes encoding the neuropeptide Corazonin (MdCrz) and its putative receptor (MdCrzR) in the House Fly, Musca domestica. The MdCrz gene contains two introns, one within the 5' untranslated region and the other within the open reading frame. The 150-amino-acid precursor consists of an N-terminal signal peptide, and mature Crz followed by Crz-associated peptide (CAP). The CAP region is highly diverged from those of other insect precursors, whereas the mature Crz is identical in other dipteran members. In situ hybridization and immunohistochemistry consistently found a group of three MdCrz-producing neurons in the dorso-lateral protocerebrum, and eight pairs of bi-lateral neurons in the ventral nerve cord in the larvae. In adults, the expression was found exclusively in a cluster of five to seven neurons per brain lobe. Comparable expression patterns observed in other dipteran species suggest conserved regulatory mechanisms of Crz expression and functions during the course of evolution. MdCrzR deduced from the full-length cDNA sequence is a 655-amino acid polypeptide that contains seven trans-membrane (TM) domains and other motifs that are characteristics of Class-A G-protein coupled receptors. Although the TMs and loops between the TMs are conserved in other CrzRs, N-terminal extracellular domain is quite dissimilar. Tissue-specific RT-PCR revealed a high level of MdCrzR expression in the larval salivary glands and a moderate level in the CNS. In adults, the receptor was expressed both in the head and body, suggesting multifunctionality of the Crz signaling system.     

Neurohormones as putative circadian clock output signals in the central nervous system of two cricket species

Antisera to the neuropeptides corazonin (Crz) and crustacean cardioactive peptide (CCAP) and to the diapause hormone (DH) react with small sets of neurones in the cephalic ganglia of the crickets Dianemobius nigrofasciatus and Allonemobius allardi. The distribution of their immunoreactivities is similar in the two species and overlaps with the locations of presumed circadian clock components in the optic lobes, protocerebrum, tritocerebrum, suboesophageal ganglion (SOG) and frontal ganglion. D. nigrofasciatus contains two Crz-immunoreactive (Crz-ir) cells in each optic lobe, six cell groups in the protocerebrum, four in the tritocerebrum, and one in SOG, whereas A. allardi harbours only five Crz-ir groups in the protocerebrum and four in the tritocerebrum. CCAP immunoreactivity occurs in both species in four protocerebrum cell clusters, four tritocerebrum cell clusters, four SOG cell clusters, one frontal ganglion cell cluster, and two optic lobe cell clusters; D. nigrofasciatus possesses two additional cells with unique links to the lamina in the optic lobe. DH-related antigens are present in four cell clusters in the optic lobe, six (D. nigrofasciatus) or eight (A. allardi) in the protocerebrum, four in the tritocerebrum, and three (A. allardi) or five (D. nigrofasciatus) in the SOG. Some of the detected cells also react with antibody to the clock protein Period (PER) or lie close to PER-ir cells. Crickets reared at two different photoperiods do not differ in the distribution and intensity of immunoreactivities. No changes have been detected during the course of diurnal light/dark cycles, possibly because the antisera react with persistent prohormones, whereas circadian fluctuations may occur at the level of their processing or of hormone release. The projection of immunoreactive fibres to several brain regions, the stomatogastric nervous system and the neurohaemal organs indicates multiple functions of the respective hormones. The work was supported by the “Research for Future” program of the Japan Society for the Promotion of Science (JSPS, 99L01205) and by the JSPS Postdoctoral Fellowship for Foreign Researchers (no. P 04197).     

Knockdown of the corazonin gene reveals its critical role in the control of gregarious characteristics in the desert locust

The two plague locusts, Schistocerca gregaria and Locusta migratoria, exhibit density-dependent phase polyphenism. Nymphs occurring at low population densities (solitarious forms) are uniformly colored and match their body color to the background color of their habitat, whereas those occurring at high population densities (gregarious) develop black patterns. An injection of the neuropeptide, corazonin (Crz) has been shown to induce black patterns in locusts and affect the classical morphometric ratio, F/C (F, hind femur length; C, maximum head width). We herein identified and cloned the CRZ genes from S. gregaria (SgCRZ) and L. migratoria. A comparative analysis of prepro-Crz sequences among insects showed that the functional peptide was well conserved; its conservation was limited to the peptide region. Silencing of the identified SgCRZ gene in gregarious S. gregaria nymphs markedly lightened their body color and shifted the adult F/C ratio toward the value typical of solitarious forms. In addition, knockdown of the gene in solitarious nymphs strongly inhibited darkening even after a transfer to crowded conditions; however, these individuals developed black patterns after being injected with the Crz as a rescue treatment. SgCRZ was constitutively expressed in the brains of S. gregaria during nymphal development in both phases. This gene was highly expressed not only in the brain in both phases, but also in the corpora allata in the gregarious phase. This conspicuous phase-dependent difference in SgCRZ gene expression may indicate a functional role in the control of phase polyphenism in this locust.