Corazonin and corazonin-like substances in the central nervous system of the Pterygote and Apterygote insects

Antisera against corazonin were used to investigate distribution of immunoreactive cells in the central nervous system (CNS) of representatives of six insect orders: Ctenolepisma lineata (Zygentoma), Locusta migratoria (Orthoptera), Oxya yezoensis (Orthoptera), Gryllus bimaculatus (Orthoptera), Pyrrhocoris apterus (Hemiptera), Arge nigrinodosa (Hymenoptera), Athalia rosae (Hymenoptera), Bombyx mori (Lepidoptera) and Anomala cuprea (Coleoptera). Corazonin-like immunoreactive (CLI) cells were detected in the brain and ventral ganglia of all insects studied except for the albino strain of L. migratoria and the beetle A. cuprea. Implantation of the brain or different ganglia from insects with detected immunoreactivity induced dark coloration in the albino locust, providing further evidence for the presence of authentic corazonins [His(7)- and Arg(7)-isoforms] in these insects. The protocerebral lateral neurosecretory cells projecting into the ipsilateral retrocerebral neurohemal organs and bilateral longitudinal tracts extending and branching throughout the entire CNS seem to be a well-conserved part of the corazonin system in insects. The bilateral longitudinal tracts were formed by species-specific numbers of bilateral interneurons segmentally distributed in the ventral ganglia. Additional immunoreactive somata, mostly interneurons, were detected in the CNS of various insects. The distribution of corazonin in the cephalic neurosecretory system and in the bilateral interneurons suggests that corazonin acts as a hormone as well as a neurotransmitter or a neuromodulator. An ancient origin of corazonin is suggested by the presence of a corazonin-like substance in the primitive insect, C. lineata. These results support previous findings on the common occurrence of corazonin among insects, except for the albino strain of L. migratoria and the Coleoptera.     

Endocrine mechanisms controlling body-color polymorphism in locusts.

The present article reviews recent published and unpublished findings on the hormonal mechanisms for the control of body-color polymorphism in locusts. Emphasis is placed on the dark color-inducing factors and their role in the induction of various types of body coloration observed under different environmental conditions. Implantation of corpora cardiaca (CC) taken from normal nymphs of Locusta migratoria induced dark color in nymphs of an albino strain. Using the albino strain for the bioassay, a neuropeptide, [His7]-corazonin, was identified as a dark color-inducing factor for L. migratoria and Schistocerca gregaria. In the former, depending upon the dose and timing of the injection, this peptide and juvenile hormone developed various body colors looking like those found in nature. The body coloration characteristic of gregarious forms was also induced in isolated albino nymphs and field-collected solitary nymphs. In S. gregaria, on the other hand, the peptide induced black patterns, but the orange or yellow background color observed in gregarious forms was not induced when the peptide was injected into solitary individuals. [His7]-corazonin also induced darkening in other grasshoppers and locusts, but not in katydids. Albino L. migratoria developed dark color when implanted with brains or CC taken from other insects belonging to 10 major insect orders, but not with those from Coleoptera. [His7]-corazonin or a similar compound is widespread among insects and plays a pivotal role in controlling body color in some species and presumably other physiological roles in other species. Arch.     

Cloning and characterization of a third isoform of corazonin in the honey bee Apis mellifera

The precursor of the insect hormone corazonin has been cloned from the honey bee Apis mellifera. The precursor predicts a novel isoform of corazonin, pQTFTYSHGWTNamide, which was confirmed by tandem mass spectrometry. Although Apis corazonin differs only by a glutamine/threonine substitution from [His7]-corazonin, it is considerably less active in the dark color inducing assay on albino locusts. Whole mount fluorescence immunohistochemistry of the central nervous system of the honey bee showed a pattern similar to the ones described for other insects. Four neurons of the lateral protocerebrum project axons towards the retrocerebral complex. It is unlikely that Apis corazonin is present in all hymenopteran species since the presence of this peptide could not be demonstrated by means of mass spectrometry in the retrocerebral complex of the red wood ant Formica rufa and the wasp Vespula saxonica. Instead, we found masses corresponding with [Arg7]- and [His7]-corazonin respectively, suggesting that some of the corazonin isoforms originated late during evolution in different insect orders.     

Nuclear magnetic resonance (NMR) studies of the dark-color-inducing neurohormone of locusts and corazonin

The dark-color-inducing neurohormone (DCIN) of locusts and corazonin of a cockroach, both 11 residue-long peptides, induce dark coloration in albino nymphs of Locusta migratoria when injected after a nymphal molt. These peptides differ at position 7 (His in DCIN and Arg in corazonin) and elicit an almost identical darkening response. The three-dimensional structures of these peptides, dissolved in dimethylsulfoxide (DMSO), were determined by NMR. Structural elements determined at atomic resolution may provide insight into the biological activity of these two neurohormones. The calculated structures of DCIN and corazonin indicate clear, prevalent conformations with similar secondary features. The generated low-energy solution structures of each show structural elements within residues Phe3 to Trp9 with a turn situated at the core of the peptide from which the sidechains of residue 7 of each peptide protrude. A calculated negative electrostatic potential surface almost completely covers both neurohormones and only the 7th residue sidechains of each peptide emerge in their entirety. Within these residues there is a partial sequence seen in several neurohormones that control various physiological functions in Arthropods: -Ser-X-Gly-Trp- (X=His in DCIN and Arg in corazonin). This partial sequence may play a role in the physiological activity of some Arthropod neurohormones.     

Structure-activity relations of the dark-colour-inducing neurohormone of locusts

The dark-colour-inducing effect of several peptides in comparison to that of the dark-colour-inducing neurohormone (DCIN, [His(7)]-corazonin) of locusts was investigated by a bioassay based on nymphs of a DCIN-deficient albino mutant of Locusta migratoria. The study was aimed at elucidating the active part of the DCIN and to explore the contribution of its amino acids to the activity. Graded doses of all peptides were injected in oil. [Arg(7)]-corazonin and DCIN were equally effective. Certain arthropod neuropeptides having the -SXGW- partial sequence (a part of the DCIN and of [Arg(7)]-corazonin; X=His and X=Arg, respectively) yielded the following findings: Scg-AKH-II (adipokinetic hormone II of Schistocerca gregaria X=Thr), Grb-AKH ( adipokinetic hormone of Gryllus bimaculatus X=Thr) and RPCH (red pigment concentrating hormone of crustaceans X=Pro) evoked a moderate darkening response, but Lom-AKH-II (adipokinetic hormone II of L. migratoria X=Ala) was ineffective. Step by step shortening of the sequence of the DCIN at the N-terminal, from pGlu-3-11DCIN to pGlu-9-11DCIN, resulted in a decreasing activity, but even pGlu-9-11DCIN induced a weak response with high doses. Shortening of the DCIN from the C-terminal revealed a moderate activity of 1-7DCIN-NH(2) and a weak activity of 1-5DCIN-NH(2). An octadecapeptide which induces dark colour in moth larvae, having the pentamer FTPRL-NH(2) at its C-terminal, evoked no darkening in the albino locusts. We conclude that although the -SXGW- partial sequence has some role in induction of darkening, for obtaining maximal effect the whole sequence of the DCIN (or of [Arg(7)]-corazonin) is necessary.     

Genetic and hormonal control of melanization in reddish–brown and albino mutants in the desert locust Schistocerca gregaria

The genetic and hormonal control of body colouration is investigated using two recessive genetic mutant strains, the reddish–brown (RB) mutant and an albino mutant, as well as a normal (pigmented) strain of the desert locust Schistocerca gregaria. The colour patterns of the RB nymphs are similar to those of a normal strain, although the intensity of the melanization is weaker in the former. Reciprocal crosses between the RB and albino mutants produce only normal phenotypes in the F₁ generation. In the F₂ generation, the normal, RB and albino phenotypes appear in a ratio of 9 : 3 : 4, indicating that two Mendelian units might determine the appearance of dark body colour and the intensity of melanization, respectively. In other words, at least two steps of regulation might be involved in the expression of body colour. Injections of [His⁷]‐corazonin, a neuropeptide inducing dark colour in this locust, fail to induce dark colour in albino nymphs but show a dose‐dependent darkening in RB nymphs in the range, 10 pmol to 1 nmol. Some RB nymphs become indistinguishable from normal individuals after injection of the peptide. Implantation of corpora cardiaca (CC) taken from RB mutants into other RB individuals induces darkening in the latter and CC from RB, albino and normal strains have similar dark colour‐inducing activity when implanted into albino Locusta migratoria. These results suggest the possibility that the RB mutant gene regulates the intensity of melanization, possibly through controlling the pathway of pigment biosynthesis associated with [His⁷]‐corazonin.     

Hormonal control of body-color polymorphism in Locusta migratoria: interaction between

The effects of injection of [His(7)]-corazonin and juvenile hormone (JH) III on the body color in L. migratoria were investigated using albino and normal (pigmented) nymphs. Most albino nymphs turned green in the fourth instar if injected with JH III during the last 2 days of the previous instar. When albino third instar nymphs injected with 10 pmol of [His(7)]-corazonin on different days were treated with 100 μg of JH III on day 3.5, they developed various body colors in the following nymphal instar: those injected with [His(7)]-corazonin during the first 2 days developed very dark green or black color, whereas some of those injected after this period turned green and their legs and ventral side of the body were variously pigmented, the coloration being similar to green solitary individuals often found in the field. Field-collected brown solitary nymphs injected with 1 nmol of [His(7)]-corazonin and kept individually, turned reddish without any black spots in the following nymphal instar when the ecdysis occurred within 1 day after injection. Injection of [His(7)]-corazonin 2 days before the following ecdysis induced black patterns on an orange background color, the coloration characteristic of gregarious forms. Similar injections into field-collected green solitary nymphs also induced black patterns but the rest of their body remained green. These results may indicate that the temporal changes in the hemolymph titers of [His(7)]-corazonin and JH play an important role in the control of body-color polymorphism in this locust.     

Corazonin in insects

Corazonin is a peptidergic neurohormone of insects that is expressed in neurosecretory neurons of the pars lateralis of the protocerebrum and transported via nervi corporis cardiaci to the storage lobes of the corpora cardiaca. This peptide occurs with a single isoform in all insects studied so far, with the exception of the Coleoptera in which no corazonin form could be detected. Very few modifications of [Arg(7)]-corazonin, originally isolated from cockroaches, are known, namely [His(7)]-corazonin which is expressed in certain locusts and the stick insect Carausius morosus, and [Thr(4), His(7)]-corazonin recently described from the honey bee Apis mellifera. In this study, we performed a comprehensive screening for corazonin in the different insect groups after detecting of a fourth isoform in a crane fly, Tipula sp. ([Gln(10)]-corazonin). [Arg(7)]-corazonin is distributed in most major lineages of insects, and is thus the ancient form which was present at the time the phylum Insecta evolved. The replacement of Arg with His at position 7 from the N-terminus occurred several times in the evolution of insects. The third isoform, [Thr(4), His(7)]-corazonin, seems to be restricted to bees (Apidae); whereas wasps (Vespidae) and a bumble bee (Apidae) express other corazonins, specifically [His(7)]-corazonin and [Tyr(3), Gln(7), Gln(10)]-corazonin, respectively. A novel corazonin form, [His(4), Gln(7)]-corazonin, was also detected in all South African members of the newly described insect order Mantophasmatodea. The [His(4), Gln(7)]-corazonin separates these species from the Namibian Mantophasmatodea which express [Arg(7)]-corazonin and can be used as a distinct character to distinguish these morphologically similar insects.     

Comparison of structure-activity relations of corazonin using two different bioassay systems

The structure-activity relations of [His(7)]-corazonin were studied using two different bioassay systems; i.e. inhibitory effect on spinning rate in the silkworm, Bombyx mori, and darkening response in albino nymphs of the migratory locust, Locusta migratoria. Deletion of the N-terminus, shortening of the peptide and single amino acid substitutions reduced activity in a similar manner except for the minimum effective dose in the two insects. The results also revealed that the residues at position 1, 3 and 5 were particularly important for biological activity. Despite the different physiological affects, the two insect species exhibited similar structure-activity relationships, suggesting that they might have similar receptor systems.     

The role of

The effect of [His(7)]-corazonin on the body color in Locusta migratoria was examined by varying the injected dose and the time of injection in both an albino and a normal (pigmented) strain. Albino nymphs injected with a high dose (100pmol) of [His(7)]-corazonin at the beginning of the third instar turned completely black in the following instar, whereas those injected with the same dose in the middle of the instar developed black patterns with an orange background color, the body coloration characteristic of normal gregarious (crowded) individuals. Injection at the end of the third instar induced a reddish color with few black spots. Irrespective of the time of injection of the peptide, most of these individuals became completely black after ecdysis to the fifth instar. A similar result was obtained with a lower dose (1pmol), although the color expressed was lighter. In the normal strain, injection of 1nmol or 100pmol into crowded third instar nymphs also caused most of them to become completely black in the fourth and fifth instars, but a lower dose apparently had no influence. These results suggest that the temporal changes in hemolymph titer of [His(7)]-corazonin are important in the expression of body color in L. migratoria.     

The dark-color inducing neuropeptide, [His(7)]-corazonin,causes a shift in morphometic characteristics towards the gregarious phase in isolated-reared (solitarious) Locusta migratoria

The neurohormone, [His⁷]-corazonin is known to induce dark color in the cuticle and epidermis of Locusta migratoria. In the present study, we examined the effects of this hormone on development and morphometrics in two strains, albino and normal, of this locust under isolated conditions. Injection of [His⁷]-corazonin induced dark color in both strains. In either strain, [His⁷]-corazonin injected at the second and third instars did not affect duration of nymphal development or the number of nymphal instars. The shape of the pronotum was more convex in isolated-reared animals than in crowd-reared ones, and injection of [His⁷]-corazonin caused isolated-reared animals to develop a less convex pronotum in the normal strain injected at a high dose (1 nmol×2) but not in the albino strain injected at a low dose (50 pmol×2). [His⁷]-corazonin injected into isolated-reared nymphs caused a shift in classical morphometric ratios (F/C and E/F; F=length of the hind femur, C=maximum width of the head, E=length of the fore wings) towards values typical for crowd-reared (gregarious) individuals of the two strains. This study demonstrated for the first time that [His⁷]-corazonin affected morphometric characteristics in L. migratoria.     

A comparison of phase-related shifts in behavior and morphometrics of an albino strain,deficient in [His(7)]-corazonin,and a normally colored Locusta migratoria strain

The albino Okinawa strain of Locusta migratoria is deficient in the neurohormone [His⁷]-corazonin. This peptide induces darkening of the cuticle, one of the typical features of gregarious locusts. As part of a broader study on the possible role of [His⁷]-corazonin in phase transition, we explored whether corazonin-deficiency might be associated with differences in behavior and morphometrics between albino and normal phenotypes of L. migratoria. Using a modification of the logistic-regression assay of behavioral phase state previously derived for Schistocerca gregaria, we found that there were strain dependent behavioral differences between crowd-reared nymphs of the albino Okinawa and the normally colored African strain, with no evidence of the albino strain being obligatorily solitarious. However, upon isolation, a shift towards more solitarious behavior occurs in both strains, even more profoundly in the Okinawa albinos. A shift could also be recorded in morphometrics. The conclusion is that the albino strain, although showing some solitarious features even when crowd-reared, is not, as has been suggested, obligatory solitarious and, as a consequence, the complete absence of corazonin is not sufficient to bring about the solitarious state.     

飞蝗变型及体色多型的内分泌控制机理

飞蝗具变型现象,散居型和群居型在形态特征、生理机能、行为及体色等方面存在明显差异。保幼激素已被证实能诱导飞蝗散居型绿色的出现。近年,从飞蝗心侧体成功地分离了一种神经肽——黑化诱导激素([His^7]-corazonin),并用白化型进行检测,证实了其对体色黑化作用的活性。不同时间对飞蝗若虫注射不同剂量的[His^7]-corazonin,能诱导出现除绿色以外的散居型体色,如浅褐色、褐色、赤褐色、黑色等;也能诱导出现群居型的体色,即黑色配以橘黄色的底色。而且,对散居型若虫注射[His^7]-corazonin能诱导其形态向群居型转换。这些研究证实[His^7]-corazonin对飞蝗的变型有着重要的控制作用,但又不是唯一的。     

An improved method of light-induced pigmentation.

An improved procedure was developed whereby a primary light signal can be intensified and made visible by activation of a pre-tyrosinase (pre-phenoloxidase) enzyme [isolated from silkworm (Bombyx mori)] by alpha-chymotrypsin; this activation results from the light-activated conversion of the inactive cis-cinnamoyl-alpha-chymotrypsin.     

Postembryonic changes in circadian photo-responsiveness rhythms of optic lobe interneurons in the cricket Gryllus bimaculatus

The photo-responsiveness of 2 groups of interneurons responding to light in the protocerebrum was investigated at 2 developmental stages, the last instar nymphs and adults, in the cricket Gryllus bimaculatus. The cricket is diurnally active during the nymphal stage but becomes nocturnal as an adult. In both adults and nymphs, light-induced responses of optic lobe light-responding interneurons that conduct light information from the optic medulla to the lobula and the cerebral lobe showed a circadian rhythm peaking during the subjective night. Amplitudes of the rhythms were not significantly different between adults and nymphs, but adults showed more stable day and night states than did nymphs. The medulla bilateral neurons that interconnect the bilateral medulla areas of the optic lobe also showed circadian rhythms in their light-induced responses in both adults and nymphs. The rhythm had a clear peak and a trough in adults, and its amplitude was significantly greater than that of nymphs. These results suggest that the 2 classes of interneurons are differentially controlled by the circadian clock. The difference might be related to their functional roles in the animal's circadian behavioral organization.     

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.     

cDNA cloning and nuclear localization of the circadian neuropeptide designated as pigment-dispersing factor PDF in the cricket Gryllus bimaculatus

Pigment-dispersing factor (PDF) was recently reported to be a principal circadian neuromodulator involved in transmitting circadian rhythms of daily locomotion in insects. In Drosophila, PDF functions in some of the neurons expressing the clock genes period, timeless, Clock, and cycle, and those clock genes in turn regulate pdf gene expression. In the present study, we cloned a cDNA encoding PDF in the brain of a nocturnal insect, the cricket Gryllus bimaculatus, and found that an isolated clone (310 bp) codes for an extraordinarily short precursor protein with no definite signal sequence, but a nuclear localization signal (NLS)-like sequence instead. The cricket PDF exhibits high sequence identity (78-94%) and similarity (89-100%) to insect PDFs and also to crustacean beta-PDH peptides. In the optic lobes of G. bimaculatus there are PDF-immunoreactive neurons in both the medulla and lamina neuropiles. Among the strongly immunoreactive lamina PDF neurons, on electron microscopy we identified cells exhibiting distinct staining that is not only cytoplasmic but also nuclear. When GFP-fused PDF precursor proteins were expressed in COS-7 cells, distinct translocation of the fusion protein into the nucleus was observed. This is the first finding of PDF peptide in the nucleus, which suggests a fundamental role of PDF peptide per se in the circadian clock system.     

The dark-colour-inducing neurohormone of locusts in relation to an albino mutant of Schistocerca gregaria

In the albino mutant of an Okinawa strain of Locusta migratoria (L.) (Orthoptera: Acrididae), albinism is caused by the absence of the dark‐colour‐inducing neurohormone (DCIN), which is present in the corpora cardiaca (CC) of normally coloured phenotypes. This study tests whether the absence of DCIN is responsible for albinism in an albino mutant of another locust, Schistocerca gregaria (Forsk.) (Orthoptera: Acrididae). This seemed feasible because a single Mendelian unit controls albinism in both species. However, implantation of CC, or injection of an extract of CC, from albino donors of S. gregaria, induce dark coloration in crowded nymph recipients of the Okinawa albino mutant of L. migratoria, as effectively as do implanted CC, or injections of extract of CC, from normal phenotype donors of S. gregaria. Therefore, DCIN is present in the albino mutant of S. gregaria, and consequently, the albinism in this mutant is not caused by its absence. Implantation of CC, or injection of extracts of CC, from albino donors of S. gregaria to conspecific albino nymphs does not induce darkening. Only extremely high doses of synthetic DCIN injected into albino nymphs of S. gregaria are effective, inducing some darkening. The dose to induce such darkening in albino nymphs of S. gregaria is 50 nmol, ≈ 5 × 10⁶ times higher than that (10 femtomol) needed to induce equivalent darkening in nymphs of the Okinawa albinos of L. migratoria. The results are discussed and some possible explanations of the observed effects outlined.     

Evidence of selection at melanin synthesis pathway loci during silkworm domestication

The domesticated silkworm (Bombyx mori) was domesticated from wild silkworm (Bombyx mandarina) more than 5,000 years ago. During domestication, body color between B. mandarina and B. mori changed dramatically. However, the molecular mechanism of the silkworm body color transition is not known. In the present study, we examined within- and between-species nucleotide diversity for eight silkworm melanin synthesis pathway genes, which play a key role in cuticular pigmentation of insects. Our results showed that the genetic diversity of B. mori was significantly lower than that of B. mandarina and 40.7% of the genetic diversity of wild silkworm was lost in domesticated silkworm. We also examined whether position effect exists among melanin synthesis pathway genes in B. mandarina and B. mori. We found that the upstream genes have significantly lower levels of genetic diversity than the downstream genes, supporting a functional constraint hypothesis (FCH) of metabolic pathway, that is, upstream enzymes are under greater selective constraint than downstream enzymes because upstream enzymes participate in biosynthesis of a number of metabolites. We also investigated whether some of the melanin synthesis pathway genes experienced selection during domestication. Neutrality test, coalescent simulation, as well as network and phylogenetic analyses showed that tyrosine hydroxylase (TH) gene was a domestication locus. Sequence analysis further suggested that a putative expression enhancer (Abd-B-binding site) in the intron of TH gene might be disrupted during domestication. TH is the rate-limiting enzyme of melanin synthesis pathway in insects. Real-time polymerase chain reaction assay did show that the relative expression levels of TH gene in B. mori were significantly lower than that in B. mandarina at three different developmental stages, which is consistent with light body color of domesticated silkworm relative to wild silkworm. Therefore, we speculated that expression change of TH gene may contribute to the body color transition from B. mandarina to B. mori. Our results emphasize the exceptional role of gene expression regulation in morphological transition of domesticated animals.     

Purification, structural characterization, and myotropic activity of a peptide related to des-Arg(9)-bradykinin from an elasmobranch fish, the little skate, Leucoraja erinacea

A bradykinin (BK)-related peptide was isolated from heat-denaturated plasma from an elasmobranch fish, the little skate, Leucoraja erinacea after incubation with porcine pancreatic kallikrein. The primary structure of the peptide (H-Gly-Ile-Thr-Ser-Trp-Leu-Pro-Phe-OH; skate BK) shows limited structural similarity to the mammalian B1 receptor agonist, des-Arg(9)-BK. The myotropic activities of synthetic skate BK, and the analog skate [Arg(9)]BK, were examined in isolated skate vascular and intestinal smooth muscle preparations. Skate BK produced a concentration-dependent constriction of the mesenteric artery (EC(50)=4.37x10(-8)M; maximum response=103.4+/-10.23% of the response to 60mM KCl) but the response to skate [Arg(9)]BK was appreciably weaker (response to 10(-6)M=73.0+/-23.4% of the response to 60mM KCl). Neither the first branchial gill arch nor the ventral aorta responded to either purified peptide. Skate BK also produced a concentration-dependent constriction of intestinal smooth muscle preparations (EC(50)=2.74x10(-7)M; maximum response 31.0+/-12.2% of the response to 10(-5)M acetylcholine). Skate [Arg(9)]BK was without effect on the intestinal preparation. The data provide evidence for the existence of the kallikrein-kinin system in a phylogenetically ancient vertebrate group and the greater potency of skate BK compared with the analog skate [Arg(9)]BK suggests that the receptor mediating vascular responses resembles the mammalian B1 receptor more closely than the B2 receptor.