Hans-Willi Honegger

Identification of the Gene Encoding Bursicon, an Insect Neuropeptide Responsible for Cuticle Sclerotization and Wing Spreading

To accommodate growth, insects must periodically replace their exoskeletons. After shedding the old cuticle, the new soft cuticle must sclerotize. Sclerotization has long been known to be controlled by the neuropeptide hormone bursicon, but its large size of 30 kDa has frustrated attempts to determine its sequence and structure. Using partial sequences obtained from purified cockroach bursicon, we identified the Drosophila melanogaster gene CG13419 as a candidate bursicon gene. CG13419 encodes a peptide with a predicted final molecular weight of 15 kDa, which likely functions as a dimer. This predicted bursicon protein belongs to the cystine knot family, which includes vertebrate transforming growth factor-beta (TGF-beta) and glycoprotein hormones. Point mutations in the bursicon gene cause defects in cuticle sclerotization and wing expansion behavior. Bioassays show that these mutants have decreased bursicon bioactivity. In situ hybridization and immunocytochemistry revealed that bursicon is co-expressed with crustacean cardioactive peptide (CCAP). Transgenic flies that lack CCAP neurons also lacked bursicon bioactivity. Our results indicate that CG13419 encodes bursicon, the last of the classic set of insect developmental hormones. It is the first member of the cystine knot family to have a defined function in invertebrates. Mutants show that the spectrum of bursicon actions is broader than formerly demonstrated.

Bursicon Functions within the Drosophila CNS to Modulate Wing Expansion Behavior, Hormone Secretion, and Cell Death

Hormones are often responsible for synchronizing somatic physiological changes with changes in behavior. Ecdysis (i.e., the shedding of the exoskeleton) in insects has served as a useful model for elucidating the molecular and cellular mechanisms of this synchronization, and has provided numerous insights into the hormonal coordination of body and behavior. An example in which the mechanisms have remained enigmatic is the neurohormone bursicon, which, after the final molt, coordinates the plasticization and tanning of the initially folded wings with behaviors that drive wing expansion. The somatic effects of the hormone are governed by bursicon that is released into the blood from neurons in the abdominal ganglion (the BAG), which die after wing expansion. How bursicon induces the behavioral programs required for wing expansion, however, has remained unknown. Here we show by targeted suppression of excitability that a pair of bursicon-immunoreactive neurons distinct from the BAG and located within the subesophageal ganglion in Drosophila (the BSEG) is involved in controlling wing expansion behaviors. Unlike the BAG, the BSEG arborize widely in the nervous system, including within the abdominal neuromeres, suggesting that, in addition to governing behavior, they also may modulate the BAG. Indeed, we show that animals lacking bursicon receptor function have deficits both in the humoral release of bursicon and in posteclosion apoptosis of the BAG. Our results reveal novel neuromodulatory functions for bursicon and support the hypothesis that the BSEG are essential for orchestrating both the behavioral and somatic processes underlying wing expansion.

Glutamate‐like immunoreactivity marks compartments of the mushroom bodies in the brain of the cricket

In the mushroom bodies of the brain of the cricket Gryllus bimaculatus, the distribution of glutamate‐like immunoreactivity is shown by using several immunocytochemical staining protocols and confocal and conventional microscopy. Glutamate‐like staining of intrinsic cells of mushroom bodies (Kenyon cells), their axons and projections, is demonstrated for the first time. Two types of Kenyon cells constituting distinct, separated populations within the perikaryal layer and in prominent neuropilar subcompartments exhibit strong (type III cells) or medium (type II cells) glutamate‐like immunoreactivity, whereas the small neurons of a central population (type I cells) lack staining above background. Type III Kenyon cells display a strong immunoreactivity similarly found in some giant neurons and in identified antennal motorneurons by using glutamate as an excitatory transmitter, indicating that also distinct populations of the Kenyon cells use glutamate as a putative transmitter. The pattern of glutamate‐like immunoreactivity in the mushroom bodies and in other parts of the brain is different from γ‐aminobutyric acid (GABA)‐like immunoreactivity (investigated for comparison). GABA‐like immunostaining is particularly prominent in the mushroom body calyces where Kenyon cells have their dendritic branchings. Differences in glutamate‐like immunostaining in Kenyon cell subpopulations, together with differences in their arborization and axonal projection patterns, indicate a functional diversity of these neurons. J. Comp. Neurol. 418:227–239, 2000.

Cardioacceleratory function of the neurohormone CCAP in the mosquito Anopheles gambiae

SUMMARY Crustacean cardioactive peptide (CCAP) is a highly conserved arthropod neurohormone that is involved in ecdysis, hormone release and the modulation of muscle contractions. Here, we determined the CCAP gene structure in the malaria mosquito Anopheles gambiae, assessed the developmental expression of CCAP and its receptor and determined the role that CCAP plays in regulating mosquito cardiac function. RACE sequencing revealed that the A. gambiae CCAP gene encodes a neuropeptide that shares 100% amino acid identity with all sequenced CCAP peptides, with the exception of Daphnia pulex. Quantitative RT-PCR showed that expression of CCAP and the CCAP receptor displays a bimodal distribution, with peak mRNA levels in second instar larvae and pupae. Injection of CCAP revealed that augmenting hemocoelic CCAP levels in adult mosquitoes increases the anterograde and retrograde heart contraction rates by up to 28%, and increases intracardiac hemolymph flow velocities by up to 33%. Partial CCAP knockdown by RNAi had the opposite effect, decreasing the mosquito heart rate by 6%. Quantitative RT-PCR experiments showed that CCAP mRNA is enriched in the head region, and immunohistochemical experiments in newly eclosed mosquitoes detected CCAP in abdominal neurons and projections, some of which innervated the heart, but failed to detect CCAP in the abdomens of older mosquitoes. Instead, in older mosquitoes CCAP was detected in the pars lateralis, the subesophageal ganglion and the corpora cardiaca. In conclusion, CCAP has a potent effect on mosquito circulatory physiology, and thus heart physiology in this dipteran insect is under partial neuronal control.

Cellular localization of bursicon using antisera against partial peptide sequences of this insect cuticle‐sclerotizing neurohormone

Bursicon is the final neurohormone released at the end of the molting cycle. It triggers the sclerotization (tanning) of the insect cuticle. Until now, its existence has been verified only by bioassays. In an attempt to identify this important neurohormone, bursicon was purified from homogenates of 2,850 nerve cords of the cockroach Periplaneta americana by using high performance liquid chromatography technology and two‐dimensional gel electrophoresis. Bursicon bioactivity was found in four distinct protein spots at approximately 30 kDa between pH 5.3 and 5.9. The protein of one of these spots at pH 5.7 was subsequently microsequenced, and five partial amino acid sequences were retrieved. Evidence is presented that two of these sequences are derived from bursicon. Antibodies raised against the two sequences labeled bursicon‐containing neurons in the central nervous systems of P. americana. One of these antisera labeled bursicon‐containing neurons in the crickets Teleogryllus commodus and Gryllus bimaculatus, and the moth Manduca sexta. A cluster of four bilaterally paired neurons in the brain of Drososphila melanogaster was also labeled. In addition, this antiserum detected three spots corresponding to bursicon in Western blots of two‐dimensional gels. The 12‐amino acid sequence detected by this antiserum, thus, seems to be conserved even among species that are distantly related. J. Comp. Neurol. 452:163–177, 2002.

The Bursicon Gene in Mosquitoes: An Unusual Example of mRNA Trans-splicing

The bursicon gene in Anopheles gambiae is encoded by two loci. Burs124 on chromosome arm 2L contains exons 1, 2, and 4, while burs3 on arm 2R contains exon 3. Exon 3 is efficiently spliced into position in the mature transcript. This unusual gene arrangement is ancient within mosquitoes, being shared by Aedes aegypti and Culex pipiens.

Bursicon, the cuticle sclerotizing hormone - comparison of its molecular mass in different insects

Abstract We have shown in a recent study that bursicon, which induces the tanning of the cuticle in freshly molted insects, is a 30 kDa protein in the meal beetle Tenebrio molitor . We now show that bursicon in the insect species Calliphora erythrocephala, Periplaneta americana, Gryllus bimaculatus and Locusta migratoria is also a protein of about 30 kDa. The determination of bursicons molecular mass was accomplished by SDS-PAGE of nervous system homogenates, subsequent division of the gel into slices, protein elution from these slices, and a test for bursicon activity of the eluted proteins in the ligated fly bioassay. Bursicon activity can only be eluted from one gel slice spanning the molecular mass around 30 kDa. Dose-response curves for bursicon of different ventral ganglia show that there are appreciable differences in the bursicon content between thoracic and abdominal ganglia and between developmental stages. These differences vary from insect to insect. An attempt to determine the molecular mass of Manduca sexta bursicon was not possible because the ligated fly bioassay does not work with homogenates of the nervous system of Manduca . Homogenates of abdominal ganglia of Homarus americanus show a positive score in the ligated fly bioassay but not those of its thoracic ganglia. The results indicate that bursicon may have a similar structure throughout the arthropods, but with distinct exceptions.

INVOLVEMENT OF THE SUBOESOPHAGEAL AND THORACIC GANGLIA IN THE CONTROL OF ANTENNAL MOVEMENTS IN CRICKETS

Abstract In crickets (Gryllus campestris, Gryllus bimaculatus) the contribution of the suboesophageal ganglia (SOG) and thoracic ganglia to the generation of antennal movements during visual tracking, walking and flight was investigated by the transection of connectives. Transection of one circumoesophageal connective abolished the movements and postures of the antenna ipsilateral to the lesion, while the contralateral antenna behaved normally. Simple antennal reflexes remained. Transection of one neck connective reduced fast components of antennal movements during tracking and walking. During flight the ipsilateral antenna could not be maintained in a prolonged forward position. Antennal movements during tracking and walking appeared normal after transection of one connective between pro- and mesothoracic ganglia. However, the antennal flight posture required uninterrupted connections between brain and mesothoracic ganglion. The ablation of more posterior ganglia had no effect on the antennal behaviours investigated. Recordings from an antennal motor nerve revealed a unilateral net excitation relayed via the SOG to the brain. Two ascending interneurones with activity closely correlated with antennal movements are candidates for such a relay function. The data show that the brain is not sufficient to generate antennal movements and postures as integral parts of several behaviours. The SOG and the thoracic ganglia are required in addition.

Identification, developmental expression, and functions of bursicon in the tobacco hawkmoth, Manduca sexta

During posteclosion, insects undergo sequential processes of wing expansion and cuticle tanning. Bursicon, a highly conserved neurohormone implicated in regulation of these processes, was characterized recently as a heterodimeric cystine knot protein in Drosophila melanogaster. Here we report the predicted precursor sequences of bursicon subunits (Masburs and Maspburs) in the moth Manduca sexta. Distinct developmental patterns of mRNA transcript and subunit‐specific protein labeling of burs and pburs as well as crustacean cardioactive peptide in neurons of the ventral nervous system were observed in pharate larval, pupal, and adult stages. A subset of bursicon neurons located in thoracic ganglia of larvae expresses ecdysis‐triggering hormone (ETH) receptors, suggesting that they are direct targets of ETH. Projections of bursicon neurons within the CNS and to neurohemal secretory sites are consistent with both central signaling and circulatory hormone functions. Intrinsic cells of the corpora cardiaca contain pburs transcripts and pburs‐like immunoreactivity, whereas burs transcripts and burs‐like immunoreactivity were absent in these cells. Recombinant bursicon induces both wing expansion and tanning, whereas synthetic eclosion hormone induces only wing expansion. J. Comp. Neurol. 506:759–774, 2008.

The antennal motor system of crickets : modulation of muscle contractions by a common inhibitor, DUM neurons, and proctolin

In the crickets Gryllus bimaculatus and Gryllus campestris, the two intrinsic antennal muscles in the scape (first antennal segment) control antennal movements in the horizontal plane. Of the 17 excitatory antennal motoneurons, three motoneurons, two fast and one slow, can be stimulated selectively and their effect on muscle contraction, i.e. antennal movement, measured. Simultaneously, either a common inhibitor (CI) neuron or two DUM neurons can be stimulated and the effect on the slow and/or fast muscle contraction measured. The activity of the common inhibitor affected only slow muscle contractions. It decreased contraction rate, increased relaxation rate and suppressed prolonged muscle tension. This effect was blocked by picrotoxin. DUM neuron stimulation affected both slow and fast contractions. It reduced slow and enhanced fast contractions but in only 10% of the experiments could this effect be detected. DUM neuron activity could be mimicked by octopamine application. Proctolin application enhanced both slow and fast contractions but did not increase muscle tension in the absence of motoneuron activity. The results are discussed in relation to the large variability of possible antennal movements during behaviors.