Reinhard Predel

A genome-wide inventory of neurohormone GPCRs in the red flour beetle Tribolium castaneum ☆

Insect neurohormones (biogenic amines, neuropeptides, and protein hormones) and their G protein-coupled receptors (GPCRs) play a central role in the control of behavior, reproduction, development, feeding and many other physiological processes. The recent completion of several insect genome projects has enabled us to obtain a complete inventory of neurohormone GPCRs in these insects and, by a comparative genomics approach, to analyze the evolution of these proteins. The red flour beetle Tribolium castaneum is the latest addition to the list of insects with a sequenced genome and the first coleopteran (beetle) to be sequenced. Coleoptera is the largest insect order and about 30% of all animal species living on earth are coleopterans. Some coleopterans are severe agricultural pests, which is also true for T. castaneum, a global pest for stored grain and other dried commodities for human consumption. In addition, T. castaneum is a model for insect development. Here, we have investigated the presence of neurohormone GPCRs in Tribolium and compared them with those from the fruit fly Drosophila melanogaster (Diptera) and the honey bee Apis mellifera (Hymenoptera). We found 20 biogenic amine GPCRs in Tribolium (21 in Drosophila; 19 in the honey bee), 48 neuropeptide GPCRs (45 in Drosophila; 35 in the honey bee), and 4 protein hormone GPCRs (4 in Drosophila; 2 in the honey bee). Furthermore, we identified the likely ligands for 45 of these 72 Tribolium GPCRs. A highly interesting finding in Tribolium was the occurrence of a vasopressin GPCR and a vasopressin peptide. So far, the vasopressin/GPCR couple has not been detected in any other insect with a sequenced genome (D. melanogaster and six other Drosophila species, Anopheles gambiae, Aedes aegypti, Bombyx mori, and A. mellifera). Tribolium lives in very dry environments. Vasopressin in mammals is the major neurohormone steering water reabsorption in the kidneys. Its presence in Tribolium, therefore, might be related to the animals need to effectively control water reabsorption. Other striking differences between Tribolium and the other two insects are the absence of the allatostatin-A, kinin, and corazonin neuropeptide/receptor couples and the duplications of other hormonal systems. Our survey of 340 million years of insect neurohormone GPCR evolution shows that neuropeptide/receptor couples can easily duplicate or disappear during insect evolution. It also shows that Drosophila is not a good representative of all insects, because several of the hormonal systems that we now find in Tribolium do not exist in Drosophila.

Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding

Maternal metabolic homeostasis exerts long-term effects on the offsprings health outcomes. Here, we demonstrate that maternal high-fat diet (HFD) feeding during lactation predisposes the offspring for obesity and impaired glucose homeostasis in mice, which is associated with an impairment of the hypothalamic melanocortin circuitry. Whereas the number and neuropeptide expression of anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP) neurons, electrophysiological properties of POMC neurons, and posttranslational processing of POMC remain unaffected in response to maternal HFD feeding during lactation, the formation of POMC and AgRP projections to hypothalamic target sites is severely impaired. Abrogating insulin action in POMC neurons of the offspring prevents altered POMC projections to the preautonomic paraventricular nucleus of the hypothalamus (PVH), pancreatic parasympathetic innervation, and impaired glucose-stimulated insulin secretion in response to maternal overnutrition. These experiments reveal a critical timing, when altered maternal metabolism disrupts metabolic homeostasis in the offspring via impairing neuronal projections, and show that abnormal insulin signaling contributes to this effect.

Peptidomics of CNS-associated neurohemal systems of adult Drosophila melanogaster: A mass spectrometric survey of peptides from individual flies

Neuropeptides are important messenger molecules that influence nearly all physiological processes. In insects, they can be released as neuromodulators within the central nervous system (CNS) or as neurohormones into the hemolymph. We analyzed the peptidome of neurohormonal release sites and associated secretory peptidergic neurons of adult Drosophila melanogaster. MALDI‐TOF mass spectrometric analyzes were performed on single organs or cell cluster from individual flies. This first peptidomic characterization in adult fruit flies revealed 32 different neuropeptides. Peptides not directly predictable from previously cloned or annotated precursor genes were sequenced by tandem mass spectrometry. These peptides turned out to be either intermediate products of neuropeptide processing or shorter versions of known peptides. We found that the peptidome of the CNS‐associated neurohemal organs is tagma‐specific in Drosophila. Abdominal neurohemal organs and their supplying peptidergic neurons contain the capa gene products periviscerokinins and pyrokinin‐1, thoracic neurohemal organs contain FMRFamides, and the neurohemal release sites of the brain contain pyrokinin‐12‐15, pyrokinin‐2, corazonin, myosuppressin, and sNPF as their major putative release products. Our results show that peptidomic approaches are well suited to study differential neuropeptide expression or posttranslational modifications in morphologically defined parts of the nervous system and in a developmental and physiological context in animals as small as Drosophila melanogaster. J. Comp. Neurol. 474:379–392, 2004.

Genomics, transcriptomics, and peptidomics of Daphnia pulex neuropeptides and protein hormones.

We report 43 novel genes in the water flea Daphnia pulex encoding 73 predicted neuropeptide and protein hormones as partly confirmed by RT-PCR. MALDI-TOF mass spectrometry identified 40 neuropeptides by mass matches and 30 neuropeptides by fragmentation sequencing. Single genes encode adipokinetic hormone, allatostatin-A, allatostatin-B, allatotropin, Ala(7)-CCAP, CCHamide, Arg(7)-corazonin, DENamides, CRF-like (DH52) and calcitonin-like (DH31) diuretic hormones, two ecdysis-triggering hormones, two FIRFamides, one insulin, two alternative splice forms of ion transport peptide (ITP), myosuppressin, neuroparsin, two neuropeptide-F splice forms, three periviscerokinins (but no pyrokinins), pigment dispersing hormone, proctolin, Met(4)-proctolin, short neuropeptide-F, three RYamides, SIFamide, two sulfakinins, and three tachykinins. There are two genes for a preprohormone containing orcomyotropin-like peptides and orcokinins, two genes for N-terminally elongated ITPs, two genes (clustered) for eclosion hormones, two genes (clustered) for bursicons alpha, beta, and two genes (clustered) for glycoproteins GPA2, GPB5, three genes for different allatostatins-C (two of them clustered) and three genes for IGF-related peptides. Detailed comparisons of genes or their products with those from insects and decapod crustaceans revealed that the D. pulex peptides are often closer related to their insect than to their decapod crustacean homologues, confirming that branchiopods, to which Daphnia belongs, are the ancestor group of insects.

Biology of the CAPA peptides in insects

Abstract.CAPA peptides have been isolated from a broad range of insect species as well as an arachnid, and can be grouped into the periviscerokinin and pyrokinin peptide families. In insects, CAPA peptides are the characteristic and most abundant neuropeptides in the abdominal neurohemal system. In many species, CAPA peptides exert potent myotropic effects on different muscles such as the heart. In others, including blood-sucking insects able to transmit serious diseases, CAPA peptides have strong diuretic or anti-diuretic effects and thus are potentially of medical importance. CAPA peptides undergo cell-type-specific sorting and packaging, and are the first insect neuropeptides shown to be differentially processed. In this review, we discuss the current knowledge on the structure, distribution, receptors and physiological actions of the CAPA peptides.

Genomics and Peptidomics of Neuropeptides and Protein Hormones Present in the Parasitic Wasp Nasonia vitripennis

Neuropeptides and protein hormones constitute a very important group of signaling molecules, regulating central physiological processes such as reproduction, development, and behavior. Using a bioinformatics approach, we screened the recently sequenced genome of the parasitic wasp, Nasonia vitripennis, for the presence of these signaling molecules and annotated 30 precursor genes encoding 51 different mature neuropeptides or protein hormones. Twenty-four of the predicted mature Nasonia neuropeptides could be experimentally confirmed by mass spectrometry. We also discovered a completely novel neuropeptide gene in Nasonia, coding for peptides containing the C-terminal sequence RYamide. This gene has orthologs in nearly all arthropods with a sequenced genome, and its expression in mosquitoes was confirmed by mass spectrometry. No precursor could be identified for N-terminally extended FMRFamides, even though their putative G protein coupled receptor (GPCR) is present in the Nasonia genome. Neither the precursor nor the putative receptor could be identified for allatostatin-B, capa, the glycoprotein hormones GPA2/GPB5, kinin, proctolin, sex peptide, and sulfakinin, arguing that these signaling systems are truly absent in the wasp. Also, antidiuretic factors, allatotropin, and NPLP-like precursors are missing in Nasonia, but here the receptors have not been identified in any insect, so far. Nasonia (Hymenoptera) has the lowest number of neuropeptide precursor genes compared to Drosophila melanogaster, Aedes aegypti (both Diptera), Bombyx mori (Lepidoptera), Tribolium castaneum (Coleoptera), Apis mellifera (Hymenoptera), and Acyrthosiphon pisum (Hemiptera). This lower number of neuropeptide genes might be related to Nasonias parasitic life.

Peptidergic neurohemal system of an insect: Mass spectrometric morphology

Neuropeptides are by far the most diverse group of messenger molecules in insects. To understand cell signaling and function, it is essential to reveal the complete neuropeptide profile of a single neuron/nerve/neurohemal organ first. In this study, matrix‐assisted laser desorption ionization time of flight (MALDI‐TOF) mass spectrometry was used to analyze the peptidergic system of an insect, focussing on the neurohemal structures. Major neurohemal organs were investigated, including the retrocerebral complex, perisympathetic organs, and all nerves supplying these organs with neurosecretions. Additionally, peripheral neurohemal release sites such as the dilator muscle of the antennal circulatory organ and lateral heart nerves were studied, as well as parts of the stomatogastric nervous system. The following neuropeptide families were analyzed: kinins, allatostatins, leucomyosuppressin, corazonin, adipokinetic hormones, myoinhibitory peptide, sulfakinins, periviscerokinins, YLSamide, VEAacid, SKNacid, proctolin, the head peptide, and pyrokinins. Beyond a contribution to a map of the distribution of neuropeptides in a neurohemal system, the following conclusions can be drawn from these experiments. (1) Nearly all abundant peaks in the different mass spectra represent peptides that have already been identified. (2) Although only adult males were used in this study, variations in the peptide abundances were observed that are possibly correlated with different physiological/developmental conditions. (3) Peptides have a body‐region–specific distribution in the neurohemal system. (4) A clear compartmentalisation of the retrocerebral complex could be observed. J. Comp. Neurol. 436:363–375, 2001.

Myoinhibitory neuropeptides in the American cockroach

A large number of myostimulatory neuropeptides from neurohaemal organs of the American cockroach have been described since 1989. These peptides, isolated from the retrocerebral complex and abdominal perisympathetic organs, are thought to be released as hormones. To study the coordinated action of these neuropeptides in the regulation of visceral muscle activity, it might be necessary to include myoinhibitors as well, however, not a single myoinhibitory neuropeptide of the American cockroach has been described so far. To fill this gap, we describe the isolation of LMS (leucomyosuppressin) and Pea-MIP (myoinhibitory peptide) from neurohaemal organs of the American cockroach. LMS was very effective in inhibiting phasic activity of all visceral muscles tested. It was found in the corpora cardiaca of different species of cockroaches, as well as in related insect groups, including mantids and termites. Pea-MIP which is strongly accumulated in the corpora cardiaca was not detected with a muscle bioassay system but when searching for tryptophane-containing peptides using a diode-array detector. This peptide caused only a moderate inhibition in visceral muscle assays. The distribution of Pea-MIP in neurohaemal organs and cells supplying these organs with Pea-MIP immunoreactive material, is described. Additionally to LMS and Pea-MIP, a member of the allatostatin peptide family, known to exhibit inhibitory properties in other insects, was tested in visceral muscle assays. This allatostatin was highly effective in inhibiting spontaneous activity of the foregut, but not of other tested visceral muscles of the American cockroach.

The Drosophila hugin gene codes for myostimulatory and ecdysis-modifying neuropeptides

In a genomic screen we isolated the Drosophila gene hugin (hug, cytology 87C1-2) by cross-hybridisation to a human glial cell line-derived neurotrophic factor cDNA. Upon cDNA sequence analysis and in vitro expression assays, the hugin gene was found to encode a signal peptide containing proprotein that was further processed in Schneider-2 cells into peptides similar to known neuropeptides. Two of the peptides were similar to FXPRL-amides (pyrokinins) and to the ecdysis-triggering hormone, respectively. The former displayed myostimulatory activity in a bioassay on the cockroach hyperneural muscle preparation, as well as in the Drosophila heart muscle assay. Hugin is expressed during the later half of embryogenesis and during larval stages in a subgroup of neurosecretory cells of the suboesophageal ganglion. Ubiquitous ectopic hugin expression resulted in larval death predominantly at or shortly after ecdysis from second to third instar, suggesting that at least one of the posttranslational cleavage products affects molting of the larva by interfering with the regulation of ecdysis.

Neuropeptidomics of the mosquito Aedes aegypti

Neuropeptidomic data were collected on the mosquito Ae. aegypti, which is considered the most tractable mosquito species for physiological and endocrine studies. The data were solely obtained by direct mass spectrometric profiling, including tandem fragmentation, of selected tissues from single specimens, which yielded a largely complete accounting of the putative bioactive neuropeptides; truncated neuropeptides with low abundance were not counted as mature peptides. Differential processing within the CNS was detected for the CAPA-precursor, and differential post-translational processing (pyroglutamate formation) was detected for AST-C and CAPA-PVK-2. For the first time in insects, we succeeded in the direct mass spectrometric profiling of midgut tissue which yielded a comprehensive and immediate overview of the peptides involved in the endocrine system of the gut. Head peptides which were earlier identified as the most abundant RFamides of Ae. aegypti, were not detected in any part of the CNS or midgut. This study provides a framework for future investigations on mosquito endocrinology and neurobiology. Given the high sequence similarity of neuropeptide precursors identified in other medically important mosquitoes, conclusions regarding the peptidome of Ae. aegypti likely are applicable to these mosquitoes.