Characterization of the gut peptidome and the function of brain-gut peptides with regard to food intake and metabolism in Drosophila melanogaster and the agricultural pest Delia radicum

Regulatory peptides, which comprise neuropeptides and peptide hormones, are cell-cell signaling molecules that control a variety of biological processes in insects and other metazoans. The insect midgut, like the mammalian digestive system, contains numerous peptide-producing endocrine cells, and the diversity of insect enteroendocrine peptides gives a hint at their relevance for metabolism, energy balance and feeding behavior. In the first study, we characterized the midgut peptidome of adult and larval Drosophila melanogaster by extraction of peptides from midgut tissue and subsequent LC-MS/MS analysis of peptide structures. By this means we identified 24 peptides originating from 9 different peptide precursors. All gut peptides were found in identical form within the CNS and thus represent brain-gut peptides. Processing of Drosophila neuropeptide hormones was previously shown to require the subtilisin-like proprotein convertase 2 (dPC2, AMON). Our results suggest a general need of AMON for gut peptide production as well. In the second study, we could expand the knowledge on peptide structures of relevant insects. We investigated the peptidome of the cabbage maggot (i.e., the larva of the cabbage root fly Delia radicum), which causes substantial agricultural damage by feeding on plant roots. By mass spectrometric analysis of CNS, neurohemal organ and gut tissue, we could characterize 38 peptides belonging to diverse insect peptide families. Moreover, we identified a new peptide with sequence similarity to the eclosion hormone precursor of several Drosophila species. Immunocytochemical characterization of peptide-producing neurons and enteroendocrine cells in cabbage maggots showed that peptide distribution was largely similar to Drosophila larvae. The observed similarities in the peptidergic systems of both species suggest that Drosophila can serve as a genetically accessible pest species model in terms of peptidergic regulation of, e.g., metabolism. In the future, our results could be of use for the development of a targeted, peptide-based management of cabbage root flies. In the third study, we analyzed the role of allatostatin A (AstA), a peptide family commonly occurring in insects (and other arthropods). Previous studies had already demonstrated a role for AstA in metabolic regulation and nutritional homeostasis of Drosophila. We addressed the question whether specific effects were connected to the activity of certain subsets of the numerous AstA-producing cells found in adult fruit flies. AstA neurons are located in different regions of the CNS. The thorax also contains a few peripheral AstA neurons. The hindgut and the posteriormost portion of the midgut are innervated by central AstA neurons. In addition, a large number of enteroendocrine AstA cells are scattered across the epithelium of the posterior midgut. Thermogenetic activation of certain AstA cells significantly reduced food intake of flies, and also considerably diminished their locomotor activity. The combination of our results with findings of a previous study suggested that two pairs of AstA-producing posterior lateral protocerebrum neurons function to promote satiety, while enteroendocrine AstA cells seem to regulate locomotor activity. In addition, our findings indicated that AstA cells might directly and indirectly influence defecation behavior, while no effect on water and ion homeostasis could be observed. Furthermore, we tested the effect of synthetic AstA peptides on isolated midguts in vitro and observed a dose-dependent inhibition of midgut motility. Downregulation of AstA receptor mRNA in the gut musculature via RNAi showed that the DAR-2 receptor mediates the myoinhibitory effect of AstA peptides. Altogether, by influencing satiety, locomotion, gut peristalsis and possibly also defecation, AstA cells appear to affect different levels of metabolism and different tissues, seemingly promoting several interrelated processes connected to food intake

Characterization of the gut peptidome and the function of brain-gut peptides with regard to food intake and metabolism in Drosophila melanogaster and the agricultural pest Delia radicum

Regulatory peptides, which comprise neuropeptides and peptide hormones, are cell-cell signaling molecules that control a variety of biological processes in insects and other metazoans. The insect midgut, like the mammalian digestive system, contains numerous peptide-producing endocrine cells, and the diversity of insect enteroendocrine peptides gives a hint at their relevance for metabolism, energy balance and feeding behavior. In the first study, we characterized the midgut peptidome of adult and larval Drosophila melanogaster by extraction of peptides from midgut tissue and subsequent LC-MS/MS analysis of peptide structures. By this means we identified 24 peptides originating from 9 different peptide precursors. All gut peptides were found in identical form within the CNS and thus represent brain-gut peptides. Processing of Drosophila neuropeptide hormones was previously shown to require the subtilisin-like proprotein convertase 2 (dPC2, AMON). Our results suggest a general need of AMON for gut peptide production as well. In the second study, we could expand the knowledge on peptide structures of relevant insects. We investigated the peptidome of the cabbage maggot (i.e., the larva of the cabbage root fly Delia radicum), which causes substantial agricultural damage by feeding on plant roots. By mass spectrometric analysis of CNS, neurohemal organ and gut tissue, we could characterize 38 peptides belonging to diverse insect peptide families. Moreover, we identified a new peptide with sequence similarity to the eclosion hormone precursor of several Drosophila species. Immunocytochemical characterization of peptide-producing neurons and enteroendocrine cells in cabbage maggots showed that peptide distribution was largely similar to Drosophila larvae. The observed similarities in the peptidergic systems of both species suggest that Drosophila can serve as a genetically accessible pest species model in terms of peptidergic regulation of, e.g., metabolism. In the future, our results could be of use for the development of a targeted, peptide-based management of cabbage root flies. In the third study, we analyzed the role of allatostatin A (AstA), a peptide family commonly occurring in insects (and other arthropods). Previous studies had already demonstrated a role for AstA in metabolic regulation and nutritional homeostasis of Drosophila. We addressed the question whether specific effects were connected to the activity of certain subsets of the numerous AstA-producing cells found in adult fruit flies. AstA neurons are located in different regions of the CNS. The thorax also contains a few peripheral AstA neurons. The hindgut and the posteriormost portion of the midgut are innervated by central AstA neurons. In addition, a large number of enteroendocrine AstA cells are scattered across the epithelium of the posterior midgut. Thermogenetic activation of certain AstA cells significantly reduced food intake of flies, and also considerably diminished their locomotor activity. The combination of our results with findings of a previous study suggested that two pairs of AstA-producing posterior lateral protocerebrum neurons function to promote satiety, while enteroendocrine AstA cells seem to regulate locomotor activity. In addition, our findings indicated that AstA cells might directly and indirectly influence defecation behavior, while no effect on water and ion homeostasis could be observed. Furthermore, we tested the effect of synthetic AstA peptides on isolated midguts in vitro and observed a dose-dependent inhibition of midgut motility. Downregulation of AstA receptor mRNA in the gut musculature via RNAi showed that the DAR-2 receptor mediates the myoinhibitory effect of AstA peptides. Altogether, by influencing satiety, locomotion, gut peristalsis and possibly also defecation, AstA cells appear to affect different levels of metabolism and different tissues, seemingly promoting several interrelated processes connected to food intake

Peptidomics and Peptide Hormone Processing in the Drosophila Midgut

Peptide hormones are key messengers in the signaling network between the nervous system, endocrine glands, energy stores and the gastrointestinal tract that regulates feeding and metabolism. Studies on the Drosophila nervous system have uncovered parallels and homologies in homeostatic peptidergic signaling between fruit flies and vertebrates. Yet, the role of enteroendocrine peptides in the regulation of feeding and metabolism has not been explored, with research hampered by the unknown identity of peptides produced by the fly's intestinal tract. We performed a peptidomic LC/MS analysis of the fruit fly midgut containing the enteroendocrine cells. By MS/MS fragmentation, we found 24 peptides from 9 different preprohormones in midgut extracts, including MIP-4 and 2 forms of AST-C. DH31, CCHamide1 and CCHamide2 are biochemically characterized for the first time. All enteroendocrine peptides represent brain-gut peptides, and apparently are processed by Drosophila prohormone convertase 2 (AMON) as suggested by impaired peptide detectability in amon mutants and localization of amon-driven GFP to enteroendocrine cells. Because of its genetic amenability and peptide diversity, Drosophila provides a good model system to study peptide signaling. The identification of enteroendocrine peptides in the fruit fly provides a platform to address functions of gut peptide hormones in the regulation of feeding and metabolism

Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)

The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva

Peptidomics and processing of regulatory peptides in the fruit fly Drosophila

More than a decade has passed since the release of the Drosophila melanogaster genome and the first predictions of fruit fly regulatory peptides (neuropeptides and peptide hormones). Since then, mass spectrometry-based methods have fuelled the chemical characterisation of regulatory peptides, from 7 Drosophila peptides in the pre-genomic area to more than 60 today. We review the development of fruit fly peptidomics, and present a comprehensive list of the regulatory peptides that have been chemically characterised until today. We also summarise the knowledge on peptide processing in Drosophila, which has strongly profited from a combination of MS-based techniques and the genetic tools available for the fruit fly. This combination has a very high potential to study the functional biology of peptide signalling on all levels, especially with the ongoing developments in quantitative MS in Drosophila

Morphology of the neurohemal organs of a <i>D. radicum</i> larva, scanning electron microscopy.

<p>A) Dorsal view of the larval nervous system consisting of the two brain hemispheres (BR) and the ventral ganglion (VG, to the right) comprising the suboesophageal, thoracic and abdominal neuromeres. The ring gland (RG) is visible to the left, attached to the brain. The stars mark the abdominal PSOs. B) Enlarged picture of the ventral ganglion. Four abdominal PSOs (stars) are visible as thickenings of the median/transverse nerve. Also two blindly-ending thoracic PSOs (arrows) are visible. Oe  =  oesophagus, Tr  =  trachea.</p

Sequences, accession numbers and tissue distribution of the peptides characterised in <i>D. radicum</i> larvae.

<p>a)Leu and Ile have the same molecular mass. Since we did not obtain distinguishing high-energy collision w-fragments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041543#pone.0041543-Nachman2" target="_blank">[76]</a>, we are unable to distinguish between these two amino acids. Therefore, Leu and Ile in the sequences above have to be considered as predicted only based on the homolog peptides from <i>Drosophila</i> or other Dipterans. Small letter c within a sequence indicates cysteines that form an intramolecular disulfide bridge.</p><p>b)tPSO  =  thoracic PSO, aPSO  =  abdominal PSO, data from direct profiling of the dorsal sheath of the adult thoracico-abdominal ganglion.</p><p>c)these peptides could be sequenced in their SPITC-labelled form.</p><p>d)Mass peak indicative of this peptide appeared consistently, but could not be fragmented. Sequence adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041543#pone.0041543-Audsley1" target="_blank">[9]</a>.</p><p>e)a peptide with similar mass but different sequence (SPKQDFMRFa, 1154.6 Da and KPNQDFMRFa, 1181.6 Da) was reported by Audsley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041543#pone.0041543-Audsley1" target="_blank">[9]</a>.</p><p>f)The y9-fragment identifying the sequence order of positions 2–3 could not be found in SPITC-labelled and unlabeled spectra. The sequence LG is assumed since a very similar tachykinin (<i>Cav</i>-TKII: GLGNNAFVGVRa) was isolated and Edman-sequenced from the blowfly <i>Calliphora vomitoria</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041543#pone.0041543-Lundquist2" target="_blank">[73]</a>.</p><p>g)Only amino acids 1–11 of APK have been fully fragmented and are sequence identical to the N-terminus of APK of <i>Drosophila melanogaster</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041543#pone.0041543-Predel2" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041543#pone.0041543-Baggerman1" target="_blank">[28]</a>. The y-fragment representing amino acids 12–16 matches the mass of amino acids 12–15 of <i>Drosophila</i> APK plus the mass of tyrosine. Therefore, we assume the listed sequence. The position of the tyrosine and the C-terminal NAPK is not confirmed by fragmentation data.</p

MS/MS spectrum of Yamide.

<p>A+B) SPITC-labelled; C) unlabeled. A+B) The fragment spectrum was divided, therefore the relative intensities vary. y-fragments are labelled with blue lines, b-fragments with green lines. Internal and a-fragments are shown as well.</p

Daniel Solander to John Ellis

Daniel Solander to John Elli

PDF and tachykinin-like immunoreactivity in the larval gut.

<p>A) PDF-immunoreactive neurites innervate the hindgut, but do not reach the midgut or Malpighian tubules. B) Tachykinin-like-immunoreactive EECs are visible in a region possibly representing the anterior-middle midgut junction (asterisk). Further immunoreactive cells are scattered throughout the posterior midgut. The most posterior midgut portion is shown enlarged in the inset. PV  =  proventriculus, aMG  =  anterior midgut, pMG  =  posterior midgut. Scale bars  = 150 µm.</p