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-1(2-15), 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.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykininrelated peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykininrelated peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.Substance P was the first peptide signaling molecule to be identified by virtue of its effects upon blood pressure and smooth muscle contraction (1) and is the archetypal member of the tachykinin family of peptides. Vertebrate tachykinins represent a large family of peptides that elicit a wide range of both central and peripheral responses (2-5). Although these peptides are structurally diverse, all contain a conserved C-terminal FXGLM-amide motif. Like other biologically active peptides, substance P is derived from a larger prohormone polypeptide (preprotachykinin A (PPT-A) 1 ) that also allows the production of several other biologically active peptides (neurokinin A, neuropeptide K, and neuropeptide ␥) (6). Three different isoforms of preprotachykinin can be produced as a result of alternative splicing of the PPT-A mRNA, which, in conjunction with alternative post-translational processing of the prohormone, allows the production of these peptides in a tissue-specific manner (7-9). A fifth mammalian tachykinin, neurokinin B, is derived from a separate gene product, preprotachykinin B (10).The tachykinin family is not confined to vertebrates, and a large number of tachykinins have now been isolated from a variety of invertebrate species such as the cockroach Leucophaea maderae (11, 12), the mosquito Culex salinarius (13), and the echiuroid worm Urechis unicinctus (14). In contrast to the vertebrate tachykinins, almost all of the invertebrate tachykinins contain a conserved C-terminal FXGXR-amide motif and, for this reason, have been termed tachykinin-related peptides (TRPs). Not...

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.

The success of insects is linked to their impressive tolerance to environmental stress, but little is known about how such responses are mediated by the neuroendocrine system. Here we show that the capability (capa) neuropeptide gene is a desiccation-and cold stressresponsive gene in diverse dipteran species. Using targeted in vivo gene silencing, physiological manipulations, stress-tolerance assays, and rationally designed neuropeptide analogs, we demonstrate that the Drosophila melanogaster capa neuropeptide gene and its encoded peptides alter desiccation and cold tolerance. Knockdown of the capa gene increases desiccation tolerance but lengthens chill coma recovery time, and injection of capa peptide analogs can reverse both phenotypes. Immunohistochemical staining suggests that capa accumulates in the capa-expressing Va neurons during desiccation and nonlethal cold stress but is not released until recovery from each stress. Our results also suggest that regulation of cellular ion and water homeostasis mediated by capa peptide signaling in the insect Malpighian (renal) tubules is a key physiological mechanism during recovery from desiccation and cold stress. This work augments our understanding of how stress tolerance is mediated by neuroendocrine signaling and illustrates the use of rationally designed peptide analogs as agents for disrupting protective stress tolerance.environmental stress | insects | neuropeptides | capa | desiccation and cold tolerance